No description available.

The tightly regulated openings of the cardiac ryanodine receptor (RyR2) help to ensure that intracellular Ca2+- release from the sarcoplasmic reticulum (SR) can only occur when heart contractions are required. Usually this process is self-regulatory, where Ca2+ both activates and inhibits release of further Ca2+ from the SR. In the progression of heart failure some of this control is lost and in rest periods Ca2+ can leak from the SR into the cytosol. Recent evidence has suggested that Zn2+- dyshomeostasis may contribute to SR Ca2+- leak but the underlying mechanism is unclear. Using single channel electrophysiological studies in combination with live cell imaging of HEK 293 and fibroblasts, this study reveals that Zn2+, along with Ca2+ and the inhibitor Mg2+, plays a physiological role in the grading of Ca2+- release via RyR2. Importantly the data reveal that pathophysiological concentrations of Zn2+ (> 100pM) within the cytosol remove the requirement of Ca2+ to activate RyR2, resulting in irregular channel activity even in the presence of Mg2+. This increase in channel open probability due to Zn2+ is known to be associated with increased Ca2+- release events such as Ca2+ sparks suggesting that Zn2+ is a regulator of the SR Ca2+-leak current. A potential source of releasable Zn2+, which could modulate RyR2 activity in cardiomyocytes, are the acidic organelles (endosomes and lysosomes). This study provides key evidence that the two pore channels (TPCs), which are expressed on the surface of these organelles, are candidate channels for ligand-gated release of Zn2+. Importantly this research demonstrates that dysregulated Zn2+ homeostasis, resulting in elevated Zn2+ within the lysosome, has severe consequences upon cellular Ca2+- release from fibroblasts, which is primarily the result of Zn2+ acting as a pore blocker of TPC2. Together these data reveal a key role of Zn2+ as a second messenger which can regulate intracellular Ca2+- release in both health and disease.

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.

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.

Adicionalmente às alterações estruturais e funcionais observadas na insuficiência cardíaca (IC), as alterações na dinâmica do Ca2+ intracelular apresentam uma relação negativa com a contratilidade e função cardíaca. Para evitar a progressão da IC, o tratamento desta síndrome, conta atualmente com intervenções multifatoriais, incluindo a terapia farmacológica, com destaque aos -bloqueadores, e a terapia não medicamentosa incluindo o treinamento físico aeróbico, que quando realizado em intensidade adequada melhora a tolerância ao esforço e a qualidade de vida do portador de insuficiência cardíaca. É bem conhecido que os -bloqueadores melhoram a função cardíaca e dinâmica do Ca2+ intracelular na IC, além de minimizar a remodelação cardíaca, no entanto, ainda existem controvérsias sobre qual o melhor -bloqueador a ser utilizado e seus efeitos específicos na regulação de Ca2+ intracelular. Quanto aos efeitos do treinamento físico aeróbico na IC, seu efeito é reconhecido na melhora da tolerância aos esforços e qualidade de vida do paciente, o que é atribuído principalmente, a ganhos vasculares e músculos-esqueléticos. Em relação à função cardíaca, em trabalho anterior do nosso laboratório, o treinamento físico aeróbico melhorou a função cardíaca associado à melhora nos transientes de cálcio do miócito cardíaco. Como tanto o treinamento físico como os -bloqueadores apresentaram seus respectivos efeitos positivos, mas diferenciados, na presente tese estudou-se o efeito associado do treinamento físico ao tratamento com -bloqueadores sobre a função cardíaca e o fluxo de cálcio no cardiomiócito. Além disso, comparou-se qual -bloqueador (metoprolol ou carvedilol) seria mais efetivo na associação com o treinamento físico. Dividimos nossa amostra em 6 grupos, todos com IC. Dentre eles, 3 foram mantidos sedentários; salina (S), metoprolol (M) e carvedilol (C) e 3 foram treinados; treinados (T), treinados e tratados com metoprolol (MT) e treinados e tratados com carvedilol (CT). Verificamos que apenas os camundongos com IC treinados melhoraram a tolerância ao esforço, medida por teste máximo em esteira rolante, o que não foi observado no tratamento farmacológico isoladamente. Quanto à função cardíaca, observamos uma melhora da função nos grupos que realizaram treinamento físico ou foram tratados com metoprolol ou carvedilol isoladamente. Nos grupos que associaram as terapias farmacológicas e nãofarmacológica, observamos uma melhora da função apenas no grupo treinado carvedilol associada a um aumento da expressão da SERCA 2, proteína esta, envolvida na recaptação do Ca2+ para o reticulo sarcoplasmático, e, portanto, relacionada ao relaxamento da bomba cardíaca, apesar de não se observar aumento no pico do transiente de cálcio em cardiomiócitos isolados. Em conjunto, os resultados sugerem uma associação preferencial entre o treinamento físico e o carvedilol o que pode trazer implicações clínicas futuras importantes para o tratamento da IC. / In heart failure (HF), structural and functional alterations in myocardium are often paralleled by, defects in intracellular Ca2+ transients.. Within therapeutical strategies for heart failure, it is worth mentioning -blockers and aerobic exercise training. It is known that -blockers improve cardiac function and Ca2+ handling in heart failure. Indeed, -blockers present cardiac antiremodeling effects, but there are many controversies concerning which - blocker is more efficient in HF treatment and which would be its specific effects in Ca2+ regulation. In relation to aerobic exercise training effects in HF, it is important to highlight its positive effects in exercise tolerance and life quality improvement associated with vascular and skeletal muscle gains. We also observed previously that exercise training improves cardiac function associated with improved calcium transients in cardiac myocytes. Taking into consideration the positive impact of both exercise training and -blockers, presently we studied the associative effect of exercise training and -blockers on cardiac function and Ca2+ handling in cardiomyocytes in sympathetic hyperactivity induced HF in mice. We further compared which -blocker (metoprolol or carvedilol) would be more effective to be associated with exercise training. HF mice were assigned into 6 different groups, being 3 of them sedentary: saline (S), metoprolol (M) and carvedilol (C) and 3 exercisetrained: trained (T), trained and treated with metoprolol (MT), and trained and treated with carvedilol (CT). We observed that only HF mice exercise-trained improved exercise tolerance evaluated by maximum exercise test on a treadmill, which was not observed in -blocker treatment alone. Both exercise training and -blockers improved cardiac function evaluated by echocardiography, respectively. When exercise training was associated with -blockers, only HF mice exercise-trained and carvedilol-treated improved cardiac function associated with increased expression of SERCA 2 protein levels, which is involved in sarcoplasmic Ca2+ reuptake and cardiac relaxation. This response was not paralleled by changes in peak Ca2+ transients in isolated cardiac myocytes. Our results provide evidence for a superior effect of exercise training associated with carvedilol for improving cardiac function in HF mice.

