Background and aim: The calcium ion (Ca2+) is an important ion involved in intracellular signalling. An increase in the free intracellular calcium concentration ([Ca2+]i) is essential for triggering insulin secretion from pancreatic beta cells. Beta cell death or disturbed insulin secretion are key factors in the pathogenesis of type 1 and type 2 diabetes respectively. A number of Ca2+ channels located on the plasma membrane or on the endoplasmic reticulum (ER) mediate Ca2+ increase in beta cells. Among the plasma membrane Ca2+ channels, members of the Transient Receptor Potential (TRP) family are currently of great interest. Transient Receptor Potential Vanilloid subtype 1 (TRPV1) is one of the 28 members of the TRP family. This ion channel is activated by heat and pungent chemicals like capsaicin. The main aim of this study was to investigate if functional TRPV1 channels are present in insulin secreting cells. Further more we examined if TRP channels could be studied by using microfluorometry in single cells. A third objective was to investigate if members of the TRP family could be identified by western blot. Methods: We used S5 cells, a highly differentiated rat insulinoma cell line, as a model of beta cells. A ratiometric fluorescence technique was used for measurement of [Ca2+]i concentration from single Fura-2 loaded cells. [Ca2+]i was measured continuously using microscope based fluorometry with the time resolution of 1 Hz. For western blot we used proteins extracted from S5 cells and human islets. The blots were probed with antibodies directed against both the N-terminal and the C-terminal end of the protein. Results: Capsaicin, an activator of TRPV1, increased [Ca2+]i in a dose-dependent manner with a half maximal effective concentration (EC50) ~ 100 nM. In nominally Ca2+ free buffer the capsaicin-induced [Ca2+]i increase was completely lost, while the intracellular depots of Ca2+ were not emptied as shown by administration of carbachol. The capsaicin-induced [Ca2+]i increase was completely blocked by capsazepine, an antagonist of TRPV1. An increase in temperature in the range of 43 – 49 °C increased [Ca2+]i, whereas temperatures < 42 °C did not. In nominally Ca2+ free medium the response to heat was reduced. Subsequent administration of carbachol showed that intracellular depots of Ca2+ were not emptied. Ruthenium red, an antagonist of TRPV1, also reduced the heat induced [Ca2+]i response. Another heat-sensitive, Ca2+ permeable protein Transient Receptor Potential Melastatin-like subtype 2 (TRPM2) was detected in S5 cells and human islets by western blot. The 171 kDa band represents the full length TRPM2 and is clearly visible in human islets, while the 95 KDa band represents the truncated form of TRPM2 and is more prominent in S5 cells. Interpretation and conclusions: Microscope based fluorometry is a powerful method for studying ion channels of the TRP family in single living cells. We found that pancreatic beta cells express functional TRPV1 channels that were activated by capsaicin and heat. TRPV1 channels of beta cells are located on the plasma membrane and not on the ER. TRP channel proteins can also be detected by the western blot technique. The ease of studying TRP channels by microfluorometry and our demonstration of functionalTRPV1 channels in beta cells paves the way for studying the role of these channels in insulin secretion and in the pathogenesis of diabetes.

Brain function implies complex information processing in neuronal circuits, critically dependent on the molecular machinery that enables signal transmission across synaptic contacts between neurons. The types of ion channels and receptors in the neuronal membranes vary with neuron types and brain regions and determine whether neuronal responses will be excitatory or inhibitory and often allow for functional synaptic plasticity which is thought to be the basis for much of the adaptability of the nervous system and for our ability to learn and store memories. The present thesis is a study of synaptic transmission in the medial preoptic nucleus (MPN), a regulatory center for several homeostatic functions but with most clearly established roles in reproductive behaviour. The latter behaviour typically shows several distinct phases with dramatically varying neuronal impulse activity and is also subject to experience-dependent modifications. It seems likely that the synapses in the MPN contribute to the behaviour by means of activity-dependent functional plasticity. Synaptic transmission in the MPN, however, has not been extensively studied and is not well understood. The present work was initiated to clarify the synaptic properties in the MPN. The aim was to achieve a better understanding of the functional properties of the MPN, but also to obtain information on the functional roles of ion channel types for neurotransmission and its plastic properties in general. The studies were carried out using a brain slice preparation from rat as well as acutely isolated neurons with adhering nerve terminals. Presynaptic nerve fibres were stimulated electrically or, in a few cases, by raised external K+ concentration, and postsynaptic responses were recorded by tight-seal perforated-patch techniques, often combined with voltage-clamp control of the post-synaptic membrane potential. Glutamate receptors of α-amino-3-hydroxy-5-methyl-4-izoxazole propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) types were identified as mediating the main excitatory synaptic signals and γ-aminobutyric acid (GABA)A receptors as mediating the main inhibitory signals. Both types of signals were suppressed by serotonin. The efficacy of AMPA-receptor-mediated transmission displayed several types of short-term plasticity, including paired-pulse potentiation and paired-pulse depression, depending on the stimulus rate and pattern. The observed plasticity was attributed to mainly presynaptic mechanisms. To clarify some of the presynaptic factors controlling synaptic efficacy, the role of presynaptic L-type Ca2+ channels, usually assumed not to directly control transmitter release, was investigated. The analysis showed that (i) L-type channels are present in GABA-containing presynaptic terminals on MPN neurons, (ii) that these channels provide a means for differential control of spontaneous and impulse-evoked GABA release and (iii) that this differential control is prominent during short-term synaptic plasticity. A model where Ca2+ influx through L-type channels may lead to reduced GABA release via effects on Ca2+-activated K+ channels, membrane potential and other Ca2+-channel types explains the observed findings. In addition, massive Ca2+ influx through L-type channels during high-frequency stimulation may contribute to increased GABA release during post-tetanic potentiation. In conclusion, the findings obtained in the present study indicate that complex neurotransmission mechanisms and different forms of synaptic plasticity contribute to the specific functional properties of the MPN.