This study investigated the neurotoxic effects of rotenone and 6-hyroxydopamine (6 OHDA), two compounds which have been implicated in Parkinson’s disease (PD). PD is a neurodegenerative disorder that results in the impairment of movement. During the disease process, a group of dopamine-containing cells in the brain region called the Substantia Nigra pars compacta (SNc), degenerate. Whilst genetic factors contribute to approximately 5% of PD cases, the causes of the remaining 95% are unknown. What does seem clear is the pivotal role of mitochondrial dysfunction as observed in post-mortem human tissue. Mitochondrial dysfunction leads to energy depletion and the generation of harmful reactive oxygen species (ROS). However, despite the fact that the involvement of mitochondria in the disease process has been well established, the cellular events that lead to, and result from, mitochondrial dysfunction remain poorly understood. Rotenone and 6 OHDA have been implicated in PD for two reasons: (1) both toxins can relatively selectively kill SNc neurons in animal models of PD, and (2) there is evidence for both compounds having a potential causative role in the etiology of the disease in humans. When 6 OHDA is injected into the brain, or rotenone applied systemically, both toxins cause degeneration of SNc neurons. This ability makes them excellent tools for studying mechanisms of PD in animal models. In addition, both toxins inhibit mitochondrial function. Despite extensive use in models of PD, the mechanisms by which each toxin cause cell damage remains elusive. The first part of this study investigated the acute responses of dopaminergic SNc neurons to rotenone exposure (5 nM – 1 µM). The experiments were conducted on brain slices obtained from rats. Electrophysiological recordings (whole-cell patch-clamp technique) were used to detect activation of specific membrane channels as well as cell firing and changes to the membrane potential. In addition, imaging of several fluorescent dyes sensitive to specific cellular events was carried out. In voltage-clamp experiments, acute rotenone (200 nM – 1 µM) application evoked a concentration-dependent outward current which was mediated by tolbutamide-sensitive KATP channels. The current was associated with a drop in cell input resistance (Rm) and, in current-clamp, membrane hyperpolarization and inhibition of spontaneous action potentials. The mechanisms by which rotenone activates KATP channels is controversial, with some studies suggesting activation by ATP depletion and others by elevated reactive oxygen species (ROS). To address this issue, experiments were conducted with high levels of ATP in the pipette solution. Since the rotenone-induced outward current was unaffected by high ATP levels, it was concluded that KATP channel activation was due to oxidative stress. Indeed, the antioxidant Trolox significantly attenuated the current response. Confirmation of elevated ROS production was obtained by recording increased mitochondrial superoxide production, using the fluorescent dye MitoSOX. In addition, rotenone evoked depolarization of mitochondrial membrane potential (ΔΨm). Measurements of intracellular Ca2+ and Na+ were performed using the fluorescent dyes Fura 2 and SBFI, respectively. Rotenone evoked increases to both [Ca2+]i and [Na+]i in a concentration-dependent manner. The rotenone-induced [Ca2+]i rise was unaffected by blocking KATP channels with Cs+. The elevation of [Ca2+]i is particularly important in relation to cell death, since [Ca2+]i overload is known to activate pathways leading to necrosis and apoptosis. There has been growing interest in the synergistic action of rotenone with other toxins/conditions which also enhance [Ca2+]i. This concept was explored in the present study by testing the relationship between the baseline [Ca2+]i level and the rotenone-induced [Ca2+]i increase. Two approaches were taken. Firstly, baseline [Ca2+]i was deliberately raised by activation of voltage-gated calcium channels. When rotenone was applied in the presence of this raised baseline calcium level, the rotenone-induced [Ca2+]i rise was significantly greater. The second approach involved post-hoc analysis of the relationship between the normal cellular variation in baseline [Ca2+]i and the rotenone-induced [Ca2+]i elevation. This analysis also revealed a dependency of the rotenone-induced [Ca2+]i elevation on the baseline calcium level. From this finding, as well as the observation that rotenone evoked ROS production, Transient Receptor Protein subtype M2 (TRPM2) channels were proposed as the likely underlying mechanism. The potentiation of the rotenone-induced [Ca2+]i rise by an elevation in baseline calcium level can be attributed to the calcium-dependence of ROS-sensitive TRPM2 channels, known to respond with increased channel opening to increased [Ca2+]i. Recent findings from our laboratory have confirmed TRPM2 involvement in rotenone toxicity, since blockade of these channels with ACA reduced the rotenone-induced [Ca2+]i rise (K. Chung, unpublished). Imaging using the fluorescent dye propidium iodide (PI) to label cells with compromised membrane integrity was also conducted in acute midbrain slices. SNc neurons were retrograde-labelled with FluoroGold and then exposed to various toxic insults. The detergent Triton-X100 caused an increase in PI labelling, whilst rotenone and high concentrations of glutamate were ineffective over the period of time investigated (up to 40 min). The second part of this study, also conducted on acute rat midbrain slices, investigated the acute responses of SNc neurons to 6 OHDA (0.2 – 2 mM) exposure. Extracellular recordings of action potential firing were conducted on SNc neurons. 