Bioethanol production from waste paper through fungal biotechnology
- Authors: Voigt, Paul George
- Date: 2010
- Subjects: Biomass energy , Cellulose -- Biodegradation , Waste paper -- Recycling , Biomass chemicals -- Economic aspects , Renewable energy sources , Fungi -- Biotechnology , Enzymes -- Biotechnology
- Language: English
- Type: Thesis , MSc , Masters
- Identifier: vital:3861 , http://hdl.handle.net/10962/d1013447
- Description: Bioethanol is likely to be a large contributor to the fuel sector of industry in the near future. Current research trends are geared towards utilizing food crops as substrate for bioethanol fermentation; however, this is the source of much controversy. Utilizing food crops for fuel purposes is anticipated to cause massive food shortages worldwide. Cellulose is the most abundant renewable resource on earth and is subject to a wide array of scientific study in order to utilize the glucose contained within it. Waste paper has a high degree of cellulose associated with it, which makes it an ideal target for cellulose biotechnology with the ultimate end goal of bioethanol production. This study focussed on producing the necessary enzymes to hydrolyse the cellulose found in waste paper and using the sugars produced to produce ethanol. The effects of various printing inks had on the production of sugars and the total envirorunental impact of the effluents produced during the production line were also examined. It was found that the fungus Trichoderma longibrachiatum DSM 769 grown in Mandel's medium with waste newspaper as the sole carbon source at 28 °C for 6 days produced extracellular cellulase enzymes with an activity of 0.203 ± 0.009 FPU.ml⁻¹, significantly higher activity as compared to other paper sources. This extracellular cellulase was used to hydrolyse waste newspaper and office paper, with office paper yielding the highest degree of sugar production with an end concentration of 5.80 ± 0.19 g/1 at 40 °C. Analysis by HPLC showed that although glucose was the major product at 4.35 ± 0.12 g/1, cellobiose was also produced in appreciable amounts (1.97 ± 0.71 g/1). The sugar solution was used as a substrate for Saccharomyces cerevisiae DSM 1333 and ethanol was produced at a level of 1.79 ± 0.26 g/1, the presence of which was confirmed by a 600 MHz NMR spectrum. It was found that cellobiose was not fermented by this strain of S. cerevisiae. Certain components of inks (the PAHs phenanthrene and naphthalene) were found to have a slight inhibitory effect (approximately 15% decrease) on the cellulase enzymes at very high concentrations (approximately 600 μg/1 in aqueous medium), while anthracene had no effect. Whole newsprint ink was shown not to sorb glucose. The environmental analysis of the effluents produced showed that in order for the effluents to be discharged into an aqueous ecosystem they would have to be diluted up to 200 times. They were also shown to have the potential to cause severe machinery damage if reused without proper treatment.
- Full Text:
- Authors: Voigt, Paul George
- Date: 2010
- Subjects: Biomass energy , Cellulose -- Biodegradation , Waste paper -- Recycling , Biomass chemicals -- Economic aspects , Renewable energy sources , Fungi -- Biotechnology , Enzymes -- Biotechnology
- Language: English
- Type: Thesis , MSc , Masters
- Identifier: vital:3861 , http://hdl.handle.net/10962/d1013447
- Description: Bioethanol is likely to be a large contributor to the fuel sector of industry in the near future. Current research trends are geared towards utilizing food crops as substrate for bioethanol fermentation; however, this is the source of much controversy. Utilizing food crops for fuel purposes is anticipated to cause massive food shortages worldwide. Cellulose is the most abundant renewable resource on earth and is subject to a wide array of scientific study in order to utilize the glucose contained within it. Waste paper has a high degree of cellulose associated with it, which makes it an ideal target for cellulose biotechnology with the ultimate end goal of bioethanol production. This study focussed on producing the necessary enzymes to hydrolyse the cellulose found in waste paper and using the sugars produced to produce ethanol. The effects of various printing inks had on the production of sugars and the total envirorunental impact of the effluents produced during the production line were also examined. It was found that the fungus Trichoderma longibrachiatum DSM 769 grown in Mandel's medium with waste newspaper as the sole carbon source at 28 °C for 6 days produced extracellular cellulase enzymes with an activity of 0.203 ± 0.009 FPU.ml⁻¹, significantly higher activity as compared to other paper sources. This extracellular cellulase was used to hydrolyse waste newspaper and office paper, with office paper yielding the highest degree of sugar production with an end concentration of 5.80 ± 0.19 g/1 at 40 °C. Analysis by HPLC showed that although glucose was the major product at 4.35 ± 0.12 g/1, cellobiose was also produced in appreciable amounts (1.97 ± 0.71 g/1). The sugar solution was used as a substrate for Saccharomyces cerevisiae DSM 1333 and ethanol was produced at a level of 1.79 ± 0.26 g/1, the presence of which was confirmed by a 600 MHz NMR spectrum. It was found that cellobiose was not fermented by this strain of S. cerevisiae. Certain components of inks (the PAHs phenanthrene and naphthalene) were found to have a slight inhibitory effect (approximately 15% decrease) on the cellulase enzymes at very high concentrations (approximately 600 μg/1 in aqueous medium), while anthracene had no effect. Whole newsprint ink was shown not to sorb glucose. The environmental analysis of the effluents produced showed that in order for the effluents to be discharged into an aqueous ecosystem they would have to be diluted up to 200 times. They were also shown to have the potential to cause severe machinery damage if reused without proper treatment.
- Full Text:
An investigation into the possible neuroprotective or neurotoxic properties of metrifonate
- Authors: Ramsunder, Adrusha
- Date: 2005 , 2013-06-11
- Subjects: Nervous system -- Degeneration -- Treatment , Neurotoxic agents , Alzheimer's disease -- Treatment , Metrifonate
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3833 , http://hdl.handle.net/10962/d1007560 , Nervous system -- Degeneration -- Treatment , Neurotoxic agents , Alzheimer's disease -- Treatment , Metrifonate
- Description: Alzheimer's disease is a progressive neurodegenerative disorder, in which there is a marked decline in neurotransmitters, especially those of the cholinergic pathways. One of the approaches to the symptomatic treatment of Alzheimer's disease is the inhibition of the breakdown of the neurotransmitter acetylcholine, using an acetylcholinesterase inhibitor. One such drug tested, is the organophosphate, metrifonate. Any drug used for the treatment of neurodegenerative disorders should preferably not induce further neurological damage. Thus, in the present study, we investigated whether or not metrifonate is neuroprotective. The in vivo and in vitro effect of this drug on free radicals generation shows that metrifonate increases the level ofthese reactive species. Lipid peroxidation induced using quinolinic acid (QA) and iron (II) and show that metrifonate increased the peroxidative damage induced by using quinolinic acid. Metrifonate is also able to induce lipid peroxidation both in vivo and in vitro. This was reduced in vitro in the presence of melatonin. Using iron (II), in vi/ro, there was no significant difference in the level of lipid peroxidation in the presence of this drug. An investigation of the activity of the mitochondrial electron transport chain and complex I of the electron transport chain in the presence of metrifonate revealed that metrifonate reduces the activity of the electron transport chain at the level of complex I. The activity of the mitochondrial electron transport chain was restored in the presence of melatonin. Pineal organ culture showed that metrifonate does not increase melatonin production. Histological and apoptosis studies show that tissue necrosis and apoptosis respectively, occur in the presence of this agent, which is reduced in the presence of melatonin. Metal binding studies were performed USing ultraviolet spectroscopy, and electrochemical analysis to examine the interaction of metrifonate with iron (II) and iron (III). No shift in the peak was observed in the ultraviolet spectrum when iron (ll) was added to metrifonate. Electrochemical studies show that there may be a very weak or no ligand formed between the metal and drug. This study shows that while drugs such as metrifonate may be beneficial in restoring cognitive function in Alzheimer's disease, it could also have the potential to enhance neurodegeneration, thus worsening the condition, in the long term. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Authors: Ramsunder, Adrusha
- Date: 2005 , 2013-06-11
- Subjects: Nervous system -- Degeneration -- Treatment , Neurotoxic agents , Alzheimer's disease -- Treatment , Metrifonate
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3833 , http://hdl.handle.net/10962/d1007560 , Nervous system -- Degeneration -- Treatment , Neurotoxic agents , Alzheimer's disease -- Treatment , Metrifonate
- Description: Alzheimer's disease is a progressive neurodegenerative disorder, in which there is a marked decline in neurotransmitters, especially those of the cholinergic pathways. One of the approaches to the symptomatic treatment of Alzheimer's disease is the inhibition of the breakdown of the neurotransmitter acetylcholine, using an acetylcholinesterase inhibitor. One such drug tested, is the organophosphate, metrifonate. Any drug used for the treatment of neurodegenerative disorders should preferably not induce further neurological damage. Thus, in the present study, we investigated whether or not metrifonate is neuroprotective. The in vivo and in vitro effect of this drug on free radicals generation shows that metrifonate increases the level ofthese reactive species. Lipid peroxidation induced using quinolinic acid (QA) and iron (II) and show that metrifonate increased the peroxidative damage induced by using quinolinic acid. Metrifonate is also able to induce lipid peroxidation both in vivo and in vitro. This was reduced in vitro in the presence of melatonin. Using iron (II), in vi/ro, there was no significant difference in the level of lipid peroxidation in the presence of this drug. An investigation of the activity of the mitochondrial electron transport chain and complex I of the electron transport chain in the presence of metrifonate revealed that metrifonate reduces the activity of the electron transport chain at the level of complex I. The activity of the mitochondrial electron transport chain was restored in the presence of melatonin. Pineal organ culture showed that metrifonate does not increase melatonin production. Histological and apoptosis studies show that tissue necrosis and apoptosis respectively, occur in the presence of this agent, which is reduced in the presence of melatonin. Metal binding studies were performed USing ultraviolet spectroscopy, and electrochemical analysis to examine the interaction of metrifonate with iron (II) and iron (III). No shift in the peak was observed in the ultraviolet spectrum when iron (ll) was added to metrifonate. Electrochemical studies show that there may be a very weak or no ligand formed between the metal and drug. This study shows that while drugs such as metrifonate may be beneficial in restoring cognitive function in Alzheimer's disease, it could also have the potential to enhance neurodegeneration, thus worsening the condition, in the long term. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
An investigation into the physico-chemical and neuroprotective properties of melatonin and 6-hydroxymelatonin
- Authors: Maharaj, Deepa Sukhdev
- Date: 2003
- Subjects: Melatonin Nervous system -- Degeneration -- Treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3768 , http://hdl.handle.net/10962/d1003246