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus are able to change their firing rate and pattern in response to small changes in external osmolality due to the involvement of osmosensitive ion channels. The firing rate and pattern determine the release of vasopressin (VP), a primary hormone regulating osmolality by controlling water excretion from the kidney. Both VP- and oxytocin (OT)-MNCs display irregular and infrequent fire when plasma osmolality is near normal, and they progressively increase the frequency of firing to fast continuous firing with increases in osmolality. VP-MNCs also respond to osmotic stimulation by adopting a phasic pattern of firing, which maximizes neuropeptide secretion. Sustained dehydration also causes structural and functional adaptations in MNCs.<p> Voltage-dependent Ca2+ channels play many important roles not only in the regulation of cell excitability but also in intracellular signal transduction, and L-type Ca2+ channel-mediated Ca2+ signals initiate intracellular signal transduction events that activate long-lasting changes in brain function and behavior. Our electrophysiological and immunocytochemical studies demonstrate that 16-24 h of water deprivation causes a significant increase in the amplitude of L-type Ca2+ current (from 55.5 ± 6.2 to 99.1 ± 10.0 pA) but not in other types of Ca2+ current. This increase occurred in both VP- and OT-MNCs. Such an increase in L-type Ca2+ current may contribute to modulation of firing rate and pattern, regulation of vasopressin release, structural adaptation in MNCs during sustained dehydration.<p> The mechanisms underlying the transition of the electrical behaviour are not completely understood. Ion channels, especially osmosensitive ion channels, play key roles in the modulation of MNC firing. A voltage-gated, 4-AP- and TEA-insensitive slowly activating outward current displayed a significant increase in about 66% of MNCs when the osmolality of the external solution was acutely increased from 295 to 325 mosmol kg-1. The responding cells showed an increase in net outward current from 12.3 ± 1.3 pA/pF to 21.4 ± 1.8 pA/pF. The reversal potential of this current was near the equilibrium for K+ and shifted with changes of K+ concentrations in external solution, suggesting that this current is a K+-selective current. The KCNQ/M current selective blockers linopirdine (150 µM) and XE991 (5 µM) suppressed this current. The IC50 of XE991 blockade was 3.9 ìM. The KCNQ/M channel openers retigabine (10 µM) and flupirtine (10 µM) significantly increased the current and shifted its activation curve toward more negative potentials. E4031, a specific blocker of ERG K+ channels, did not significantly block this current. The results from immunocytochemistry suggest that MNCs express KCNQ2, KCNQ3, KCNQ4, and KCNQ5, but not KCNQ1. These data suggest that this osmosensitive current could be a KCNQ/M current. Studies using single unit extracellular recording in hypothalamic explants showed that 10 µM XE991 increased MNC firing rate and that 20 µM retigabine decreased firing rate or caused a cessation of firing. These data suggest that a KCNQ/M current contributes to the regulation of MNC firing. KCNQ/M channels play key roles in regulating neuronal excitability in many types of central neurons. Slow activation of this current during firing might suppress activity by hyperpolarizing the cells and thus contribute to a transition between fast continuous and burst firing.<p> Our studies will be beneficial to understand the mechanisms that control VP and OT in response to acute changes in osmolality and also the mechanisms underlying MNC adaptation during sustained dehydration.

Ca2+, both as a first and a second messenger, is closely involved in the modulation and regulation of numerous important cellular events, such as cell proliferation, differentiation and cell death. Fine-tuned Ca2+ signaling is achieved by its reversible or irreversible binding to a repertoire of Ca2+ signaling molecules. Among them, the extracellular calcium sensing receptor (CaSR) senses Ca2+ concentration ([Ca2+]o) in the milieu outside of cells where Ca2+ serves as a first messenger. An array of naturally-occurring mutations in CaSR has been found in patients with inherited disorders of Ca2+ homeostasis, leading to abnormal intracellular responses toward [Ca2+]o. In the present study, we have computationally predicted and experimentally characterized the metal-binding properties of five Ca2+-binding sites within CaSR and the accompanying metal--induced conformational changes by using two complementary methods-the grafting approach and the subdomain approach. Based on our results, a model has been proposed to explain the distinct CaSR-mediated responses toward abnormally ¡°high¡± or ¡°low¡± extracellular Ca2+ levels. In addition, we predicted and verified the interaction between CaSR with the most ubiquitously expressed four EF-hand-containing intracellular Ca2+ sensor protein, calmodulin (CaM). Our results demonstrate that the C-terminal CaM-binding domain of the CaSR is essential for proper intracellular Ca2+ response to external signals. Furthermore, we have applied the grafting approach to study the metal-binding properties and oligomeric state of the single EF-hand containing protein, STIM1. Our studies confirmed that the single EF-hand motif in STIM1, which resides in an equilibratium between its monomeric and dimeric forms, was capable of binding Ca2+ with a dissociation constant comparable to the ER Ca2+ concentration, suggesting it could function as a ER Ca2+ sensor responsible for sensing the Ca2+ filling state of ER.