6 OHDA evoked rapid inhibition of firing in a similar manner to dopamine (100 µM). In the presence of D2 dopamine receptor blocker sulpiride, the inhibition of firing evoked by 6 OHDA was delayed, and an initial increase of firing was observed. Blockade of the dopamine transporter with nomifensine reduced the 6 OHDA-induced inhibition of firing, and prevented the persistent inhibition of firing after 6 OHDA washout. For comparison, the response to 6 OHDA of non-dopaminergic neurons in the subthalamic nucleus was also studied. In the subthalamic nucleus, 6 OHDA evoked an increase of spontaneous action potential firing. Rapid application of 6 OHDA (using the picospritz application technique) in voltage-clamp recorded SNc neurons evoked an outward current, similar to that observed after dopamine application. In the presence of sulpiride, 6 OHDA induced an inward current, consistent with the initial increase of firing activity observed in extracellular recordings. Microfluorometric experiments with Fura 2, showed that 6 OHDA evokes an increase in [Ca2+]i. Loading cells with the fluorescent dye Lucifer Yellow enabled visualization of 6 OHDA-induced swelling of the cell body and damage to proximal dendrites. Imaging of SNc neurons loaded with dextran-rhodamine revealed 6 OHDA-induced damage of distal dendrites. The last part of the study was performed on organotypic cultures obtained from slices of the ventral midbrain. These cultures were prepared from newborn transgenic mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase-promoter. This fluorescent marker enabled easy identification of dopamine-containing cells (including SNc neurons). Only preliminary experiments were carried out using this preparation. GFP-positive neurons did not show the classic membrane hyperpolarization in response to dopamine. For comparison, recordings from GFP-positive SNc neurons in acute slices obtained from age-matched animals did show a typical hyperpolarizing response to dopamine. GFP-neurons from organotypic cultures also lacked the Ih current – another characteristic feature of SNc neurons in vivo or in acute brain slices. In addition, atypical responses to CNQX (blocker of NMDA receptors) and baclofen (blocker of GABAB receptors) application were identified in GFP-positive neurons. These results demonstrate that the culturing process used in this study alters the functional ‘phenotype’ of dopaminergic neurons, a change which needs to be considered in future studies using this preparation. Chronic exposure of organotypic cultures to low concentration of rotenone (50 nM) evoked a delayed increase of PI labelling indicative of cell death, however technical limitations prevented detection of PI co-localization with GFP was observed. In conclusion, this study identified several key aspects of 6 OHDA and rotenone toxicity in SNc neurons. The most significant novel findings include evidence for ROS activation of KATP channels, presumed involvement of TRPM2 channels in rotenone-induced [Ca2+]i rise, and dopamine-analogous effects of 6 OHDA. The controversial role of KATP channels in neuroprotection was addressed. Findings from this study suggest therapies targeting this channel alone would be of little benefit. The proposed involvement of TRPM2 channels in rotenone-induced [Ca2+]i overload in SNc neurons is particularly interesting as it provides a mechanism for synergism between rotenone and other factors that disrupt [Ca2+]i homeostasis.

Hair disorders cause significant distress. The main, but limited, treatment for hair loss is minoxidil, an ATP-sensitive potassium (KATP) channel opener whose mechanism of stimulation is unclear. The regulatory component of KATP channels has three forms: SUR1, SUR2A and SUR2B which all respond to different molecules. Minoxidil only opens SUR2B channels, though SUR1 and SUR2B are present in human hair follicles. To expand our understanding, the red deer hair follicle model was used initially. Deer follicles expressed the same KATP channel genes as human follicles when growing (anagen), but no channels were detected in resting follicles. This reinforces the importance of KATP channels in active hair growth and the usefulness of the deer model. To assess whether SUR1 KATP channels are actually involved in human hair growth, the effects of a selective SUR1 channel opener, NNC55-9216, on scalp follicle growth in organ culture was examined. NNC55-9216 stimulated anagen; its effect was augmented by minoxidil. This creates the potential for more effective pharmaceuticals to treat hair loss via SUR1 channels, either alone or in combination with minoxidil. The dermal papilla plays a crucial regulatory role in hair follicle activity determining the type of hair produced. Minoxidil had no effect on dermal papilla cell proliferation, but altered the profile of proteins produced when assessed by proteomics. Further research into the roles of KATP channels and greater understanding of the significance of these protein changes should enhance our knowledge of hair biology and help the development of new, improved therapies for hair pathologies.