- Description: Until the beginning of this decade the antioxidant, melatonin, had been considered as little more than a tranquilizing hormone, responsible for regulating certain circadian and circannual rhythms. However, it is the discovery of melatonin as a free radical scavenger that has generated the most interest in recent years. The reduction of melatonin with age has been associated with neurodegenerative diseases such as Alzheimer’s disease (AD)and therefore, melatonin has been implicated to have an important clinical role in neuroprotection. Thus, for several years melatonin has attracted increasing attention from the general press with many advertisements touting this indoleamine to act as an aphrodisiac, rejuvenator, protector against diseases and a general wonder drug. However, melatonin formulations appear with no labelling for the correct storage conditions, dosage and side effects, as well as no control for purity and self-medicating with an unregulated product. In addition, there is much controversy surrounding the antioxidative properties of the indolemaine, 6-hydroxymelatonin (6-OHM). Therefore, the first part of this study aims to elucidate the physico-chemical and various stability characteristics of the pineal antioxidant, melatonin, while the second part is devoted to investigating the neuroprotective properties of the primary hepatic metabolite of melatonin, 6-OHM. The physical properties of melatonin were determined using various chemical techniques. This information served to both characterize and confirm the identity of melatonin raw material used in this study. In addition, this information serves to be essential as the physical properties of melatonin have not been reported in detail in literature, to date. Thereafter, using a validated high performance liquid chromatography (HPLC) method, the various physico-chemical and stability characteristics of melatonin were determined. Melatonin was shown to be extremely lipophilic, while the hygroscopic study indicates that melatonin raw material is extremely hygroscopic at temperatures above 40°C, whereas melatonin tablets are hygroscopic when left out of the original container. This study highlights the need for consumers to be aware of the proper storage of melatonin tablets to improve the stability and ensure long term integrity of the compound. Since, melatonin is most often administered orally, thus exposing it to a large variations in pH, within the gastrointestinal tract, it was decided to investigate the stability of melatonin over a range of pH’s and temperatures. The findings imply that melatonin is relatively stable at body temperature when ingested orally and that orally administered slow release preparations of melatonin should be relatively stable and therefore exhibit favourable bioavailability. However melatonin was shown to be unstable in solution. This provides important information and a challenge to the formulators of this drug substance in a liquid dosage form. An assessment of the photostability of melatonin dosage forms using International Committee on Harmonization (ICH) conditions revealed melatonin to be light sensitive and thus indicates a need for careful consideration of the packaging of these drug products. In addition a detailed assessment of the photochemistry and photoproducts formed during the UV photodegradation of melatonin is reported. Melatonin is shown to rapidly degrade in the presence of UV light, with the presence of oxygen accelerating the photodegradation. N1-acetyl-N2-formyl-5-methoxykynurenamine(AFMK) and 6-OHM were identified as the major photoproducts formed and these agents have been shown previously to retain antioxidant activity. One of the concerns of using melatonin in sunscreens is its photostability. However, it is reported in this study that the degraded solution of melatonin still possesses equipotent free radical scavenging ability as melatonin, despite the absence of melatonin in solution. In addition, melatonin is shown to reduce UV-induced oxidative stress in rat skin homogenate. Thus, these results make melatonin a likely candidate for inclusion in sunscreen preparations. Neuronal damage due to oxidative stress has been implicated in several neurodegenerative disorders. 6-OHM is not only formed as the major hepatic metabolite of melatonin, but also when melatonin reacts with toxic radicals as well as UV light. Thus the second part of the study aims to elucidate and further characterize the mechanism behind 6-OHM’s neuroprotection. The results show 6-OHM to be a more potent singlet oxygen and superoxide anion scavenger than melatonin. In addition, the results show 6-OHM to offer protection against, oxidative stress and lipid peroxidation induced by several neurotoxins in the rat brain and hippocampus. The hippocampus is an important region of the brain responsible for the formation of memory and any agent that induces stress in this area has detrimental effects and could lead to various types of dementia. Such agents include quinolinic acid (QA) and iron (II). Histological studies undertaken reveal that 6-OHM is able to protect hippocampal neurons against QA and iron (II) induced necrotic cell death. Immunohistochemical investigations showed that QA moderately induces apoptotic cell death in the hippocampus which is inhibited by both melatonin and 6-OHM. The study sought to elucidate possible mechanisms by which 6-OHM exerts its neuroprotective capabilities and the results show 6-OHM to inhibit the action of cyanide on the mitochondrial electron transport chain (ETC), one of the most common sources of free radicals. In addition, 6-OHM treatment alone, increased ETC activity above basal control levels and the results show 6-OHM to increase complex I activity in the mitochondrial ETC. Electrochemical, ultraviolet/visible spectroscopy (UV/Vis) and HPLC assessment show that an interaction exists between 6-OHM and iron (III) and 6-OHM is able to reduce iron (III) to a more biologically usable form viz. iron (II) which can be incorporated into important biomolecules such as heme. One dire consequence of this interaction is the ready provision of iron (II) to drive the Fenton reaction. However the biological and histological assessments show 6-OHM to prevent iron (II)-induced lipid peroxidation and necrotic cell death and thus, provide evidence of its antioxidant properties. The results also show 6-OHM to promote Hsp70 induction in the hippocampus. Heat shock proteins, especially Hsp 70 plays a role in cytoprotection by capturing denatured proteins and facilitating the refolding of these proteins once the stress has been relieved. 6-OHM treatment alone and together with QA was shown to increase the level of expression of Hsp70, both inducible and cognate forms of the protein. This suggests that 6-OHM helps to protect against cellular protein damage induced by any form of stress the cell may encounter. Melatonin treatment alone and in combination with QA was shown to prevent increases in the level of Hsp70 in the hippocampus, indicating that melatonin was able to reduce oxidative stress induced by QA such that Hsp70 expression was not required. The discovery of neuroprotective agents, such as melatonin and 6-OHM, is becoming important considering the rapid rise in the elderly population and the proportionate increase in neurological disorders. The findings of this study indicate the need for important information regarding the correct storage conditions and stability characteristics of melatonin dosage forms. In addition, the results indicate that 6-OHM has a definite role to play as an antioxidant. Thus further research may favour the use of these agents in the treatment of several neurodegenerative disorders.
- Full Text:
- Authors: Maharaj, Deepa Sukhdev
- Date: 2003
- Subjects: Melatonin Nervous system -- Degeneration -- Treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3768 , http://hdl.handle.net/10962/d1003246
- Description: Until the beginning of this decade the antioxidant, melatonin, had been considered as little more than a tranquilizing hormone, responsible for regulating certain circadian and circannual rhythms. However, it is the discovery of melatonin as a free radical scavenger that has generated the most interest in recent years. The reduction of melatonin with age has been associated with neurodegenerative diseases such as Alzheimer’s disease (AD)and therefore, melatonin has been implicated to have an important clinical role in neuroprotection. Thus, for several years melatonin has attracted increasing attention from the general press with many advertisements touting this indoleamine to act as an aphrodisiac, rejuvenator, protector against diseases and a general wonder drug. However, melatonin formulations appear with no labelling for the correct storage conditions, dosage and side effects, as well as no control for purity and self-medicating with an unregulated product. In addition, there is much controversy surrounding the antioxidative properties of the indolemaine, 6-hydroxymelatonin (6-OHM). Therefore, the first part of this study aims to elucidate the physico-chemical and various stability characteristics of the pineal antioxidant, melatonin, while the second part is devoted to investigating the neuroprotective properties of the primary hepatic metabolite of melatonin, 6-OHM. The physical properties of melatonin were determined using various chemical techniques. This information served to both characterize and confirm the identity of melatonin raw material used in this study. In addition, this information serves to be essential as the physical properties of melatonin have not been reported in detail in literature, to date. Thereafter, using a validated high performance liquid chromatography (HPLC) method, the various physico-chemical and stability characteristics of melatonin were determined. Melatonin was shown to be extremely lipophilic, while the hygroscopic study indicates that melatonin raw material is extremely hygroscopic at temperatures above 40°C, whereas melatonin tablets are hygroscopic when left out of the original container. This study highlights the need for consumers to be aware of the proper storage of melatonin tablets to improve the stability and ensure long term integrity of the compound. Since, melatonin is most often administered orally, thus exposing it to a large variations in pH, within the gastrointestinal tract, it was decided to investigate the stability of melatonin over a range of pH’s and temperatures. The findings imply that melatonin is relatively stable at body temperature when ingested orally and that orally administered slow release preparations of melatonin should be relatively stable and therefore exhibit favourable bioavailability. However melatonin was shown to be unstable in solution. This provides important information and a challenge to the formulators of this drug substance in a liquid dosage form. An assessment of the photostability of melatonin dosage forms using International Committee on Harmonization (ICH) conditions revealed melatonin to be light sensitive and thus indicates a need for careful consideration of the packaging of these drug products. In addition a detailed assessment of the photochemistry and photoproducts formed during the UV photodegradation of melatonin is reported. Melatonin is shown to rapidly degrade in the presence of UV light, with the presence of oxygen accelerating the photodegradation. N1-acetyl-N2-formyl-5-methoxykynurenamine(AFMK) and 6-OHM were identified as the major photoproducts formed and these agents have been shown previously to retain antioxidant activity. One of the concerns of using melatonin in sunscreens is its photostability. However, it is reported in this study that the degraded solution of melatonin still possesses equipotent free radical scavenging ability as melatonin, despite the absence of melatonin in solution. In addition, melatonin is shown to reduce UV-induced oxidative stress in rat skin homogenate. Thus, these results make melatonin a likely candidate for inclusion in sunscreen preparations. Neuronal damage due to oxidative stress has been implicated in several neurodegenerative disorders. 6-OHM is not only formed as the major hepatic metabolite of melatonin, but also when melatonin reacts with toxic radicals as well as UV light. Thus the second part of the study aims to elucidate and further characterize the mechanism behind 6-OHM’s neuroprotection. The results show 6-OHM to be a more potent singlet oxygen and superoxide anion scavenger than melatonin. In addition, the results show 6-OHM to offer protection against, oxidative stress and lipid peroxidation induced by several neurotoxins in the rat brain and hippocampus. The hippocampus is an important region of the brain responsible for the formation of memory and any agent that induces stress in this area has detrimental effects and could lead to various types of dementia. Such agents include quinolinic acid (QA) and iron (II). Histological studies undertaken reveal that 6-OHM is able to protect hippocampal neurons against QA and iron (II) induced necrotic cell death. Immunohistochemical investigations showed that QA moderately induces apoptotic cell death in the hippocampus which is inhibited by both melatonin and 6-OHM. The study sought to elucidate possible mechanisms by which 6-OHM exerts its neuroprotective capabilities and the results show 6-OHM to inhibit the action of cyanide on the mitochondrial electron transport chain (ETC), one of the most common sources of free radicals. In addition, 6-OHM treatment alone, increased ETC activity above basal control levels and the results show 6-OHM to increase complex I activity in the mitochondrial ETC. Electrochemical, ultraviolet/visible spectroscopy (UV/Vis) and HPLC assessment show that an interaction exists between 6-OHM and iron (III) and 6-OHM is able to reduce iron (III) to a more biologically usable form viz. iron (II) which can be incorporated into important biomolecules such as heme. One dire consequence of this interaction is the ready provision of iron (II) to drive the Fenton reaction. However the biological and histological assessments show 6-OHM to prevent iron (II)-induced lipid peroxidation and necrotic cell death and thus, provide evidence of its antioxidant properties. The results also show 6-OHM to promote Hsp70 induction in the hippocampus. Heat shock proteins, especially Hsp 70 plays a role in cytoprotection by capturing denatured proteins and facilitating the refolding of these proteins once the stress has been relieved. 6-OHM treatment alone and together with QA was shown to increase the level of expression of Hsp70, both inducible and cognate forms of the protein. This suggests that 6-OHM helps to protect against cellular protein damage induced by any form of stress the cell may encounter. Melatonin treatment alone and in combination with QA was shown to prevent increases in the level of Hsp70 in the hippocampus, indicating that melatonin was able to reduce oxidative stress induced by QA such that Hsp70 expression was not required. The discovery of neuroprotective agents, such as melatonin and 6-OHM, is becoming important considering the rapid rise in the elderly population and the proportionate increase in neurological disorders. The findings of this study indicate the need for important information regarding the correct storage conditions and stability characteristics of melatonin dosage forms. In addition, the results indicate that 6-OHM has a definite role to play as an antioxidant. Thus further research may favour the use of these agents in the treatment of several neurodegenerative disorders.