Adicionalmente às alterações estruturais e funcionais observadas na insuficiência cardíaca (IC), as alterações na dinâmica do Ca2+ intracelular apresentam uma relação negativa com a contratilidade e função cardíaca. Para evitar a progressão da IC, o tratamento desta síndrome, conta atualmente com intervenções multifatoriais, incluindo a terapia farmacológica, com destaque aos -bloqueadores, e a terapia não medicamentosa incluindo o treinamento físico aeróbico, que quando realizado em intensidade adequada melhora a tolerância ao esforço e a qualidade de vida do portador de insuficiência cardíaca. É bem conhecido que os -bloqueadores melhoram a função cardíaca e dinâmica do Ca2+ intracelular na IC, além de minimizar a remodelação cardíaca, no entanto, ainda existem controvérsias sobre qual o melhor -bloqueador a ser utilizado e seus efeitos específicos na regulação de Ca2+ intracelular. Quanto aos efeitos do treinamento físico aeróbico na IC, seu efeito é reconhecido na melhora da tolerância aos esforços e qualidade de vida do paciente, o que é atribuído principalmente, a ganhos vasculares e músculos-esqueléticos. Em relação à função cardíaca, em trabalho anterior do nosso laboratório, o treinamento físico aeróbico melhorou a função cardíaca associado à melhora nos transientes de cálcio do miócito cardíaco. Como tanto o treinamento físico como os -bloqueadores apresentaram seus respectivos efeitos positivos, mas diferenciados, na presente tese estudou-se o efeito associado do treinamento físico ao tratamento com -bloqueadores sobre a função cardíaca e o fluxo de cálcio no cardiomiócito. Além disso, comparou-se qual -bloqueador (metoprolol ou carvedilol) seria mais efetivo na associação com o treinamento físico. Dividimos nossa amostra em 6 grupos, todos com IC. Dentre eles, 3 foram mantidos sedentários; salina (S), metoprolol (M) e carvedilol (C) e 3 foram treinados; treinados (T), treinados e tratados com metoprolol (MT) e treinados e tratados com carvedilol (CT). Verificamos que apenas os camundongos com IC treinados melhoraram a tolerância ao esforço, medida por teste máximo em esteira rolante, o que não foi observado no tratamento farmacológico isoladamente. Quanto à função cardíaca, observamos uma melhora da função nos grupos que realizaram treinamento físico ou foram tratados com metoprolol ou carvedilol isoladamente. Nos grupos que associaram as terapias farmacológicas e nãofarmacológica, observamos uma melhora da função apenas no grupo treinado carvedilol associada a um aumento da expressão da SERCA 2, proteína esta, envolvida na recaptação do Ca2+ para o reticulo sarcoplasmático, e, portanto, relacionada ao relaxamento da bomba cardíaca, apesar de não se observar aumento no pico do transiente de cálcio em cardiomiócitos isolados. Em conjunto, os resultados sugerem uma associação preferencial entre o treinamento físico e o carvedilol o que pode trazer implicações clínicas futuras importantes para o tratamento da IC. / In heart failure (HF), structural and functional alterations in myocardium are often paralleled by, defects in intracellular Ca2+ transients.. Within therapeutical strategies for heart failure, it is worth mentioning -blockers and aerobic exercise training. It is known that -blockers improve cardiac function and Ca2+ handling in heart failure. Indeed, -blockers present cardiac antiremodeling effects, but there are many controversies concerning which - blocker is more efficient in HF treatment and which would be its specific effects in Ca2+ regulation. In relation to aerobic exercise training effects in HF, it is important to highlight its positive effects in exercise tolerance and life quality improvement associated with vascular and skeletal muscle gains. We also observed previously that exercise training improves cardiac function associated with improved calcium transients in cardiac myocytes. Taking into consideration the positive impact of both exercise training and -blockers, presently we studied the associative effect of exercise training and -blockers on cardiac function and Ca2+ handling in cardiomyocytes in sympathetic hyperactivity induced HF in mice. We further compared which -blocker (metoprolol or carvedilol) would be more effective to be associated with exercise training. HF mice were assigned into 6 different groups, being 3 of them sedentary: saline (S), metoprolol (M) and carvedilol (C) and 3 exercisetrained: trained (T), trained and treated with metoprolol (MT), and trained and treated with carvedilol (CT). We observed that only HF mice exercise-trained improved exercise tolerance evaluated by maximum exercise test on a treadmill, which was not observed in -blocker treatment alone. Both exercise training and -blockers improved cardiac function evaluated by echocardiography, respectively. When exercise training was associated with -blockers, only HF mice exercise-trained and carvedilol-treated improved cardiac function associated with increased expression of SERCA 2 protein levels, which is involved in sarcoplasmic Ca2+ reuptake and cardiac relaxation. This response was not paralleled by changes in peak Ca2+ transients in isolated cardiac myocytes. Our results provide evidence for a superior effect of exercise training associated with carvedilol for improving cardiac function in HF mice.