This study investigated the neurotoxic effects of rotenone and 6-hyroxydopamine (6 OHDA), two compounds which have been implicated in Parkinson’s disease (PD). PD is a neurodegenerative disorder that results in the impairment of movement. During the disease process, a group of dopamine-containing cells in the brain region called the Substantia Nigra pars compacta (SNc), degenerate. Whilst genetic factors contribute to approximately 5% of PD cases, the causes of the remaining 95% are unknown. What does seem clear is the pivotal role of mitochondrial dysfunction as observed in post-mortem human tissue. Mitochondrial dysfunction leads to energy depletion and the generation of harmful reactive oxygen species (ROS). However, despite the fact that the involvement of mitochondria in the disease process has been well established, the cellular events that lead to, and result from, mitochondrial dysfunction remain poorly understood. Rotenone and 6 OHDA have been implicated in PD for two reasons: (1) both toxins can relatively selectively kill SNc neurons in animal models of PD, and (2) there is evidence for both compounds having a potential causative role in the etiology of the disease in humans. When 6 OHDA is injected into the brain, or rotenone applied systemically, both toxins cause degeneration of SNc neurons. This ability makes them excellent tools for studying mechanisms of PD in animal models. In addition, both toxins inhibit mitochondrial function. Despite extensive use in models of PD, the mechanisms by which each toxin cause cell damage remains elusive. The first part of this study investigated the acute responses of dopaminergic SNc neurons to rotenone exposure (5 nM – 1 µM). The experiments were conducted on brain slices obtained from rats. Electrophysiological recordings (whole-cell patch-clamp technique) were used to detect activation of specific membrane channels as well as cell firing and changes to the membrane potential. In addition, imaging of several fluorescent dyes sensitive to specific cellular events was carried out. In voltage-clamp experiments, acute rotenone (200 nM – 1 µM) application evoked a concentration-dependent outward current which was mediated by tolbutamide-sensitive KATP channels. The current was associated with a drop in cell input resistance (Rm) and, in current-clamp, membrane hyperpolarization and inhibition of spontaneous action potentials. The mechanisms by which rotenone activates KATP channels is controversial, with some studies suggesting activation by ATP depletion and others by elevated reactive oxygen species (ROS). To address this issue, experiments were conducted with high levels of ATP in the pipette solution. Since the rotenone-induced outward current was unaffected by high ATP levels, it was concluded that KATP channel activation was due to oxidative stress. Indeed, the antioxidant Trolox significantly attenuated the current response. Confirmation of elevated ROS production was obtained by recording increased mitochondrial superoxide production, using the fluorescent dye MitoSOX. In addition, rotenone evoked depolarization of mitochondrial membrane potential (ΔΨm). Measurements of intracellular Ca2+ and Na+ were performed using the fluorescent dyes Fura 2 and SBFI, respectively. Rotenone evoked increases to both [Ca2+]i and [Na+]i in a concentration-dependent manner. The rotenone-induced [Ca2+]i rise was unaffected by blocking KATP channels with Cs+. The elevation of [Ca2+]i is particularly important in relation to cell death, since [Ca2+]i overload is known to activate pathways leading to necrosis and apoptosis. There has been growing interest in the synergistic action of rotenone with other toxins/conditions which also enhance [Ca2+]i. This concept was explored in the present study by testing the relationship between the baseline [Ca2+]i level and the rotenone-induced [Ca2+]i increase. Two approaches were taken. Firstly, baseline [Ca2+]i was deliberately raised by activation of voltage-gated calcium channels. When rotenone was applied in the presence of this raised baseline calcium level, the rotenone-induced [Ca2+]i rise was significantly greater. The second approach involved post-hoc analysis of the relationship between the normal cellular variation in baseline [Ca2+]i and the rotenone-induced [Ca2+]i elevation. This analysis also revealed a dependency of the rotenone-induced [Ca2+]i elevation on the baseline calcium level. From this finding, as well as the observation that rotenone evoked ROS production, Transient Receptor Protein subtype M2 (TRPM2) channels were proposed as the likely underlying mechanism. The potentiation of the rotenone-induced [Ca2+]i rise by an elevation in baseline calcium level can be attributed to the calcium-dependence of ROS-sensitive TRPM2 channels, known to respond with increased channel opening to increased [Ca2+]i. Recent findings from our laboratory have confirmed TRPM2 involvement in rotenone toxicity, since blockade of these channels with ACA reduced the rotenone-induced [Ca2+]i rise (K. Chung, unpublished). Imaging using the fluorescent dye propidium iodide (PI) to label cells with compromised membrane integrity was also conducted in acute midbrain slices. SNc neurons were retrograde-labelled with FluoroGold and then exposed to various toxic insults. The detergent Triton-X100 caused an increase in PI labelling, whilst rotenone and high concentrations of glutamate were ineffective over the period of time investigated (up to 40 min). The second part of this study, also conducted on acute rat midbrain slices, investigated the acute responses of SNc neurons to 6 OHDA (0.2 – 2 mM) exposure. Extracellular recordings of action potential firing were conducted on SNc neurons. 6 OHDA evoked rapid inhibition of firing in a similar manner to dopamine (100 µM). In the presence of D2 dopamine receptor blocker sulpiride, the inhibition of firing evoked by 6 OHDA was delayed, and an initial increase of firing was observed. Blockade of the dopamine transporter with nomifensine reduced the 6 OHDA-induced inhibition of firing, and prevented the persistent inhibition of firing after 6 OHDA washout. For comparison, the response to 6 OHDA of non-dopaminergic neurons in the subthalamic nucleus was also studied. In the subthalamic nucleus, 6 OHDA evoked an increase of spontaneous action potential firing. Rapid application of 6 OHDA (using the picospritz application technique) in voltage-clamp recorded SNc neurons evoked an outward current, similar to that observed after dopamine application. In the presence of sulpiride, 6 OHDA induced an inward current, consistent with the initial increase of firing activity observed in extracellular recordings. Microfluorometric experiments with Fura 2, showed that 6 OHDA evokes an increase in [Ca2+]i. Loading cells with the fluorescent dye Lucifer Yellow enabled visualization of 6 OHDA-induced swelling of the cell body and damage to proximal dendrites. Imaging of SNc neurons loaded with dextran-rhodamine revealed 6 OHDA-induced damage of distal dendrites. The last part of the study was performed on organotypic cultures obtained from slices of the ventral midbrain. These cultures were prepared from newborn transgenic mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase-promoter. This fluorescent marker enabled easy identification of dopamine-containing cells (including SNc neurons). Only preliminary experiments were carried out using this preparation. GFP-positive neurons did not show the classic membrane hyperpolarization in response to dopamine. For comparison, recordings from GFP-positive SNc neurons in acute slices obtained from age-matched animals did show a typical hyperpolarizing response to dopamine. GFP-neurons from organotypic cultures also lacked the Ih current – another characteristic feature of SNc neurons in vivo or in acute brain slices. In addition, atypical responses to CNQX (blocker of NMDA receptors) and baclofen (blocker of GABAB receptors) application were identified in GFP-positive neurons. These results demonstrate that the culturing process used in this study alters the functional ‘phenotype’ of dopaminergic neurons, a change which needs to be considered in future studies using this preparation. Chronic exposure of organotypic cultures to low concentration of rotenone (50 nM) evoked a delayed increase of PI labelling indicative of cell death, however technical limitations prevented detection of PI co-localization with GFP was observed. In conclusion, this study identified several key aspects of 6 OHDA and rotenone toxicity in SNc neurons. The most significant novel findings include evidence for ROS activation of KATP channels, presumed involvement of TRPM2 channels in rotenone-induced [Ca2+]i rise, and dopamine-analogous effects of 6 OHDA. The controversial role of KATP channels in neuroprotection was addressed. Findings from this study suggest therapies targeting this channel alone would be of little benefit. The proposed involvement of TRPM2 channels in rotenone-induced [Ca2+]i overload in SNc neurons is particularly interesting as it provides a mechanism for synergism between rotenone and other factors that disrupt [Ca2+]i homeostasis.