- Full Text:
An investigation into the possible neuroprotective role of antidepressant drugs
- Authors: Steiner, Claire
- Date: 2002
- Subjects: Antidepressants -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3792 , http://hdl.handle.net/10962/d1003270 , Antidepressants -- Research
- Description: Antidepressants are widely used in the treatment of depressive illnesses associated with neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. Neuroprotection in such disorders is of vital importance in order to delay the progression of the primary disorder. The pathology of neurodegeneration is not fully understood. It is however widely accepted that oxidative stress and excitotoxicity play a major role. Brain tissue is rich in phospholipids, which are especially prone to oxidation due to the high level of oxygen utilization in the brain. In addition, the brain lacks defence mechanisms to protect it against the wrath of free radicals. Presently, there is a wide variety of antidepressant drugs available. These range from the original tricyclic antidepressants to the newer selective serotonin reuptake inhibitors. It is not known whether antidepressant drugs, old or new, offer neuroprotection or how the existing state and/or the progression of neurodegeneration, is influenced by these agents. The present study was undertaken to determine how nortriptyline, trimipramine and fluoxetine affect neurodegeneration. Initial in vitro and in vivo studies show that all three of the antidepressants (0-1mM) studied provide neuroprotection from quinolinic acid induced lipid peroxidation. A histological investigation supported these findings by showing that a marginal degree of neuroprotection is apparent when treating animals with antidepressants (10mg/kg) before and following quinolinic acid intrastriatal injection. Further studies were undertaken in an attempt to determine the mode of neuroprotective action of the agents studied. An in vitro study of superoxide anion induced lipid peroxidation indicates that these agents do not act as antioxidants. The influence of the antidepressants on tryptophan 2,3-dioxygenase activity was assessed, based on the understanding that inhibition of this enzyme results in increased levels of the known antioxidant indoleamine, melatonin. Nortriptyline hydrochloride is seen to inhibit tryptophan 2,3-dioxygenase activity and as such it is possible that this antidepressant can indirectly provide neuroprotection by increasing available melatonin. Electrochemical and UV/visible studies show that trimipramine maleate interacts with free iron (II) and iron (III) ions. Free metal ions can catalyse the formation of damaging free radicals. Through interaction with trimipramine maleate, these ions will be unavailable to the system and thus cannot contribute to oxidative stress. The findings of this study indicate that antidepressants may be able to provide neuroprotection to neuronal cells. The mode of such neuroprotective actions need to be further examined so that patients suffering from depression coexisting with neurodegenerative diseases can be safely and effectively treated.
- Full Text:
- Authors: Steiner, Claire
- Date: 2002
- Subjects: Antidepressants -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3792 , http://hdl.handle.net/10962/d1003270 , Antidepressants -- Research
- Description: Antidepressants are widely used in the treatment of depressive illnesses associated with neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. Neuroprotection in such disorders is of vital importance in order to delay the progression of the primary disorder. The pathology of neurodegeneration is not fully understood. It is however widely accepted that oxidative stress and excitotoxicity play a major role. Brain tissue is rich in phospholipids, which are especially prone to oxidation due to the high level of oxygen utilization in the brain. In addition, the brain lacks defence mechanisms to protect it against the wrath of free radicals. Presently, there is a wide variety of antidepressant drugs available. These range from the original tricyclic antidepressants to the newer selective serotonin reuptake inhibitors. It is not known whether antidepressant drugs, old or new, offer neuroprotection or how the existing state and/or the progression of neurodegeneration, is influenced by these agents. The present study was undertaken to determine how nortriptyline, trimipramine and fluoxetine affect neurodegeneration. Initial in vitro and in vivo studies show that all three of the antidepressants (0-1mM) studied provide neuroprotection from quinolinic acid induced lipid peroxidation. A histological investigation supported these findings by showing that a marginal degree of neuroprotection is apparent when treating animals with antidepressants (10mg/kg) before and following quinolinic acid intrastriatal injection. Further studies were undertaken in an attempt to determine the mode of neuroprotective action of the agents studied. An in vitro study of superoxide anion induced lipid peroxidation indicates that these agents do not act as antioxidants. The influence of the antidepressants on tryptophan 2,3-dioxygenase activity was assessed, based on the understanding that inhibition of this enzyme results in increased levels of the known antioxidant indoleamine, melatonin. Nortriptyline hydrochloride is seen to inhibit tryptophan 2,3-dioxygenase activity and as such it is possible that this antidepressant can indirectly provide neuroprotection by increasing available melatonin. Electrochemical and UV/visible studies show that trimipramine maleate interacts with free iron (II) and iron (III) ions. Free metal ions can catalyse the formation of damaging free radicals. Through interaction with trimipramine maleate, these ions will be unavailable to the system and thus cannot contribute to oxidative stress. The findings of this study indicate that antidepressants may be able to provide neuroprotection to neuronal cells. The mode of such neuroprotective actions need to be further examined so that patients suffering from depression coexisting with neurodegenerative diseases can be safely and effectively treated.
- Full Text:
An investigation of the neuroprotective effects of estrogen in a model of quinolinic acid-induced neurodegeneration
- Authors: Heron, Paula Michelle
- Date: 2002
- Subjects: Estrogen , Quinolinic acid , Nervous system -- Degeneration
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3759 , http://hdl.handle.net/10962/d1003237 , Estrogen , Quinolinic acid , Nervous system -- Degeneration
- Description: The hippocampus, located in the medial temporal lobe, is an important region of the brain responsible for the formation of memory. Thus, any agent that induces stress in this area has detrimental effects and could lead to various types of dementia. Such agents include the neurotoxin, Quinolinic acid. Quinolinic acid (QUIN) is a neurotoxic metabolite of the tryptophan-kynurenine pathway and is an endogenous glutamate agonist that selectively injures and kills vulnerable neurons via the activation of the NMDA class of excitatory amino acid receptors. Estrogen is a female hormone that is responsible for reproduction. However, in the last decade estrogen has been shown to exhibit a wide range of actions on the brain, including neuroprotection. Estrogen has been shown to exhibit intrinsic antioxidant activity and protects cultured neurons against oxidative cell death. This is achieved by estrogen’s ability to scavenge free radicals, which is dependent on the presence of the hydroxyl group at the C3 position on the A ring of the steroid molecule. Numerous studies have shown that estrogen protects neurons against various toxic substances and may play a role in delaying the onset of neurodegenerative diseases, such as Alzheimer’s disease. Neuronal damage due to oxidative stress has been implicated in several neurodegenerative disorders. The detection and measurement of lipid peroxidation is the evidence most frequently cited to support the involvement of free radical reactions in toxicology and in human disease. The study aims to elucidate and further characterise the mechanism behind estrogen’s neuroprotection, using QUIN as a model of neurotoxicity. Initial studies confirm estrogen’s ability to scavenge potent free radicals. In addition, the results show that estrogen forms an interaction with iron (II) and also acts at the NMDA receptor as an agonist. Both mechanisms reduce the ability of QUIN to cause damage to neurons, since QUIN-induced toxicity is dependent on the activation of the NMDA receptor and the formation of a complex with iron (II) to induce lipid peroxidation. Heat shock proteins, especially Hsp 70 play a role in cytoprotection by capturing denatured proteins and facilitating the refolding of these proteins once the stress has been relieved. Estrogen has been shown to increase the level of expression of Hsp70, both inducible and cognate forms of the protein. This suggests that estrogen helps to protect against cellular protein damage induced by any form of stress the cell may encounter. The discovery of neuroprotective agents, such as estrogen, is becoming important as accumulating evidence indicates a protective role in vivo. Thus further research may favour the use of these agents in the treatment of several neurodegenerative disorders. Considering how devastating diseases, such as Alzheimer’s disease, are to a patient and the patient’s families, the discovery of new protective agents are a matter of urgency.
- Full Text:
- Authors: Heron, Paula Michelle
- Date: 2002
- Subjects: Estrogen , Quinolinic acid , Nervous system -- Degeneration
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3759 , http://hdl.handle.net/10962/d1003237 , Estrogen , Quinolinic acid , Nervous system -- Degeneration
- Description: The hippocampus, located in the medial temporal lobe, is an important region of the brain responsible for the formation of memory. Thus, any agent that induces stress in this area has detrimental effects and could lead to various types of dementia. Such agents include the neurotoxin, Quinolinic acid. Quinolinic acid (QUIN) is a neurotoxic metabolite of the tryptophan-kynurenine pathway and is an endogenous glutamate agonist that selectively injures and kills vulnerable neurons via the activation of the NMDA class of excitatory amino acid receptors. Estrogen is a female hormone that is responsible for reproduction. However, in the last decade estrogen has been shown to exhibit a wide range of actions on the brain, including neuroprotection. Estrogen has been shown to exhibit intrinsic antioxidant activity and protects cultured neurons against oxidative cell death. This is achieved by estrogen’s ability to scavenge free radicals, which is dependent on the presence of the hydroxyl group at the C3 position on the A ring of the steroid molecule. Numerous studies have shown that estrogen protects neurons against various toxic substances and may play a role in delaying the onset of neurodegenerative diseases, such as Alzheimer’s disease. Neuronal damage due to oxidative stress has been implicated in several neurodegenerative disorders. The detection and measurement of lipid peroxidation is the evidence most frequently cited to support the involvement of free radical reactions in toxicology and in human disease. The study aims to elucidate and further characterise the mechanism behind estrogen’s neuroprotection, using QUIN as a model of neurotoxicity. Initial studies confirm estrogen’s ability to scavenge potent free radicals. In addition, the results show that estrogen forms an interaction with iron (II) and also acts at the NMDA receptor as an agonist. Both mechanisms reduce the ability of QUIN to cause damage to neurons, since QUIN-induced toxicity is dependent on the activation of the NMDA receptor and the formation of a complex with iron (II) to induce lipid peroxidation. Heat shock proteins, especially Hsp 70 play a role in cytoprotection by capturing denatured proteins and facilitating the refolding of these proteins once the stress has been relieved. Estrogen has been shown to increase the level of expression of Hsp70, both inducible and cognate forms of the protein. This suggests that estrogen helps to protect against cellular protein damage induced by any form of stress the cell may encounter. The discovery of neuroprotective agents, such as estrogen, is becoming important as accumulating evidence indicates a protective role in vivo. Thus further research may favour the use of these agents in the treatment of several neurodegenerative disorders. Considering how devastating diseases, such as Alzheimer’s disease, are to a patient and the patient’s families, the discovery of new protective agents are a matter of urgency.