Acid-sensing ion channels (ASIC) are a class of ligand gated plasma membrane ion channels that are activated by low extracellular pH. During ischemia, ASIC1a are activated and contribute to the demise of neurons. Pharmacological block of ASIC1a provides neuroprotection at delayed time points. However, no endogenous receptors have been implicated in the modulation of ASIC1a activity. The hypothesis presented is that sigma receptor activation inhibits ASIC1a function and ASIC1a-induced [Ca²?]i elevations during acidosis and ischemia, which may be a mechanism by which sigma ligands provide neuroprotection following stroke. This hypothesis is based on the following observations: First, sigma receptors regulate multiple ion channels that become activated during ischemia. Second, ASIC1a remain functionally active hours beyond the ischemic insult and sigma receptors have been shown to be neuroprotective at delayed time points following stroke. Ratiometric Ca²+ fluorometry and whole-cell patch clamp experiments showed that sigma-1 receptor activation depresses elevations in [Ca²+]i and membrane currents mediated by ASIC1a channels in cortical neurons. Furthermore, most of the elevations in [Ca²+]i triggered by acidosis are the result of Ca²+ channels opening downstream of ASIC1a activation. Stimulation of sigma-1 receptors effectively suppressed these secondary Ca²+ fluxes both by inhibiting ASIC1a and the other channels directly. The signaling cascade linking sigma-1 receptors and ASIC1a was determined to involve a pertussis toxin-sensitive G protein and A-Kinase Anchoring Protein 150/calcineurin complex, which resulted in a decrease of acid-induced [Ca²+]i elevations and ASIC1a-mediated currents. Furthermore, immunohistochemical studies confirmed that sigma-1 receptors, ASIC1a and AKAP150 colocalize in the plasma membrane of cortical neuron cell bodies and in the dendritic processes of these cells. Additionally, concurrent exposure to acidosis and ischemia resulted in synergistic potentiation of [Ca²+]i dysregulation. Although ASIC1a activation does not induce long-lived priming of synaptic vesicles for release, channel activation does have a temporal effect on ischemia-mediated [Ca²+]i increases after ischemia onset. Moreover, presynaptic ASIC1a channels promote synaptic transmission during ischemia by overcoming block of neurotransmission and thus enhance postsynaptic [Ca²+]i elevations. Sigma-1 receptor activation decreased ischemia-mediated Ca²+ dysregulation at pH values of 7.4 - 6.0 and prevented the synergistic interaction between ischemia and acidosis.