This study investigated the neurotoxic effects of rotenone and 6-hyroxydopamine (6 OHDA), two compounds which have been implicated in Parkinson’s disease (PD). PD is a neurodegenerative disorder that results in the impairment of movement. During the disease process, a group of dopamine-containing cells in the brain region called the Substantia Nigra pars compacta (SNc), degenerate. Whilst genetic factors contribute to approximately 5% of PD cases, the causes of the remaining 95% are unknown. What does seem clear is the pivotal role of mitochondrial dysfunction as observed in post-mortem human tissue. Mitochondrial dysfunction leads to energy depletion and the generation of harmful reactive oxygen species (ROS). However, despite the fact that the involvement of mitochondria in the disease process has been well established, the cellular events that lead to, and result from, mitochondrial dysfunction remain poorly understood. Rotenone and 6 OHDA have been implicated in PD for two reasons: (1) both toxins can relatively selectively kill SNc neurons in animal models of PD, and (2) there is evidence for both compounds having a potential causative role in the etiology of the disease in humans. When 6 OHDA is injected into the brain, or rotenone applied systemically, both toxins cause degeneration of SNc neurons. This ability makes them excellent tools for studying mechanisms of PD in animal models. In addition, both toxins inhibit mitochondrial function. Despite extensive use in models of PD, the mechanisms by which each toxin cause cell damage remains elusive. The first part of this study investigated the acute responses of dopaminergic SNc neurons to rotenone exposure (5 nM – 1 µM). The experiments were conducted on brain slices obtained from rats. Electrophysiological recordings (whole-cell patch-clamp technique) were used to detect activation of specific membrane channels as well as cell firing and changes to the membrane potential. In addition, imaging of several fluorescent dyes sensitive to specific cellular events was carried out. In voltage-clamp experiments, acute rotenone (200 nM – 1 µM) application evoked a concentration-dependent outward current which was mediated by tolbutamide-sensitive KATP channels. The current was associated with a drop in cell input resistance (Rm) and, in current-clamp, membrane hyperpolarization and inhibition of spontaneous action potentials. The mechanisms by which rotenone activates KATP channels is controversial, with some studies suggesting activation by ATP depletion and others by elevated reactive oxygen species (ROS). To address this issue, experiments were conducted with high levels of ATP in the pipette solution. Since the rotenone-induced outward current was unaffected by high ATP levels, it was concluded that KATP channel activation was due to oxidative stress. Indeed, the antioxidant Trolox significantly attenuated the current response. Confirmation of elevated ROS production was obtained by recording increased mitochondrial superoxide production, using the fluorescent dye MitoSOX. In addition, rotenone evoked depolarization of mitochondrial membrane potential (ΔΨm). Measurements of intracellular Ca2+ and Na+ were performed using the fluorescent dyes Fura 2 and SBFI, respectively. Rotenone evoked increases to both [Ca2+]i and [Na+]i in a concentration-dependent manner. The rotenone-induced [Ca2+]i rise was unaffected by blocking KATP channels with Cs+. The elevation of [Ca2+]i is particularly important in relation to cell death, since [Ca2+]i overload is known to activate pathways leading to necrosis and apoptosis. There has been growing interest in the synergistic action of rotenone with other toxins/conditions which also enhance [Ca2+]i. This concept was explored in the present study by testing the relationship between the baseline [Ca2+]i level and the rotenone-induced [Ca2+]i increase. Two approaches were taken. Firstly, baseline [Ca2+]i was deliberately raised by activation of voltage-gated calcium channels. When rotenone was applied in the presence of this raised baseline calcium level, the rotenone-induced [Ca2+]i rise was significantly greater. The second approach involved post-hoc analysis of the relationship between the normal cellular variation in baseline [Ca2+]i and the rotenone-induced [Ca2+]i elevation. This analysis also revealed a dependency of the rotenone-induced [Ca2+]i elevation on the baseline calcium level. From this finding, as well as the observation that rotenone evoked ROS production, Transient Receptor Protein subtype M2 (TRPM2) channels were proposed as the likely underlying mechanism. The potentiation of the rotenone-induced [Ca2+]i rise by an elevation in baseline calcium level can be attributed to the calcium-dependence of ROS-sensitive TRPM2 channels, known to respond with increased channel opening to increased [Ca2+]i. Recent findings from our laboratory have confirmed TRPM2 involvement in rotenone toxicity, since blockade of these channels with ACA reduced the rotenone-induced [Ca2+]i rise (K. Chung, unpublished). Imaging using the fluorescent dye propidium iodide (PI) to label cells with compromised membrane integrity was also conducted in acute midbrain slices. SNc neurons were retrograde-labelled with FluoroGold and then exposed to various toxic insults. The detergent Triton-X100 caused an increase in PI labelling, whilst rotenone and high concentrations of glutamate were ineffective over the period of time investigated (up to 40 min). The second part of this study, also conducted on acute rat midbrain slices, investigated the acute responses of SNc neurons to 6 OHDA (0.2 – 2 mM) exposure. Extracellular recordings of action potential firing were conducted on SNc neurons. 