- Full Text:
An investigation into the possible neuroprotective role of melatonin in copper-loading
- Authors: Parmar, Paresh H
- Date: 2001
- Subjects: Melatonin , Copper , Nervous system -- Degeneration -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3783 , http://hdl.handle.net/10962/d1003261
- Description: Copper is an extremely toxic metal in biological systems and thus, its availability to the system, must be effectively and efficiently controlled. Copper is vital for life, as it is essential for critical enzymes in biological systems. It is free copper in the biological systems that is toxic, as free copper induces free radical generation, which disrupts lipid membranes, interacts with DNA causing mutations, and eventually leads to cell death. Wilson’s disease is a inherited copper disease, which results in hepatolenticular disease. Copper is unable to be excreted, and thus accumulates, eventually spilling over into the bloodstream from the liver, and “poisons” the patient. The Wilson’s disease patient leads a short life, due to neurological and hepatological problems. There is no cure for Wilson’s disease, only chelation therapy using potent chelators such as penicillamine and EDTA. Zinc, in high doses, can be used to compete with copper absorption. This has proved to be the only successful therapy at present. This study investigates the possible use of melatonin as a copper binder/chelator. Melatonin has been shown to interact with copper in vitro. By binding/chelating to copper, melatonin may inhibit copper-induced free radical generation, and thus prevent copper from interacting with DNA to cause mutations and act as a cytotoxin. In vivo studies on copper (2mg/kg) administered for 2-weeks and 6-weeks were carried out on Wistar rats. The potential of melatonin (12mg/kg) to prevent copper-induced cellular damage was investigated. The results indicate that melatonin does not protect the lipid membranes from copper-induced lipid peroxidation. In vitro investigations using 1mM, 5mM and 10mM copper and 5mM melatonin, show that melatonin prevents copper-induced lipid peroxidation at a copper concentration of 1mM (p<0.001). The 5mM and 10mM copper induces less lipid peroxidation, compared to the 1mM copper. It has been reported that metal ions, antioxidants and chelating agents can influence peroxide decomposition during the assay. Melatonin (5mM) administration does not significantly prevent copper-induced lipid peroxidation at 5mM and 10mM copper. It is possible that due to melatonin’s relatively low concentration, it is unable to inhibit lipid peroxidation induced by the copper. The chemical nature of the interaction between melatonin and copper was also investigated, using NMR, IR and electrochemistry techniques. The NMR and IR techniques show that melatonin coordinates with Cu²⁺ and not Cu¹⁺, at the carbonyl group of melatonin. The electrochemistry experiments using cyclic voltammetry and adsorptive stripping voltammetry, show that melatonin forms a strong bond with Cu¹⁺. Cu²⁺ prefers binding to oxygen, and that is clearly seen in the NMR and IR. Cu¹⁺ prefers binding to nitrogen and then oxygen, and this is seen in the electrochemistry, as Cu¹⁺ is forced to bind through one of the nitrogens on the melatonin. Previously, it has been shown that melatonin binds/chelates with Cu²⁺. Histochemical investigations show that copper administration for 2-weeks and 6-weeks, causes extensive mitochondrial damage in liver and kidney’s proximal convoluted tubule epithelium cells. Melatonin (12mg/kg) co-administration with copper for 2-weeks and 6-weeks did not significantly protect the mitochondria from copper-induced damage. Copper-specific stains (rhodanine, silver sulphide and rubeanic acid) were used to stain liver, brain and kidney tissue samples. Rhodanine and silver sulphide were equally sensitive in staining copper in the 2-week samples, but not at all in the 6-week samples. This could not be explained. Rubeanic acid was ineffective in all samples tested. Thus, it appears that specific copper stains cannot be used in making a definitive diagnosis in cases of copper overload, and that specific copper stains do not always correlate with a high concentration of copper present in tissues. Pineal organ culture was used to determine the effect of copper administration on pineal indole synthesis. Exogenous (³H) tryptophan was administered to the pineal organ cultures, and the level of (³H) pineal indoles synthesised, were measured. Pineals from 2-week and 6-week copper/melatonin treated animals exhibited paradoxical 5- methoxytryptophol (ML) levels, as compared to the 2-week and 6-week copper treated animals. The 2-week copper/melatonin administered animals, showed a decrease in the ML level (p<0.01), and the copper/melatonin administered for 6-weeks, showed an increase in the ML levels (p<0.01). This indicates that melatonin interacts with the HIOMT enzyme. Pineals from 6-week copper/melatonin treated animals, as compared to the 6-week copper treated animals, showed an increase in N-acetylserotonin levels. This indicates that melatonin prevents the inhibition of the NAT enzyme. The final experiment was to determine in vitro, the effect of Cu²⁺ and Cu¹⁺ administration, on mitochondrial electron transport chain. Rat liver homogenate was incubated with and solutions of Cu²⁺ (10mM) and Cu¹⁺ (10mM) and melatonin (10mM). Cu²⁺ administration caused an inhibition of the electron transport at t=0 and t=60, whereas Cu¹⁺ administration at t=0 caused an inhibition of electron transport, but at t=60, Cu¹⁺ administration stimulated electron transport. Melatonin administered with Cu²⁺, resulted in an inhibition of the electron transport chain at t=0 and t=60. The findings of this study indicate that melatonin might have a potentially beneficial effect in copper overloading, by binding/chelating copper.
- Full Text:
- Authors: Parmar, Paresh H
- Date: 2001
- Subjects: Melatonin , Copper , Nervous system -- Degeneration -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3783 , http://hdl.handle.net/10962/d1003261
- Description: Copper is an extremely toxic metal in biological systems and thus, its availability to the system, must be effectively and efficiently controlled. Copper is vital for life, as it is essential for critical enzymes in biological systems. It is free copper in the biological systems that is toxic, as free copper induces free radical generation, which disrupts lipid membranes, interacts with DNA causing mutations, and eventually leads to cell death. Wilson’s disease is a inherited copper disease, which results in hepatolenticular disease. Copper is unable to be excreted, and thus accumulates, eventually spilling over into the bloodstream from the liver, and “poisons” the patient. The Wilson’s disease patient leads a short life, due to neurological and hepatological problems. There is no cure for Wilson’s disease, only chelation therapy using potent chelators such as penicillamine and EDTA. Zinc, in high doses, can be used to compete with copper absorption. This has proved to be the only successful therapy at present. This study investigates the possible use of melatonin as a copper binder/chelator. Melatonin has been shown to interact with copper in vitro. By binding/chelating to copper, melatonin may inhibit copper-induced free radical generation, and thus prevent copper from interacting with DNA to cause mutations and act as a cytotoxin. In vivo studies on copper (2mg/kg) administered for 2-weeks and 6-weeks were carried out on Wistar rats. The potential of melatonin (12mg/kg) to prevent copper-induced cellular damage was investigated. The results indicate that melatonin does not protect the lipid membranes from copper-induced lipid peroxidation. In vitro investigations using 1mM, 5mM and 10mM copper and 5mM melatonin, show that melatonin prevents copper-induced lipid peroxidation at a copper concentration of 1mM (p<0.001). The 5mM and 10mM copper induces less lipid peroxidation, compared to the 1mM copper. It has been reported that metal ions, antioxidants and chelating agents can influence peroxide decomposition during the assay. Melatonin (5mM) administration does not significantly prevent copper-induced lipid peroxidation at 5mM and 10mM copper. It is possible that due to melatonin’s relatively low concentration, it is unable to inhibit lipid peroxidation induced by the copper. The chemical nature of the interaction between melatonin and copper was also investigated, using NMR, IR and electrochemistry techniques. The NMR and IR techniques show that melatonin coordinates with Cu²⁺ and not Cu¹⁺, at the carbonyl group of melatonin. The electrochemistry experiments using cyclic voltammetry and adsorptive stripping voltammetry, show that melatonin forms a strong bond with Cu¹⁺. Cu²⁺ prefers binding to oxygen, and that is clearly seen in the NMR and IR. Cu¹⁺ prefers binding to nitrogen and then oxygen, and this is seen in the electrochemistry, as Cu¹⁺ is forced to bind through one of the nitrogens on the melatonin. Previously, it has been shown that melatonin binds/chelates with Cu²⁺. Histochemical investigations show that copper administration for 2-weeks and 6-weeks, causes extensive mitochondrial damage in liver and kidney’s proximal convoluted tubule epithelium cells. Melatonin (12mg/kg) co-administration with copper for 2-weeks and 6-weeks did not significantly protect the mitochondria from copper-induced damage. Copper-specific stains (rhodanine, silver sulphide and rubeanic acid) were used to stain liver, brain and kidney tissue samples. Rhodanine and silver sulphide were equally sensitive in staining copper in the 2-week samples, but not at all in the 6-week samples. This could not be explained. Rubeanic acid was ineffective in all samples tested. Thus, it appears that specific copper stains cannot be used in making a definitive diagnosis in cases of copper overload, and that specific copper stains do not always correlate with a high concentration of copper present in tissues. Pineal organ culture was used to determine the effect of copper administration on pineal indole synthesis. Exogenous (³H) tryptophan was administered to the pineal organ cultures, and the level of (³H) pineal indoles synthesised, were measured. Pineals from 2-week and 6-week copper/melatonin treated animals exhibited paradoxical 5- methoxytryptophol (ML) levels, as compared to the 2-week and 6-week copper treated animals. The 2-week copper/melatonin administered animals, showed a decrease in the ML level (p<0.01), and the copper/melatonin administered for 6-weeks, showed an increase in the ML levels (p<0.01). This indicates that melatonin interacts with the HIOMT enzyme. Pineals from 6-week copper/melatonin treated animals, as compared to the 6-week copper treated animals, showed an increase in N-acetylserotonin levels. This indicates that melatonin prevents the inhibition of the NAT enzyme. The final experiment was to determine in vitro, the effect of Cu²⁺ and Cu¹⁺ administration, on mitochondrial electron transport chain. Rat liver homogenate was incubated with and solutions of Cu²⁺ (10mM) and Cu¹⁺ (10mM) and melatonin (10mM). Cu²⁺ administration caused an inhibition of the electron transport at t=0 and t=60, whereas Cu¹⁺ administration at t=0 caused an inhibition of electron transport, but at t=60, Cu¹⁺ administration stimulated electron transport. Melatonin administered with Cu²⁺, resulted in an inhibition of the electron transport chain at t=0 and t=60. The findings of this study indicate that melatonin might have a potentially beneficial effect in copper overloading, by binding/chelating copper.