A desnutrição proteica continua sendo um dos principais problemas nutricionais do mundo. Trabalhos de nosso laboratório e de outros autores evidenciam que entre as alterações presentes na desnutrição proteica, está a alteração do tecido hemopoético, com modificações em componentes da matriz extracelular, alterações no ciclo celular da célula tronco/progenitora hemopoética, redução da produção de precursores hemopoéticos, tanto na série eritrocitária como na série leucocitária, levando a anemia e leucopenia. Os mecanismos de participação do Ca2+ nas células da medula óssea são pouco conhecidos, porém, sabe-se que ele atua no processo de hemopoese. Têm sido descrito que elevações da concentração de Ca2+ citoplasmático induzem a proliferação e diferenciação de células mielóides. A ação dessa via em indivíduos desnutridos também é pouco conhecida. Este estudo tem como objetivo avaliar o estabelecimento da celularidade medular in vitro, bem como investigar mecanismos moleculares envolvidos na proliferação e diferenciação dessa celularidade, além de avaliar a ação do cálcio na presença da interleucina-3 em células-tronco hemopoéticas murinas e sua modulação para avaliar alterações na via das MAPKs. Camundongos C57BL/6, machos e adultos foram submetidos à desnutrição proteica e, após a perda de aproximadamente 20% de seu peso corporal, as células da medula óssea foram colhidas. Essas células foram imunofenotipadas, além de reagirem com anticorpos específicos para caracterização da célula-tronco hemopoética e proteínas da via de sinalização de cálcio intracelular. Observamos que a celularidade do estroma medular em cultura de longa duração de animais desnutridos é alterada, principalmente em células de origem mesenquimal, que aparecem em maior número em desnutridos ao longo dos dias de cultura. Além disso, as ondas de cálcio intracelular estavam diminuídas em animais desnutridos, bem como as proteínas p-PKC, p-PLCy, CAMKII, p-AKT e p-STAT5 não respondem ao estímulo de IL-3, levando a uma deficiência da expressão das MAPK: ERK 1/2, JNK e p38. A desnutrição proteica pode causar alterações na celularidade estromal da medula óssea e na diferenciação das células tronco hemopoéticas pela via das MAPKs estimulada por IL-3. / Protein malnutrition remains one of the world\'s major nutritional problems. Studies from our laboratory and others shown that alterations in protein malnutrition include hemopoietic tissue alterations, changes in extracellular matrix components, changes in the hemopoietic stem/progenitor cell tissue, reduction in the production of hemopoietic precursors, in the erythroid series as in the mieloyd series, leading to anemia and leukopenia. Mechanisms of Ca2+ participation in bone marrow cells are poorly understood, but no hemopoiesis has been developed. Elevations of cytoplasmic Ca2+ concentration in proliferation and differentiation of myeloid cells were included. Such an action through malnourished animals is also a little known. This study aims to evaluate the establishment of cellularity in vitro as well as investigate the molecular involvement in cell proliferation and differentiation, as well as to evaluate the action of calcium in the presence of IL-3 in hemopoietic stem cells and its modulation by analytical evaluations in the MAPKs pathway. C57BL/6, male adult mices were subjected to protein restriction and, after loss of approximately 20% of their body weight, bone marrow cells were harvested. These were immunophenotyped in addition to specific activation terms for the hemopoietic stem cell and intracellular signaling pathway proteins. We observed that the bone marrow cells in long-term culture of malnourished animals is altered, mainly in cells of mesenchymal origin, which appears in greater numbers in undernourished throughout the days of culture. In addition, as intracellular calcium waves decreased in malnourished animals, as well as the p-PKC, p-PLC, CAMKII, p-AKT and p-STAT5 proteins did not respond to IL-3, sugesting expression of the expression of MAPK: ERK 1/2, JNK and p38. Protein malnutrition may have changes in bone marrow capacity and differentiation of hemopoietic stem cells through IL-3-stimulated MAPKs.