6 OHDA evoked rapid inhibition of firing in a similar manner to dopamine (100 µM). In the presence of D2 dopamine receptor blocker sulpiride, the inhibition of firing evoked by 6 OHDA was delayed, and an initial increase of firing was observed. Blockade of the dopamine transporter with nomifensine reduced the 6 OHDA-induced inhibition of firing, and prevented the persistent inhibition of firing after 6 OHDA washout. For comparison, the response to 6 OHDA of non-dopaminergic neurons in the subthalamic nucleus was also studied. In the subthalamic nucleus, 6 OHDA evoked an increase of spontaneous action potential firing. Rapid application of 6 OHDA (using the picospritz application technique) in voltage-clamp recorded SNc neurons evoked an outward current, similar to that observed after dopamine application. In the presence of sulpiride, 6 OHDA induced an inward current, consistent with the initial increase of firing activity observed in extracellular recordings. Microfluorometric experiments with Fura 2, showed that 6 OHDA evokes an increase in [Ca2+]i. Loading cells with the fluorescent dye Lucifer Yellow enabled visualization of 6 OHDA-induced swelling of the cell body and damage to proximal dendrites. Imaging of SNc neurons loaded with dextran-rhodamine revealed 6 OHDA-induced damage of distal dendrites. The last part of the study was performed on organotypic cultures obtained from slices of the ventral midbrain. These cultures were prepared from newborn transgenic mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase-promoter. This fluorescent marker enabled easy identification of dopamine-containing cells (including SNc neurons). Only preliminary experiments were carried out using this preparation. GFP-positive neurons did not show the classic membrane hyperpolarization in response to dopamine. For comparison, recordings from GFP-positive SNc neurons in acute slices obtained from age-matched animals did show a typical hyperpolarizing response to dopamine. GFP-neurons from organotypic cultures also lacked the Ih current – another characteristic feature of SNc neurons in vivo or in acute brain slices. In addition, atypical responses to CNQX (blocker of NMDA receptors) and baclofen (blocker of GABAB receptors) application were identified in GFP-positive neurons. These results demonstrate that the culturing process used in this study alters the functional ‘phenotype’ of dopaminergic neurons, a change which needs to be considered in future studies using this preparation. Chronic exposure of organotypic cultures to low concentration of rotenone (50 nM) evoked a delayed increase of PI labelling indicative of cell death, however technical limitations prevented detection of PI co-localization with GFP was observed. In conclusion, this study identified several key aspects of 6 OHDA and rotenone toxicity in SNc neurons. The most significant novel findings include evidence for ROS activation of KATP channels, presumed involvement of TRPM2 channels in rotenone-induced [Ca2+]i rise, and dopamine-analogous effects of 6 OHDA. The controversial role of KATP channels in neuroprotection was addressed. Findings from this study suggest therapies targeting this channel alone would be of little benefit. The proposed involvement of TRPM2 channels in rotenone-induced [Ca2+]i overload in SNc neurons is particularly interesting as it provides a mechanism for synergism between rotenone and other factors that disrupt [Ca2+]i homeostasis.

This study investigated the neurotoxic effects of rotenone and 6-hyroxydopamine (6 OHDA), two compounds which have been implicated in Parkinson’s disease (PD). PD is a neurodegenerative disorder that results in the impairment of movement. During the disease process, a group of dopamine-containing cells in the brain region called the Substantia Nigra pars compacta (SNc), degenerate. Whilst genetic factors contribute to approximately 5% of PD cases, the causes of the remaining 95% are unknown. What does seem clear is the pivotal role of mitochondrial dysfunction as observed in post-mortem human tissue. Mitochondrial dysfunction leads to energy depletion and the generation of harmful reactive oxygen species (ROS). However, despite the fact that the involvement of mitochondria in the disease process has been well established, the cellular events that lead to, and result from, mitochondrial dysfunction remain poorly understood. Rotenone and 6 OHDA have been implicated in PD for two reasons: (1) both toxins can relatively selectively kill SNc neurons in animal models of PD, and (2) there is evidence for both compounds having a potential causative role in the etiology of the disease in humans. When 6 OHDA is injected into the brain, or rotenone applied systemically, both toxins cause degeneration of SNc neurons. This ability makes them excellent tools for studying mechanisms of PD in animal models. In addition, both toxins inhibit mitochondrial function. Despite extensive use in models of PD, the mechanisms by which each toxin cause cell damage remains elusive. The first part of this study investigated the acute responses of dopaminergic SNc neurons to rotenone exposure (5 nM – 1 µM). The experiments were conducted on brain slices obtained from rats. Electrophysiological recordings (whole-cell patch-clamp technique) were used to detect activation of specific membrane channels as well as cell firing and changes to the membrane potential. In addition, imaging of several fluorescent dyes sensitive to specific cellular events was carried out. In voltage-clamp experiments, acute rotenone (200 nM – 1 µM) application evoked a concentration-dependent outward current which was mediated by tolbutamide-sensitive KATP channels. The current was associated with a drop in cell input resistance (Rm) and, in current-clamp, membrane hyperpolarization and inhibition of spontaneous action potentials. The mechanisms by which rotenone activates KATP channels is controversial, with some studies suggesting activation by ATP depletion and others by elevated reactive oxygen species (ROS). To address this issue, experiments were conducted with high levels of ATP in the pipette solution. Since the rotenone-induced outward current was unaffected by high ATP levels, it was concluded that KATP channel activation was due to oxidative stress. Indeed, the antioxidant Trolox significantly attenuated the current response. Confirmation of elevated ROS production was obtained by recording increased mitochondrial superoxide production, using the fluorescent dye MitoSOX. In