- Full Text:
Regulation of tryptophan-2,3-dioxygenase and pineal indoleamines by selected tryptophan derivatives and antidepressants
- Authors: Walsh, Harold Archibold
- Date: 1997
- Subjects: Antidepressants Tryptophan -- Physiological effect Tryptophan -- Therapeutic use Depression, Mental -- Treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4017 , http://hdl.handle.net/10962/d1004077
- Description: The regulation of tryptophan-2,3-dioxygenase (TDO) (EC 1.13.1.12) and, to a lesser extent, pineal indoleamines, both in vitro and in vivo, is examined in this study. Rat liver TDO is a cytosolic enzyme which plays a crucial role in the regulation of circulating tryptophan (TRP) levels. Stimulation of this enzyme by heme enhances the catabolism of TRP, making less TRP available for uptake into the brain and other tissues, and for protein synthesis. At pH 7, the enzyme has an approximate Km of 100μM, is subject to substrate inhibition immediately beyond Sopt([S] at Vmax), and response of the enzyme is cooperative in both uninhibited and inhibited regions. Hill analysis of the uninhibited region reveals a biphasic plot and two classes of binding sites. Negative cooperativity is brought about through deprotonation of the enzyme. Substrate iphibition also occurs at both acidic and basic pH values with concomitant shifts in Sopt. The results obtained indicate that substrate inhibition could be an additional mechanism whereby the flux through the TRP-kynurenine pathway is regulated. TDO is subject to a diurnal rhythm, with peak activity during the pre-dark period and the loweSt activity towards the end of the dark period. It is possible that the enzyme controls the synthesis of the neurotransmitter serotonin (5-HT), and that the circadian rhythm in TDO activity is due to the endogenous rhythm of melatonin (aMT) production by the pineal gland. In the present study, aMT displaces TRP from bovine serum albumin (BSA) in vitro, and it is therefore possible for the indoleamine to regulate the availability of TRP for uptake into the brain for conversion to its derivatives. Chronic intraperitoneal administration of aMT affects physiological hepatic parameters in rats, such as TDO activity and stromal fatty acid composition, whilst no observable effect is demonstrable with respect to protein synthesis, nucleic acid metabolism, membrane fatty acid composition and pineal indole biosynthesis. On the other hand, chronic treatment of rats with antidepressants, the tricyclic desmethylimipramine (DMI) and the selective serotonin reuptake inhibitor (SSRI), fluoxetine, reveals significant negative alterations in TDO concentrations and pineal indole amine synthesis. Combining aMT with any of these two drugs normalises the activity of the hepatic enzyme. DMI is found to be an effective inhibitor of TDO in the micromolar range in vitro, and also affects total enzyme concentrations in vivo. Fluoxetine has no effect on TDO in vitro, but in vivo also reduces total enzyme levels in the liver. However, the SSRI does not affect conjugation between apo- and holoenzyme. Instead, it decreases extant holoenzyme levels. Indoleamine synthesis by the pineal gland, in organ culture, is altered by both antidepressants, although in different ways. DMI increases N-acetylserotonin levels and reduces the output of methoxyindole acetic acid and meth6xytryptophol. Fluoxetine treatment markedly reduces aMT concentrations and also brings about high levels of the 5-HT catabolites, 5-hydroxytryptophol and 5-hydroxyindole acetic acid. Insulin also lowers aMT synthesis significantly in pineal organ cultures, via a mechamsm that involves inhibition of the enzyme, N-acetyl transferase, that regulates aMT synthesis. The effects of insulin on pineal indole metabolism are due to the observation that a carbohydrate rich diet which induces insulin release elevates plasma TRP and brain 5-HT, but has no effect on pineal TRP and indole amine synthesis. It could thus be possible for insulin to have an effect on the pineal, since the latter is outside the blood brain barrier. The finilings of this study support the biogenic amine deficiency hypothesis, implicating some of the major biogenic amines such as noradrenaline (NA), 5-HT and aMT in depression. There is believed to be a deficiency of NA and 5-HT at their respective synapses in the depressed state. The drug DMI could act, firstly, by inhibiting TDO and thus increasing plasma TRP levels, and could, secondly, stimulate NA release and inhibit NA reuptake at the pineal membrane. The combined effect would be to enhance aMT synthesis, with eventual remission. Fluoxetine, on the other hand, appears to utilize a slightly different mode of action to DMI, which seems to focus on the preservation of 5-HT. The fact that aMT counteracts the effects of both antidepressants, and restores the activity of TDO to that of the controls, is also consistent with the observation that the therapeutic action of drugs such as these coincides willi the restoration of normal plasma levels of the neurohormone in depressives. In view of the biogenic amine deficiency hypothesis of depression and the contentious claim that TDO is the major peripheral determinant of brain TRP, brain 5-HT and ultimately aMT, the regulation of TDO is investigated and discussed. The study concludes that TDO activity is regulated by a number of endogenous compounds which are mainly derivatives of TRP, such as aMT and oxidized nicotinamide adenine dinucleotide and exogenous substances, of which DMI and fluoxetine are but two. In addition, modulation of IDO activity in depression appears to be an important aspect of antidepressant action. aMT, the product of the pineal gland, also has the potential to increase plasma TRP and hence forebrain TRP levels, and ultimately 5-HT concentrations, firstly by displacing TRP from serum albumin and secondly by inhibiting TDO.
- Full Text:
- Authors: Walsh, Harold Archibold
- Date: 1997
- Subjects: Antidepressants Tryptophan -- Physiological effect Tryptophan -- Therapeutic use Depression, Mental -- Treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4017 , http://hdl.handle.net/10962/d1004077
- Description: The regulation of tryptophan-2,3-dioxygenase (TDO) (EC 1.13.1.12) and, to a lesser extent, pineal indoleamines, both in vitro and in vivo, is examined in this study. Rat liver TDO is a cytosolic enzyme which plays a crucial role in the regulation of circulating tryptophan (TRP) levels. Stimulation of this enzyme by heme enhances the catabolism of TRP, making less TRP available for uptake into the brain and other tissues, and for protein synthesis. At pH 7, the enzyme has an approximate Km of 100μM, is subject to substrate inhibition immediately beyond Sopt([S] at Vmax), and response of the enzyme is cooperative in both uninhibited and inhibited regions. Hill analysis of the uninhibited region reveals a biphasic plot and two classes of binding sites. Negative cooperativity is brought about through deprotonation of the enzyme. Substrate iphibition also occurs at both acidic and basic pH values with concomitant shifts in Sopt. The results obtained indicate that substrate inhibition could be an additional mechanism whereby the flux through the TRP-kynurenine pathway is regulated. TDO is subject to a diurnal rhythm, with peak activity during the pre-dark period and the loweSt activity towards the end of the dark period. It is possible that the enzyme controls the synthesis of the neurotransmitter serotonin (5-HT), and that the circadian rhythm in TDO activity is due to the endogenous rhythm of melatonin (aMT) production by the pineal gland. In the present study, aMT displaces TRP from bovine serum albumin (BSA) in vitro, and it is therefore possible for the indoleamine to regulate the availability of TRP for uptake into the brain for conversion to its derivatives. Chronic intraperitoneal administration of aMT affects physiological hepatic parameters in rats, such as TDO activity and stromal fatty acid composition, whilst no observable effect is demonstrable with respect to protein synthesis, nucleic acid metabolism, membrane fatty acid composition and pineal indole biosynthesis. On the other hand, chronic treatment of rats with antidepressants, the tricyclic desmethylimipramine (DMI) and the selective serotonin reuptake inhibitor (SSRI), fluoxetine, reveals significant negative alterations in TDO concentrations and pineal indole amine synthesis. Combining aMT with any of these two drugs normalises the activity of the hepatic enzyme. DMI is found to be an effective inhibitor of TDO in the micromolar range in vitro, and also affects total enzyme concentrations in vivo. Fluoxetine has no effect on TDO in vitro, but in vivo also reduces total enzyme levels in the liver. However, the SSRI does not affect conjugation between apo- and holoenzyme. Instead, it decreases extant holoenzyme levels. Indoleamine synthesis by the pineal gland, in organ culture, is altered by both antidepressants, although in different ways. DMI increases N-acetylserotonin levels and reduces the output of methoxyindole acetic acid and meth6xytryptophol. Fluoxetine treatment markedly reduces aMT concentrations and also brings about high levels of the 5-HT catabolites, 5-hydroxytryptophol and 5-hydroxyindole acetic acid. Insulin also lowers aMT synthesis significantly in pineal organ cultures, via a mechamsm that involves inhibition of the enzyme, N-acetyl transferase, that regulates aMT synthesis. The effects of insulin on pineal indole metabolism are due to the observation that a carbohydrate rich diet which induces insulin release elevates plasma TRP and brain 5-HT, but has no effect on pineal TRP and indole amine synthesis. It could thus be possible for insulin to have an effect on the pineal, since the latter is outside the blood brain barrier. The finilings of this study support the biogenic amine deficiency hypothesis, implicating some of the major biogenic amines such as noradrenaline (NA), 5-HT and aMT in depression. There is believed to be a deficiency of NA and 5-HT at their respective synapses in the depressed state. The drug DMI could act, firstly, by inhibiting TDO and thus increasing plasma TRP levels, and could, secondly, stimulate NA release and inhibit NA reuptake at the pineal membrane. The combined effect would be to enhance aMT synthesis, with eventual remission. Fluoxetine, on the other hand, appears to utilize a slightly different mode of action to DMI, which seems to focus on the preservation of 5-HT. The fact that aMT counteracts the effects of both antidepressants, and restores the activity of TDO to that of the controls, is also consistent with the observation that the therapeutic action of drugs such as these coincides willi the restoration of normal plasma levels of the neurohormone in depressives. In view of the biogenic amine deficiency hypothesis of depression and the contentious claim that TDO is the major peripheral determinant of brain TRP, brain 5-HT and ultimately aMT, the regulation of TDO is investigated and discussed. The study concludes that TDO activity is regulated by a number of endogenous compounds which are mainly derivatives of TRP, such as aMT and oxidized nicotinamide adenine dinucleotide and exogenous substances, of which DMI and fluoxetine are but two. In addition, modulation of IDO activity in depression appears to be an important aspect of antidepressant action. aMT, the product of the pineal gland, also has the potential to increase plasma TRP and hence forebrain TRP levels, and ultimately 5-HT concentrations, firstly by displacing TRP from serum albumin and secondly by inhibiting TDO.
- Full Text:
The effect of appetite suppressants on pineal function
- Authors: Mchunu, Bongani Isaac
- Date: 1994
- Subjects: Pineal gland -- Research , Pineal gland -- Secretions , Appetite depressants -- Physiological effect
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4038 , http://hdl.handle.net/10962/d1004098 , Pineal gland -- Research , Pineal gland -- Secretions , Appetite depressants -- Physiological effect
- Description: The pineal gland has become the subject of considerable investigation as it provides a productive experimental model for studying circadian rhythms and regulation of end organs. In the rat, the pineal gland provides a convenient model for investigating the noradrenergic receptor system and the effects of various drugs on this system. The effect of appetite suppressants on the rat pineal gland function is described. Appetite suppressants increase melatonin synthesis in organ cultures of rat pineal glands. This effect appears to be mediated by noradrenaline acting on β-adrenoceptors on the pinealocyte membrane. When β-adrenoceptors are blocked, the appetite suppressant-induced rise in melatonin synthesis is prevented. Depletion of noradrenaline in sympathetic nerve terminals also prevented the appetite suppressant-induced rise in melatonin synthesis. Activation of β-adrenoceptors is followed by a rise in N-acetyltransferase activity via a cyclic adenosine monophosphate second messenger system. The effect of appetite suppressants on the activity of liver tryptophan pyrrolase was also investigated. The activity of this enzyme is an important determinant of tryptophan availability to the brain and consequently of brain serotonin levels. The results show that appetite suppressants inhibit both holoenzyme and total enzyme activities of tryptophan pyrrolase. This finding suggests that appetite suppressants may act by inhibiting tryptophan pyrrolase activity thereby increasing brain serotonin, a phenomenon known to be associated with anorexia. There are two possible mechanisms by which appetite suppressants inhibit tryptophan pyrrolase activity. Firstly, these agents, being drugs of dependence, may increase liver NADPH concentrations which inhibit pyrrolase activity. Secondly, appetite suppressants may act on the pineal gland to stimulate melatonin synthesis. Melatonin inhibits pyrrolase activity in a dose-dependent manner. This inhibition will elevate plasma tryptophan levels which result in a rise in brain serotonin synthesis. The present study suggests a possible relationship between the pineal gland and appetite centres in the hypothalamus. Melatonin may have a direct effect on appetite centres since food restriction is associated with an increased melatonin binding in the hypothalamus. If this possible relationship can be extended, melatonin can open new possibilities for the control of food intake and consequently, of pathological obesity.