addition, rotenone evoked depolarization of mitochondrial membrane potential (ΔΨm). Measurements of intracellular Ca2+ and Na+ were performed using the fluorescent dyes Fura 2 and SBFI, respectively. Rotenone evoked increases to both [Ca2+]i and [Na+]i in a concentration-dependent manner. The rotenone-induced [Ca2+]i rise was unaffected by blocking KATP channels with Cs+. The elevation of [Ca2+]i is particularly important in relation to cell death, since [Ca2+]i overload is known to activate pathways leading to necrosis and apoptosis. There has been growing interest in the synergistic action of rotenone with other toxins/conditions which also enhance [Ca2+]i. This concept was explored in the present study by testing the relationship between the baseline [Ca2+]i level and the rotenone-induced [Ca2+]i increase. Two approaches were taken. Firstly, baseline [Ca2+]i was deliberately raised by activation of voltage-gated calcium channels. When rotenone was applied in the presence of this raised baseline calcium level, the rotenone-induced [Ca2+]i rise was significantly greater. The second approach involved post-hoc analysis of the relationship between the normal cellular variation in baseline [Ca2+]i and the rotenone-induced [Ca2+]i elevation. This analysis also revealed a dependency of the rotenone-induced [Ca2+]i elevation on the baseline calcium level. From this finding, as well as the observation that rotenone evoked ROS production, Transient Receptor Protein subtype M2 (TRPM2) channels were proposed as the likely underlying mechanism. The potentiation of the rotenone-induced [Ca2+]i rise by an elevation in baseline calcium level can be attributed to the calcium-dependence of ROS-sensitive TRPM2 channels, known to respond with increased channel opening to increased [Ca2+]i. Recent findings from our laboratory have confirmed TRPM2 involvement in rotenone toxicity, since blockade of these channels with ACA reduced the rotenone-induced [Ca2+]i rise (K. Chung, unpublished). Imaging using the fluorescent dye propidium iodide (PI) to label cells with compromised membrane integrity was also conducted in acute midbrain slices. SNc neurons were retrograde-labelled with FluoroGold and then exposed to various toxic insults. The detergent Triton-X100 caused an increase in PI labelling, whilst rotenone and high concentrations of glutamate were ineffective over the period of time investigated (up to 40 min). The second part of this study, also conducted on acute rat midbrain slices, investigated the acute responses of SNc neurons to 6 OHDA (0.2 – 2 mM) exposure. Extracellular recordings of action potential firing were conducted on SNc neurons. 6 OHDA evoked rapid inhibition of firing in a similar manner to dopamine (100 µM). In the presence of D2 dopamine receptor blocker sulpiride, the inhibition of firing evoked by 6 OHDA was delayed, and an initial increase of firing was observed. Blockade of the dopamine transporter with nomifensine reduced the 6 OHDA-induced inhibition of firing, and prevented the persistent inhibition of firing after 6 OHDA washout. For comparison, the response to 6 OHDA of non-dopaminergic neurons in the subthalamic nucleus was also studied. In the subthalamic nucleus, 6 OHDA evoked an increase of spontaneous action potential firing. Rapid application of 6 OHDA (using the picospritz application technique) in voltage-clamp recorded SNc neurons evoked an outward current, similar to that observed after dopamine application. In the presence of sulpiride, 6 OHDA induced an inward current, consistent with the initial increase of firing activity observed in extracellular recordings. Microfluorometric experiments with Fura 2, showed that 6 OHDA evokes an increase in [Ca2+]i. Loading cells with the fluorescent dye Lucifer Yellow enabled visualization of 6 OHDA-induced swelling of the cell body and damage to proximal dendrites. Imaging of SNc neurons loaded with dextran-rhodamine revealed 6 OHDA-induced damage of distal dendrites. The last part of the study was performed on organotypic cultures obtained from slices of the ventral midbrain. These cultures were prepared from newborn transgenic mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase-promoter. This fluorescent marker enabled easy identification of dopamine-containing cells (including SNc neurons). Only preliminary experiments were carried out using this preparation. GFP-positive neurons did not show the classic membrane hyperpolarization in response to dopamine. For comparison, recordings from GFP-positive SNc neurons in acute slices obtained from age-matched animals did show a typical hyperpolarizing response to dopamine. GFP-neurons from organotypic cultures also lacked the Ih current – another characteristic feature of SNc neurons in vivo or in acute brain slices. In addition, atypical responses to CNQX (blocker of NMDA receptors) and baclofen (blocker of GABAB receptors) application were identified in GFP-positive neurons. These results demonstrate that the culturing process used in this study alters the functional ‘phenotype’ of dopaminergic neurons, a change which needs to be considered in future studies using this preparation. Chronic exposure of organotypic cultures to low concentration of rotenone (50 nM) evoked a delayed increase of PI labelling indicative of cell death, however technical limitations prevented detection of PI co-localization with GFP was observed. In conclusion, this study identified several key aspects of 6 OHDA and rotenone toxicity in SNc neurons. The most significant novel findings include evidence for ROS activation of KATP channels, presumed involvement of TRPM2 channels in rotenone-induced [Ca2+]i rise, and dopamine-analogous effects of 6 OHDA. The controversial role of KATP channels in neuroprotection was addressed. Findings from this study suggest therapies targeting this channel alone would be of little benefit. The proposed involvement of TRPM2 channels in rotenone-induced [Ca2+]i overload in SNc neurons is particularly interesting as it provides a mechanism for synergism between rotenone and other factors that disrupt [Ca2+]i homeostasis.

No description available.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--Stellenbosch University, 2008. / Background and purpose. To limit ischaemic injury, rapid restoration of coronary blood flow is required, which will in turn reduce infarct size. However, reperfusion itself causes myocyte death – a phenomenon termed lethal reperfusion-induced injury, which limits protection of the ischaemic myocardium. Thus the reperfusion of irreversibly damaged myocytes may accelerate the process of cell necrosis. Additive protection of the ischaemic myocardium in the form of adjunct therapy remains a topic of intensive research. Levosimendan, a calcium sensitizing agent with positive inotropic effects has in several studies been found to alleviate the damaging effects of reperfusion injury. Levosimendan has been shown to be a KATP channel opener. These channels have been implicated to play an important role in ischaemic preconditioning (IPC). With this knowledge, the aim of this study was to determine whether levosimendan and IPC have certain cardioprotective mechanisms in common and whether protection with pharmacological preconditioning could be elicited with levosimendan. In this study, we investigated whether: 1) the isolated guinea pig heart could be protected by ischaemic preconditioning (IPC) and postconditioning (IPostC), 2) the heart could be pharmacologically pre- and postconditioned, using levosimendan (LPC & LPostC), 3) a combination of IPC & LPC had an additive protective effect on the heart, 4) the KATP (both mitochondrial and sarcolemmal) channels are involved in this protection and 5) the pro-survival kinases of the RISK (reperfusion injury salvage kinase) pathway are involved. Experimental approach. Isolated perfused guinea pig hearts were subjected to three different IPC protocols (1x5, 2x5 and 3x5 minutes of ischaemia) or levosimendan (0.1μM) preconditioning, before coronary artery occlusion (CAO – 40min@36.5ºC), followed by 30 minutes of reperfusion. Hearts were also subjected to a combination of IPC & LPC, to establish whether they had additive protective effects. In addition, hearts were pre-treated with levosimendan directly before induction of sustained ischaemia (without washout of the drug – levosimendan pre-treatment (LPT)) for 10min. With the postconditioning protocol, iii the hearts were subjected to 3x30second cycles of ischaemia/reperfusion or levosimendan/vehicle. In a separate series of experiments, hearts were treated with KATP channel blockers (for both sarcolemmal & mitochondrial), before LPC, LPT and LPostC. The endpoints that were measured were: cardiac reperfusion function, myocardial infarct size and RISK pathway expression and phosphorylation (PKB/Akt and extracellular signal-regulated kinase – ERK42/44). Results. IPC, IPostC, LPC & LPostC decreased myocardial infarct size significantly compared with their controls (21.9±2.2%, 21.4±2.2%, 20.6±3.1% and 20.6±1.8% respectively vs. 46.4±1.8% for controls, p<0.05). The combination of IPC & LPC had no additive protective effect. Pre-treating the hearts with levosimendan (without washout), before index ischaemia, proved to be the most effective method of cardioprotection (infarct size: 5.8±0.9% vs. 46.4±1.8% for controls, p<0.001). With LPT a significant increase (p < 0.05 vs. control) in phosphorylation of ER42/44 was also observed. An increase in the activity of one of the RISK pathway kinases, ERK42/44 seems to be one of the reasons for LPT’s efficacy. Treating the hearts with KATP channel blockers before subjecting them to LPC, LPT & LPostC abolished the protective effects induced by levosimendan, suggesting a role for the sarcolemmal and mitochondrial KATP channels in levosimendan-induced cardioprotection. Conclusions and implications. 1) Isolated guinea pig hearts could be pre- and postconditioned within the setting of ischaemia, 2) Hearts could be pharmacologically pre- and postconditioned with levosimendan, 3) levosimendan pre-treatment is the most effective way to reduce infarct size, possibly acting by increasing the phosphorylation of ERK42/44, 4) Myocardial protection was not increased by combining IPC & LPC (suggesting similar mechanisms of protection), 5) LPC, LPT and LPostC were abolished by both sarcolemmal and mitochondrial KATP channel blockers. .LPC and especially LPT, could be useful before elective cardiac surgery while LPostC may be considered after acute coronary artery events.