- Full Text:
- Authors: Mchunu, Bongani Isaac
- Date: 1994
- Subjects: Pineal gland -- Research , Pineal gland -- Secretions , Appetite depressants -- Physiological effect
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4038 , http://hdl.handle.net/10962/d1004098 , Pineal gland -- Research , Pineal gland -- Secretions , Appetite depressants -- Physiological effect
- Description: The pineal gland has become the subject of considerable investigation as it provides a productive experimental model for studying circadian rhythms and regulation of end organs. In the rat, the pineal gland provides a convenient model for investigating the noradrenergic receptor system and the effects of various drugs on this system. The effect of appetite suppressants on the rat pineal gland function is described. Appetite suppressants increase melatonin synthesis in organ cultures of rat pineal glands. This effect appears to be mediated by noradrenaline acting on β-adrenoceptors on the pinealocyte membrane. When β-adrenoceptors are blocked, the appetite suppressant-induced rise in melatonin synthesis is prevented. Depletion of noradrenaline in sympathetic nerve terminals also prevented the appetite suppressant-induced rise in melatonin synthesis. Activation of β-adrenoceptors is followed by a rise in N-acetyltransferase activity via a cyclic adenosine monophosphate second messenger system. The effect of appetite suppressants on the activity of liver tryptophan pyrrolase was also investigated. The activity of this enzyme is an important determinant of tryptophan availability to the brain and consequently of brain serotonin levels. The results show that appetite suppressants inhibit both holoenzyme and total enzyme activities of tryptophan pyrrolase. This finding suggests that appetite suppressants may act by inhibiting tryptophan pyrrolase activity thereby increasing brain serotonin, a phenomenon known to be associated with anorexia. There are two possible mechanisms by which appetite suppressants inhibit tryptophan pyrrolase activity. Firstly, these agents, being drugs of dependence, may increase liver NADPH concentrations which inhibit pyrrolase activity. Secondly, appetite suppressants may act on the pineal gland to stimulate melatonin synthesis. Melatonin inhibits pyrrolase activity in a dose-dependent manner. This inhibition will elevate plasma tryptophan levels which result in a rise in brain serotonin synthesis. The present study suggests a possible relationship between the pineal gland and appetite centres in the hypothalamus. Melatonin may have a direct effect on appetite centres since food restriction is associated with an increased melatonin binding in the hypothalamus. If this possible relationship can be extended, melatonin can open new possibilities for the control of food intake and consequently, of pathological obesity.
- Full Text:
An investigation into cholinergic interactions in the rat pineal gland
- Authors: Eason, Jason Shane
- Date: 1993
- Subjects: Pineal gland -- Research , Acetylcholine -- Receptors
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4048 , http://hdl.handle.net/10962/d1004109 , Pineal gland -- Research , Acetylcholine -- Receptors
- Description: The mammalian pineal gland is mainly innervated by the sympathetic nervous system which modulates the activity of indole pathway enzymes and the secretion of pineal hormones. Recently researchers have demonstrated and characterized the presence of muscarinic cholinergic receptors in the pineal gland. However the role of these receptors remains unclear. In an attempt to investigate the role of cholinergic receptors in the pineal gland, a number of studies were carried out on the various steps in the indole metabolic pathway, using various agents which act on the cholinergic system. Investigations using pineal organ cultures showed that stimulation of these muscarinic cholinergic receptor sites with a parasympathomimetic agent, a rise in levels of aHT occurred without a concomitant increase in aMT levels. Further organ culture experiments using the cholinergic agonist acetylcholine and anticholinesterase agent physostigmine, produced a similar rise in aHT without altering aMT levels. This acetylcholine-induced rise in aHT levels were not altered by the ganglion blocking agent hexamethonium whilst the antimuscarinic agent atropine prevented the acetylcholine-induced rise in aHT levels. Thesefindings suggest that cholinergic agents may play a role in regulating indoleamine synthesis in the pineal gland. Cyclic-AMP assay studies showed that acetylcholine increases pineal cAMP levels significantly and does not influence the isoproterenol-induced cAMP rise in the pineal gland. The cAMP regulator cAMP-phosphodiesterase (cAMP-PDE) was found to increase significantly in the presence of the anticholinesterase agent physostigmine. NAT enzyme studies revealed that physostigmine does not affect NAT enzyme levels significantly and HIOMT studies showed that this agent does not inhibit HIOMT activity. The mechanism by which acetylcholine and physostigmine are able to cause a increase in aHT and not aMT levels needs to be researched further. Acetylcholinesterase enzyme assay studies revealed that the AChE enzyme undergoes a diurnal rhythm in the pineal gland with activity being higher during the day and lower at night. Investigations using the drug reserpine showed that this rhythm is not under the control of the sympathetic nervous system. Further research needs to be done however, in determining whether or not this enzyme is present in the pineal gland to regulate the levels of acetylcholine interacting with muscarinic receptors in the gland, or for some other reason. Choline acetyltransferase studies demonstrate the presence of the enzyme in the rat brain cerebral cortex as well as showing that melatonin increases ChAT enzyme activity in this tissue. This suggests that melatonin plays a role in cholinergic transmission there. ChAT activity could not be measured in the pineal gland however. Muscarinic receptor binding studies also carried out on rat brain cerebral cortex show that melatonin enhances cholinergic receptor affinity and receptor number in this tissue. In summary, data presented herein concur with proposals that: i) the cholinergic system affects the indole metabolic pathway by causing a rise in aRT but not aMT levels. ii) cholinergic agonist acetylcholine causes cAMP levels to rise with a concomitant increase in cAMP-PDE levels. iii) the enzyme acetylcholinesterase undergoes a diurnal rhythm in the pineal gland which is not under the control of the sympathetic nervous system. iv) the activity of the enzyme choline acetyltransferase is increased by melatonin in the rat brain cerebral cortex suggesting that melatonin facilitates cholinergic transmission in this tissue. v) melatonin enhances cholinergic receptor affinity and receptor number in the cerebral cortex of rat brain.
- Full Text:
- Authors: Eason, Jason Shane
- Date: 1993
- Subjects: Pineal gland -- Research , Acetylcholine -- Receptors
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4048 , http://hdl.handle.net/10962/d1004109 , Pineal gland -- Research , Acetylcholine -- Receptors
- Description: The mammalian pineal gland is mainly innervated by the sympathetic nervous system which modulates the activity of indole pathway enzymes and the secretion of pineal hormones. Recently researchers have demonstrated and characterized the presence of muscarinic cholinergic receptors in the pineal gland. However the role of these receptors remains unclear. In an attempt to investigate the role of cholinergic receptors in the pineal gland, a number of studies were carried out on the various steps in the indole metabolic pathway, using various agents which act on the cholinergic system. Investigations using pineal organ cultures showed that stimulation of these muscarinic cholinergic receptor sites with a parasympathomimetic agent, a rise in levels of aHT occurred without a concomitant increase in aMT levels. Further organ culture experiments using the cholinergic agonist acetylcholine and anticholinesterase agent physostigmine, produced a similar rise in aHT without altering aMT levels. This acetylcholine-induced rise in aHT levels were not altered by the ganglion blocking agent hexamethonium whilst the antimuscarinic agent atropine prevented the acetylcholine-induced rise in aHT levels. Thesefindings suggest that cholinergic agents may play a role in regulating indoleamine synthesis in the pineal gland. Cyclic-AMP assay studies showed that acetylcholine increases pineal cAMP levels significantly and does not influence the isoproterenol-induced cAMP rise in the pineal gland. The cAMP regulator cAMP-phosphodiesterase (cAMP-PDE) was found to increase significantly in the presence of the anticholinesterase agent physostigmine. NAT enzyme studies revealed that physostigmine does not affect NAT enzyme levels significantly and HIOMT studies showed that this agent does not inhibit HIOMT activity. The mechanism by which acetylcholine and physostigmine are able to cause a increase in aHT and not aMT levels needs to be researched further. Acetylcholinesterase enzyme assay studies revealed that the AChE enzyme undergoes a diurnal rhythm in the pineal gland with activity being higher during the day and lower at night. Investigations using the drug reserpine showed that this rhythm is not under the control of the sympathetic nervous system. Further research needs to be done however, in determining whether or not this enzyme is present in the pineal gland to regulate the levels of acetylcholine interacting with muscarinic receptors in the gland, or for some other reason. Choline acetyltransferase studies demonstrate the presence of the enzyme in the rat brain cerebral cortex as well as showing that melatonin increases ChAT enzyme activity in this tissue. This suggests that melatonin plays a role in cholinergic transmission there. ChAT activity could not be measured in the pineal gland however. Muscarinic receptor binding studies also carried out on rat brain cerebral cortex show that melatonin enhances cholinergic receptor affinity and receptor number in this tissue. In summary, data presented herein concur with proposals that: i) the cholinergic system affects the indole metabolic pathway by causing a rise in aRT but not aMT levels. ii) cholinergic agonist acetylcholine causes cAMP levels to rise with a concomitant increase in cAMP-PDE levels. iii) the enzyme acetylcholinesterase undergoes a diurnal rhythm in the pineal gland which is not under the control of the sympathetic nervous system. iv) the activity of the enzyme choline acetyltransferase is increased by melatonin in the rat brain cerebral cortex suggesting that melatonin facilitates cholinergic transmission in this tissue. v) melatonin enhances cholinergic receptor affinity and receptor number in the cerebral cortex of rat brain.
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The regulation of Serotonin N-acetyltransferase in the rat pineal gland
- Authors: Olivieri, Gianfranco
- Date: 1993
- Subjects: Serotonin -- Research Pineal gland Acetyltransferases
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4051 , http://hdl.handle.net/10962/d1004112
- Description: The synthesis of the pineal hormone, melatonin, is finely regulated by the pineal enzyme serotonin N-acetyltransferase (NAT). In the absence of light, the activity of NAT is markedly enhanced by the release of nor-adrenaline from sympathetic nerve endings in the pineal. Exposure of animals to light during darkness causes a sudden and dramatic reduction in the activity of NAT. The present study investigated a possible mechanism for this sudden decline in NAT activity. These investigations included the determination of the effects of S-adenosylmethionine (SAM), adenosine nucleotides and calcium on NAT activity. In vitro experiments using SAM showed that pineals pre-incubated with SAM prior to adrenergic stimulation did not significantly alter NAT activity or pineal indoleamine metabolism. However, measurement of pineal cyclic AMP showed that SAM exposure reduced the adrenergic-induced rise in pineal cyclic AMP. Experiments using adenosine 5'-monophosphate (5'-AMP) showed that this nucleotide enhanced both dark- and isoproterenol-induced NAT activity. Adenosine 5'-triphosphate (A TP), on the other hand, reduced NAT activity with a concomitant reduction in pineal indoleamine metabolism. Exposure of isoproterenol-stimulated pineals in organ culture to propranolol resulted in a marked rise in ATP and adenosine 5'-diphosphate (ADP) synthesis accompanied by a decline in 5'-AMP levels as compared with pineals treated with isoproterenol alone. This then implies that exposure of animals to light could cause a change in pineal nucleotide levels. Since nucleotide levels are also controlled by calcium, experiments were carried out to determine the effect of calcium on pineal NAT activity. These experiments showed that ethyleneglycol-bis-N,N,N,N,-tetraacetic acid (EGTA) enhanced NAT activity whilst calcium reduced the activity in pineal homogenates, implying that calcium may act directly on NAT to regulate its activity. Exposure of pineal glands in organ culture to the calmodulin antagonist R24571 caused a rise in pineal cyclic AMP levels with a concomitant decrease in cAMP-phosphodiesterase activity. This was, however, accompanied by a decline in Nacetyl serotonin and melatonin synthesis. These findings implicate a number of factors in the regulation of pineal NAT activity. A mechanism for the regulation of pineal NAT is proposed.
- Full Text:
- Authors: Olivieri, Gianfranco
- Date: 1993
- Subjects: Serotonin -- Research Pineal gland Acetyltransferases
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4051 , http://hdl.handle.net/10962/d1004112
- Description: The synthesis of the pineal hormone, melatonin, is finely regulated by the pineal enzyme serotonin N-acetyltransferase (NAT). In the absence of light, the activity of NAT is markedly enhanced by the release of nor-adrenaline from sympathetic nerve endings in the pineal. Exposure of animals to light during darkness causes a sudden and dramatic reduction in the activity of NAT. The present study investigated a possible mechanism for this sudden decline in NAT activity. These investigations included the determination of the effects of S-adenosylmethionine (SAM), adenosine nucleotides and calcium on NAT activity. In vitro experiments using SAM showed that pineals pre-incubated with SAM prior to adrenergic stimulation did not significantly alter NAT activity or pineal indoleamine metabolism. However, measurement of pineal cyclic AMP showed that SAM exposure reduced the adrenergic-induced rise in pineal cyclic AMP. Experiments using adenosine 5'-monophosphate (5'-AMP) showed that this nucleotide enhanced both dark- and isoproterenol-induced NAT activity. Adenosine 5'-triphosphate (A TP), on the other hand, reduced NAT activity with a concomitant reduction in pineal indoleamine metabolism. Exposure of isoproterenol-stimulated pineals in organ culture to propranolol resulted in a marked rise in ATP and adenosine 5'-diphosphate (ADP) synthesis accompanied by a decline in 5'-AMP levels as compared with pineals treated with isoproterenol alone. This then implies that exposure of animals to light could cause a change in pineal nucleotide levels. Since nucleotide levels are also controlled by calcium, experiments were carried out to determine the effect of calcium on pineal NAT activity. These experiments showed that ethyleneglycol-bis-N,N,N,N,-tetraacetic acid (EGTA) enhanced NAT activity whilst calcium reduced the activity in pineal homogenates, implying that calcium may act directly on NAT to regulate its activity. Exposure of pineal glands in organ culture to the calmodulin antagonist R24571 caused a rise in pineal cyclic AMP levels with a concomitant decrease in cAMP-phosphodiesterase activity. This was, however, accompanied by a decline in Nacetyl serotonin and melatonin synthesis. These findings implicate a number of factors in the regulation of pineal NAT activity. A mechanism for the regulation of pineal NAT is proposed.
- Full Text:
In vitro effects of three organic calcium channel blockers on the rat pineal gland
- Authors: Brown, Clint
- Date: 1992
- Subjects: Calcium -- Antagonists , Pineal gland -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3745 , http://hdl.handle.net/10962/d1003223 , Calcium -- Antagonists , Pineal gland -- Research
- Description: The calcium signal has emerged as an imponant component of intracellular regulation. Pineal function was thought to be slowed by the prominent calcification seen with increasing age, but recently it has been shown that calcium plays a crucial role in the adrenergic regulation of the gland. Beta-adrenoceptor stimulation increases melatonin (aMT) synthesis by increasing the activity of cyclic 3 '-5' adenosine mono phosphate (cAMP). Cyclic-AMP regulates the production of the pineal hormone, melatonin, from serotonin via the rate-limiting enzyme N-acetyltransferase (NAT). Increased intracellular cAMP is essential to the adrenergic induction of NAT. Noradrenaline(NA)also elevates pinealocyte cyclic guanosine monophosphate (cGMP). Adrenergic regulation of these cyclic nucleotides involves both α₁ - and β-adrenoceptors. Beta-adrenoceptor stimulation is an absolute requirement. Alphal-adrenoceptor activation, which is ineffective alone, serves to amplify the β-stimulated cAMP and cGMP responses via a positive effect on a Ca²⁺⁻/ phospholipiddependent protein kinase (Protein kinase-C) and a net influx of Ca²⁺ into the pinealocyte. Previous studies suggest the use of organic calcium channel blockers (CCBs) as probes of calcium-mediated processes. Applying this concept, the study set out to investigate the influence of a representative of each of the structurally diverse groups of calcium channel blockers viz. verapamil, diltiazem and nifedipine, and to examine their effect on β-adrenoceptor stimulation. It used the β-agonist isoprenaline (ISO) and the mixed [α₁/β]agonist noradrenaline (NA), for its combined [α₁/β]adrenoceptor stimulation, on agonist-induced increases in the production of radio-labelled aMT and N-acetylserotonin(aHT) -measured as the sum of N-acetylated product- from [¹⁴C] serotonin. This was done using organ cultures of rat pineal glands. It was speciously assumed that this drug paradigm would allow the determination of Ca²⁺ influx and/or the blocking thereof in the reported potentiation by using ISO as a non Ca²⁺ -entry stimulating agonist, compared with NA and its Ca²⁺ -entry stimulating properties. Surprisingly, all 3 CCB's potentiated the effect of NA. Only diltiazem was found not to potentiate the effect of ISO. In an attempt to uncover the reason for these results, the study moved toward a mechanistic approach,focusing in an antecedent manner on the various steps in the indole metabolic pathway to identify the point at which the change occurred, and hence possibly elucidate the mechanism responsible for the paradoxical increase. Experiments which assayed the levels of NAT, under the same drug conditions, showed the paradoxical increase to be already evident at this stage. Secondary experiments confirmed that NA stimulation of the pineal is dependent on Ca²⁺, both in organ culture and with NAT: the Ca²⁺ chelator EGTA abolished adrenergically-induced stimulation, while Ca²⁺ added after EGTA, restored the enzyme activity. The ionophore A23187 (which is able to transport Ca²⁺ directly into the pinealocyte via a mechanism which differs from the α₁ - mechanism) when used in conjunction with ISO or NA, was able to potentiate the responses of these two agonists relative to control values (agonist-alone), but by itself had no effect. With the enzyme NAT critically dependent upon cAMP for its induction, it was decided to determine the levels of cAMP and then those of its regulator, cAMP-phosphodiesterase (cAMP-PDE). This reasoning was prompted by reports of anti-calmodulin activity shown by the CCBs, in addition to their channel blocking effects. By binding to calmodulin (CaM), the CCBs are reportedly able to inhibit the CaM-dependent activation of cAMP-PDE. Following NA stimulation, verapamil caused a significant decrease in cAMP-PDE levels and an increase in cAMP. The other CCBs showed a similar trend. Glands stimulated with ISO in the presence of verapamil and nifedipine showed no significant differences in cAMP or cAMP-PDE levels. Diltiazem, however, was found to decrease the effect of ISO on cAMP while causing a concomitant increase in cAMP-PDE. This i) supported a possible hypothesis that the observed enhancement is a result of cAMP levels remaining elevated due to an inhibition of cAMP-PDE by the CCEs and ii) pointed to the possible presence of a CaM-sensitive PDE within the rat pineal gland. To test this hypothesis, two drugs which are more specific in their actions on CaM effects were chosen to see if the earlier results could be mimicked and thereby confirmed. Glands stimulated with NA in the presence of the specific CaM inhibitor R 24571 showed increased NAT activity and [¹⁴C]-aMT production. cAMP-PDE levels were clearly down, thus corroborating the possibility of cAMP-PDE inhibition. Glands incubated in the presence of M&B 22948, a CaM-sensitive PDE inhibitor, showed similar increases in NAT activity and [¹⁴C]-aMT. These findings therefore support the initial results and although indirect, confirm the hypothesis that the paradoxical increase following predominantly NA stimulation could be a result of cAMP levels remaining elevated, due to inhibition by the CCEs of the CaM-dependent activation of its regulator cAMP-PDE. In summary, data presented herein concur with proposals that: i) the CCEs are not specific enough to be used as tools to research Ca²⁺ -mediated events, as they appear to have sites of action other than the voltage operated channel (VOC); eg. binding to calmodulin, ii) there are functional differences between the CCEs as shown by diltiazem in this series of experiments, iii) there is a CaM-sensitive-PDE present in the pineal.