3-Hydroxy-3-Methylglutaryl Coenzyme A (HMG CoA) reductase is a 97 kDa glycoprotein located in the endoplasmic reticulum responsible for cholesterol biosynthesis in mammalian liver and intestine. HMG CoA reductase inhibitors (statins) (e.g. simvastatin, mevastatin and parvastatin) are used clinically to treat and prevent coronary artery diseases by reducing plasma LDL-cholesterol level. Recent studies have demonstrated that statins can provide beneficial effects (pleiotropic effects) beyond its lipid-lowering activity. However, the modulatory effects of statins on ion channels activities have not been fully explored. Hence, this study is designed to demonstrate the existence of the HMG CoA reductase in various human isolate cardiovascular preparations and the modulatory effect(s) of simvastatin on both large-conductance calcium-activated (BKCa) and ATP-sensitive (IKATP) potassium channels of porcine isolated coronary vascular smooth muscle cells. / In conclusion, our results demonstrated the biochemical existence of HMG CoA reductase in various human isolated cardiovascular preparations and porcine isolated coronary artery. Simvastatin modulates the BKCa and IKATP channels of the porcine isolated coronary artery via different and multiple cellular mechanisms. / In this study, we demonstrated the biochemical existence of the HMG CoA reductase in various human isolated cardiovascular preparations and porcine isolated coronary artery. In addition, we demonstrated that simvastatin modulates both the BKCa channels and IKATP channels of porcine isolated coronary artery via different mechanisms. Acute application of simvastatin (100 nM) slightly enhanced whereas simvastatin (&ge; 1 muM) inhibited the BKCa amplitude of porcine coronary artery smooth muscle cells. The classical HMG CoA reductase-mevalonate cascade is important in mediating the inhibitory effect of simvastatin observed at low concentrations (1 and 3 muM), whereas an increased PKC-delta protein expression and activation is important in simvastatin (10 muM)-mediated inhibition of BKCa channels. In contrast, the basal activity of the IKATP channels was not affected by simvastatin (1, 3 and 10 muM). However, acute application of simvastatin (1, 3 and 10 muM) inhibited the opening of the IKATP channels by cromakalim and pinacidil in a PP2A-dependent manner (sensitive to okadaic acid, a PP2A inhibitor). The okadaic acid-sensitive, simvastatin-mediated inhibitory effect on IKATP channel is mediated by an activation of AMPK in a Ca2+-dependent manner. Activation of AMPK probably increased the activity of the Na+/K+ ATPase and subsequently caused an influx of glucose via the SGLT1 down the Na + concentration gradient for the ouabain-sensitive, glucose-dependent activation of PP2A. / Seto, Sai Wang. / Adviser: Yiu-Wa Kwan. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3456. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 221-254). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

<p>Insulin release from single islets of Langerhans is pulsatile. The secretory activities of the islets in the pancreas are coordinated resulting in plasma insulin oscillations. Nutrients amplitude-regulate the insulin pulses without influencing their frequency. Diabetic patients show an abnormal plasma insulin pattern, but the cause of the disturbance remains to be elucidated. Ithe present thesis the influence of the cytoplasmic calcium concentratio([Ca²⁺]_i) and cell metabolism on pulsatile insulin release was examined in single islets of Langerhans from <i>ob/ob</i>-mice. Glucose stimulation of insulin release involves closure of ATP-sensitive K⁺ channels (K_ATP channels), depolarization, and Ca²⁺ influx in β-cells. In the presence of 11 mM glucose, pulsatile insulin secretion occurs in synchrony with oscillations i[Ca²⁺]_i. When [Ca²⁺]_i is low and stable, e.g. under basal conditions, low amplitude insulin pulses are still observed. When [Ca²⁺]_i is elevated and non-oscillating, e.g. when the β-cells are depolarized by potassium, high amplitude insulin pulses are observed. The frequency of the insulin pulses under these conditions is similar to that observed when [Ca²⁺]_i oscillations are present. By permanently opening or closing the K_ATP channels with diazoxide or tolbutamide, respectively, it was investigated if glucose can modulate pulsatile insulin secretion when it does not influence the channel activity. Under these conditions, [Ca²⁺]_i remained stable whereas the amplitude of the insulin pulses increased with sugar stimulation without change in the frequency. Metabolic inhibition blunted but did not prevent the insulin pulses. The results indicate that oscillations in metabolism can generate pulsatile insulin release when [Ca²⁺]_i is stable. However, under physiological conditions, pulsatile secretion is driven by oscillations in metabolism and [Ca²⁺]_i, acting in synergy.</p>

Insulin release from single islets of Langerhans is pulsatile. The secretory activities of the islets in the pancreas are coordinated resulting in plasma insulin oscillations. Nutrients amplitude-regulate the insulin pulses without influencing their frequency. Diabetic patients show an abnormal plasma insulin pattern, but the cause of the disturbance remains to be elucidated. Ithe present thesis the influence of the cytoplasmic calcium concentratio([Ca2+]i) and cell metabolism on pulsatile insulin release was examined in single islets of Langerhans from ob/ob-mice. Glucose stimulation of insulin release involves closure of ATP-sensitive K+ channels (KATP channels), depolarization, and Ca2+ influx in β-cells. In the presence of 11 mM glucose, pulsatile insulin secretion occurs in synchrony with oscillations i[Ca2+]i. When [Ca2+]i is low and stable, e.g. under basal conditions, low amplitude insulin pulses are still observed. When [Ca2+]i is elevated and non-oscillating, e.g. when the β-cells are depolarized by potassium, high amplitude insulin pulses are observed. The frequency of the insulin pulses under these conditions is similar to that observed when [Ca2+]i oscillations are present. By permanently opening or closing the KATP channels with diazoxide or tolbutamide, respectively, it was investigated if glucose can modulate pulsatile insulin secretion when it does not influence the channel activity. Under these conditions, [Ca2+]i remained stable whereas the amplitude of the insulin pulses increased with sugar stimulation without change in the frequency. Metabolic inhibition blunted but did not prevent the insulin pulses. The results indicate that oscillations in metabolism can generate pulsatile insulin release when [Ca2+]i is stable. However, under physiological conditions, pulsatile secretion is driven by oscillations in metabolism and [Ca2+]i, acting in synergy.