- Full Text:
- Authors: Brown, Clint
- Date: 1992
- Subjects: Calcium -- Antagonists , Pineal gland -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3745 , http://hdl.handle.net/10962/d1003223 , Calcium -- Antagonists , Pineal gland -- Research
- Description: The calcium signal has emerged as an imponant component of intracellular regulation. Pineal function was thought to be slowed by the prominent calcification seen with increasing age, but recently it has been shown that calcium plays a crucial role in the adrenergic regulation of the gland. Beta-adrenoceptor stimulation increases melatonin (aMT) synthesis by increasing the activity of cyclic 3 '-5' adenosine mono phosphate (cAMP). Cyclic-AMP regulates the production of the pineal hormone, melatonin, from serotonin via the rate-limiting enzyme N-acetyltransferase (NAT). Increased intracellular cAMP is essential to the adrenergic induction of NAT. Noradrenaline(NA)also elevates pinealocyte cyclic guanosine monophosphate (cGMP). Adrenergic regulation of these cyclic nucleotides involves both α₁ - and β-adrenoceptors. Beta-adrenoceptor stimulation is an absolute requirement. Alphal-adrenoceptor activation, which is ineffective alone, serves to amplify the β-stimulated cAMP and cGMP responses via a positive effect on a Ca²⁺⁻/ phospholipiddependent protein kinase (Protein kinase-C) and a net influx of Ca²⁺ into the pinealocyte. Previous studies suggest the use of organic calcium channel blockers (CCBs) as probes of calcium-mediated processes. Applying this concept, the study set out to investigate the influence of a representative of each of the structurally diverse groups of calcium channel blockers viz. verapamil, diltiazem and nifedipine, and to examine their effect on β-adrenoceptor stimulation. It used the β-agonist isoprenaline (ISO) and the mixed [α₁/β]agonist noradrenaline (NA), for its combined [α₁/β]adrenoceptor stimulation, on agonist-induced increases in the production of radio-labelled aMT and N-acetylserotonin(aHT) -measured as the sum of N-acetylated product- from [¹⁴C] serotonin. This was done using organ cultures of rat pineal glands. It was speciously assumed that this drug paradigm would allow the determination of Ca²⁺ influx and/or the blocking thereof in the reported potentiation by using ISO as a non Ca²⁺ -entry stimulating agonist, compared with NA and its Ca²⁺ -entry stimulating properties. Surprisingly, all 3 CCB's potentiated the effect of NA. Only diltiazem was found not to potentiate the effect of ISO. In an attempt to uncover the reason for these results, the study moved toward a mechanistic approach,focusing in an antecedent manner on the various steps in the indole metabolic pathway to identify the point at which the change occurred, and hence possibly elucidate the mechanism responsible for the paradoxical increase. Experiments which assayed the levels of NAT, under the same drug conditions, showed the paradoxical increase to be already evident at this stage. Secondary experiments confirmed that NA stimulation of the pineal is dependent on Ca²⁺, both in organ culture and with NAT: the Ca²⁺ chelator EGTA abolished adrenergically-induced stimulation, while Ca²⁺ added after EGTA, restored the enzyme activity. The ionophore A23187 (which is able to transport Ca²⁺ directly into the pinealocyte via a mechanism which differs from the α₁ - mechanism) when used in conjunction with ISO or NA, was able to potentiate the responses of these two agonists relative to control values (agonist-alone), but by itself had no effect. With the enzyme NAT critically dependent upon cAMP for its induction, it was decided to determine the levels of cAMP and then those of its regulator, cAMP-phosphodiesterase (cAMP-PDE). This reasoning was prompted by reports of anti-calmodulin activity shown by the CCBs, in addition to their channel blocking effects. By binding to calmodulin (CaM), the CCBs are reportedly able to inhibit the CaM-dependent activation of cAMP-PDE. Following NA stimulation, verapamil caused a significant decrease in cAMP-PDE levels and an increase in cAMP. The other CCBs showed a similar trend. Glands stimulated with ISO in the presence of verapamil and nifedipine showed no significant differences in cAMP or cAMP-PDE levels. Diltiazem, however, was found to decrease the effect of ISO on cAMP while causing a concomitant increase in cAMP-PDE. This i) supported a possible hypothesis that the observed enhancement is a result of cAMP levels remaining elevated due to an inhibition of cAMP-PDE by the CCEs and ii) pointed to the possible presence of a CaM-sensitive PDE within the rat pineal gland. To test this hypothesis, two drugs which are more specific in their actions on CaM effects were chosen to see if the earlier results could be mimicked and thereby confirmed. Glands stimulated with NA in the presence of the specific CaM inhibitor R 24571 showed increased NAT activity and [¹⁴C]-aMT production. cAMP-PDE levels were clearly down, thus corroborating the possibility of cAMP-PDE inhibition. Glands incubated in the presence of M&B 22948, a CaM-sensitive PDE inhibitor, showed similar increases in NAT activity and [¹⁴C]-aMT. These findings therefore support the initial results and although indirect, confirm the hypothesis that the paradoxical increase following predominantly NA stimulation could be a result of cAMP levels remaining elevated, due to inhibition by the CCEs of the CaM-dependent activation of its regulator cAMP-PDE. In summary, data presented herein concur with proposals that: i) the CCEs are not specific enough to be used as tools to research Ca²⁺ -mediated events, as they appear to have sites of action other than the voltage operated channel (VOC); eg. binding to calmodulin, ii) there are functional differences between the CCEs as shown by diltiazem in this series of experiments, iii) there is a CaM-sensitive-PDE present in the pineal.
- Full Text:
Neuropharmacological interactions in the rat pineal gland a study of antidepressant drugs
- Authors: Banoo, Shabir
- Date: 1992
- Subjects: Antidepressants -- Research , Pineal gland -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3744 , http://hdl.handle.net/10962/d1003222 , Antidepressants -- Research , Pineal gland -- Research
- Description: The rat pineal gland provides a convenient model for investigating nor adrenergic receptor neurotransmission and the effects of various drugs on these processes in health and disease. The effect of a variety of antidepressant drugs on rat pineal gland function following acute and chronic administration is described. Antidepressants from several different classes increase melatonin synthesis in rat pineal gland cultures when administered acutely. This effect appears to be mediated by noradrenaline acting on postsynaptic β-adrenoceptors. Activation of these receptors, in turn, activates the enzyme serotonin N-acetyltransferase via a cyclic adenosine monophosphate (cAMP) second messenger system. Serotonin N-acetyltransferase catalyses the rate-limiting conversion of serotonin to melatonin. Blockade of postsynaptic β-adrenoceptors prevents the antidepressant-induced increase in melatonin synthesis. The possibility that atypical antidepressants as well as those that selectively inhibit serotonin reuptake may increase melatonin synthesis via a β-adrenoceptor mechanism is discussed. In contrast, however, antidepressants from different classes have variable effects on rat pineal gland function when administered repeatedly. Chronic treatment with antidepressants that selectively inhibit noradrenaline reuptake appear to down-regulate the β-adrenoceptor system while, simultaneously, increasing melatonin output. Atypical antidepressants and those that selectively inhibit serotonin reuptake appear to be without these effects when administered repeatedly. The pineal gland of normal rats may therefore not represent a suitable model for evaluating the biochemical effects of chronic antidepressant treatment. In an attempt to investigatc pineal gland function in rats with "model depression" , antidepressants were administered to chronically reserpinized rats. Treatment with reserpine produced an increase in the density of pineal β-adrenoceptors. In addition, pineal cyclic AMP accumulation and N-acetyltransferase activity were increased in reserpinized rats following exogenous catecholamine stimulation. Reserpine, by depleting intraneuronal catecholamine stores, prevented the nocturnal induction of N-acetyltransferase activity and reduced the synthesis of melatonin in pineal gland cultures. A variety of antidepressants, irrespective of their acute pharmacological actions, reversed these effects when administered chronically to resepinized rats. Acute antidepressant administration was not associated with a reversal of the reserpine-induced effects. These findings provide additional evidence against the hypothesis that antidepressant drugs act by reducing noradrenergic neurotransmission and casts doubt on the importance of β-adrenoceptor down-regulation in the mechanism of antidepressant action. The possibility that the pineal gland of the reserpinized rat may represent an alternative model for evaluating antidepressant therapies is discussed.
- Full Text:
- Authors: Banoo, Shabir
- Date: 1992
- Subjects: Antidepressants -- Research , Pineal gland -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3744 , http://hdl.handle.net/10962/d1003222 , Antidepressants -- Research , Pineal gland -- Research
- Description: The rat pineal gland provides a convenient model for investigating nor adrenergic receptor neurotransmission and the effects of various drugs on these processes in health and disease. The effect of a variety of antidepressant drugs on rat pineal gland function following acute and chronic administration is described. Antidepressants from several different classes increase melatonin synthesis in rat pineal gland cultures when administered acutely. This effect appears to be mediated by noradrenaline acting on postsynaptic β-adrenoceptors. Activation of these receptors, in turn, activates the enzyme serotonin N-acetyltransferase via a cyclic adenosine monophosphate (cAMP) second messenger system. Serotonin N-acetyltransferase catalyses the rate-limiting conversion of serotonin to melatonin. Blockade of postsynaptic β-adrenoceptors prevents the antidepressant-induced increase in melatonin synthesis. The possibility that atypical antidepressants as well as those that selectively inhibit serotonin reuptake may increase melatonin synthesis via a β-adrenoceptor mechanism is discussed. In contrast, however, antidepressants from different classes have variable effects on rat pineal gland function when administered repeatedly. Chronic treatment with antidepressants that selectively inhibit noradrenaline reuptake appear to down-regulate the β-adrenoceptor system while, simultaneously, increasing melatonin output. Atypical antidepressants and those that selectively inhibit serotonin reuptake appear to be without these effects when administered repeatedly. The pineal gland of normal rats may therefore not represent a suitable model for evaluating the biochemical effects of chronic antidepressant treatment. In an attempt to investigatc pineal gland function in rats with "model depression" , antidepressants were administered to chronically reserpinized rats. Treatment with reserpine produced an increase in the density of pineal β-adrenoceptors. In addition, pineal cyclic AMP accumulation and N-acetyltransferase activity were increased in reserpinized rats following exogenous catecholamine stimulation. Reserpine, by depleting intraneuronal catecholamine stores, prevented the nocturnal induction of N-acetyltransferase activity and reduced the synthesis of melatonin in pineal gland cultures. A variety of antidepressants, irrespective of their acute pharmacological actions, reversed these effects when administered chronically to resepinized rats. Acute antidepressant administration was not associated with a reversal of the reserpine-induced effects. These findings provide additional evidence against the hypothesis that antidepressant drugs act by reducing noradrenergic neurotransmission and casts doubt on the importance of β-adrenoceptor down-regulation in the mechanism of antidepressant action. The possibility that the pineal gland of the reserpinized rat may represent an alternative model for evaluating antidepressant therapies is discussed.
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Regulation of the indoleamines by sex steroids
- Authors: Awah, Edmund Kpabi
- Date: 1992
- Subjects: Steroids -- Research , Steroid drugs -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4053 , http://hdl.handle.net/10962/d1004114 , Steroids -- Research , Steroid drugs -- Research
- Description: Alteration of serum tryptophan leads to parallel alterations in brain tryptophan levels. Such changes in brain tryptophan levels has been shown to lead to mood disturbances. The primary enzyme responsible for altering serum tryptophan levels is the liver cytosolic enzyme, tryptophan pyrrolase. Activation of this enzyme is responsible for the enhanced catabolism of circulating tryptophan. The purpose of the present study was firstly to establish whether there is a link between sex steroids and tryptophan pyrrolase activity especially since sex steroids are also known to cause mood disturbances and secondly to determine the effects of sex steroids on brain indolamine metabolism. The results show that all three sex steroids induce the activity of tryptophan pyrrolase implying that they decrease serum tryptophan levels by the activation of tryptophan pyrrolase, thus making less tryptophan available for uptake by the brain. It was also shown that the sex steroids enhance the uptake of ¹⁴C-tryptophan by brain synatopsomes. In addition, the sex steroids influenced the pattern of metabolism of serotonin by organ cultures of rat pineal glands. It is possible that the sex steroids regulate the availability and uptake of indoleamines in the brain.
- Full Text:
- Authors: Awah, Edmund Kpabi
- Date: 1992
- Subjects: Steroids -- Research , Steroid drugs -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4053 , http://hdl.handle.net/10962/d1004114 , Steroids -- Research , Steroid drugs -- Research
- Description: Alteration of serum tryptophan leads to parallel alterations in brain tryptophan levels. Such changes in brain tryptophan levels has been shown to lead to mood disturbances. The primary enzyme responsible for altering serum tryptophan levels is the liver cytosolic enzyme, tryptophan pyrrolase. Activation of this enzyme is responsible for the enhanced catabolism of circulating tryptophan. The purpose of the present study was firstly to establish whether there is a link between sex steroids and tryptophan pyrrolase activity especially since sex steroids are also known to cause mood disturbances and secondly to determine the effects of sex steroids on brain indolamine metabolism. The results show that all three sex steroids induce the activity of tryptophan pyrrolase implying that they decrease serum tryptophan levels by the activation of tryptophan pyrrolase, thus making less tryptophan available for uptake by the brain. It was also shown that the sex steroids enhance the uptake of ¹⁴C-tryptophan by brain synatopsomes. In addition, the sex steroids influenced the pattern of metabolism of serotonin by organ cultures of rat pineal glands. It is possible that the sex steroids regulate the availability and uptake of indoleamines in the brain.
- Full Text:
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