Few studies have quantified resting mouse muscle metabolism and even fewer studies have separated the contribution of sarcoplasmic reticulum (SR) Ca2+ pumping to resting metabolic rate. Furthermore, the studies that have attempted to quantify the contribution of Ca2+ pumping have used indirect methods to inhibit SR Ca2+ ATPase activity. The purpose of this study is to directly quantify resting muscle oxygen consumption and the contribution of SR Ca2+ pumping to resting oxygen consumption in mouse hindlimb muscles by using CPA to specifically inhibit Ca2+ pump activity in intact muscles at rest. The TIOX system was used to measure resting muscle VO2 of extensor digitorum longus (EDL) and soleus (SOL) muscles at 30oC and 20oC. C57BL mice aged 8-12 weeks were used with an average whole body mass of 23.8 g and EDL and SOL dry weights averaging 1.88 mg and 1.8 mg, respectively. All muscle VO2 measurements are expressed per gram dry weight. There were no differences (P>0.1) in resting muscle VO2 between EDL and SOL muscles at either 30oC (EDL, 2.05 µL/g/s; SOL, 2.27 µL/g/s) or 20oC (EDL, 0.62 µL/g/s; SOL, 0.71 µL/g/s). The average Q10 (3.1) was determined from EDL and SOL VO2 measures at 20oC and 30oC. The contribution of Ca2+ pumping by the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) was measured at 30oC using a range of CPA concentrations (1-15 µM) . There was a concentration-dependent effect of CPA on oxygen consumption with increasing CPA concentrations up to 10 µM resulting in progressively greater reductions in muscle oxygen consumption. Specifically, 1, 5, 10, and 15 µM CPA caused an 11, 35.4, 49.5, and 50.3% reduction in VO2. There were no differences (P>0.1) between 10 and 15 µM CPA indicating that 10 µM CPA induces maximal inhibition of SERCA in isolated muscle preparations. The results indicate that the Ca2+ pumping by SERCA is responsible for ~50% of oxygen consumption in resting mouse EDL and SOL muscle. This is the first study to use a direct inhibitor of SERCA to quantify the contribution of Ca2+ cycling to resting oxygen consumption and therefore is a more accurate reflection of the actual contribution of SERCA to resting muscle oxygen consumption compared to previous findings. These results suggest that SERCA energy consumption accounts for a large portion of resting muscle metabolism and may represent a potential therapeutic target for metabolic alterations to oppose obesity.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus are able to change their firing rate and pattern in response to small changes in external osmolality due to the involvement of osmosensitive ion channels. The firing rate and pattern determine the release of vasopressin (VP), a primary hormone regulating osmolality by controlling water excretion from the kidney. Both VP- and oxytocin (OT)-MNCs display irregular and infrequent fire when plasma osmolality is near normal, and they progressively increase the frequency of firing to fast continuous firing with increases in osmolality. VP-MNCs also respond to osmotic stimulation by adopting a phasic pattern of firing, which maximizes neuropeptide secretion. Sustained dehydration also causes structural and functional adaptations in MNCs.<p> Voltage-dependent Ca2+ channels play many important roles not only in the regulation of cell excitability but also in intracellular signal transduction, and L-type Ca2+ channel-mediated Ca2+ signals initiate intracellular signal transduction events that activate long-lasting changes in brain function and behavior. Our electrophysiological and immunocytochemical studies demonstrate that 16-24 h of water deprivation causes a significant increase in the amplitude of L-type Ca2+ current (from 55.5 ± 6.2 to 99.1 ± 10.0 pA) but not in other types of Ca2+ current. This increase occurred in both VP- and OT-MNCs. Such an increase in L-type Ca2+ current may contribute to modulation of firing rate and pattern, regulation of vasopressin release, structural adaptation in MNCs during sustained dehydration.<p> The mechanisms underlying the transition of the electrical behaviour are not completely understood. Ion channels, especially osmosensitive ion channels, play key roles in the modulation of MNC firing. A voltage-gated, 4-AP- and TEA-insensitive slowly activating outward current displayed a significant increase in about 66% of MNCs when the osmolality of the external solution was acutely increased from 295 to 325 mosmol kg-1. The responding cells showed an increase in net outward current from 12.3 ± 1.3 pA/pF to 21.4 ± 1.8 pA/pF. The reversal potential of this current was near the equilibrium for K+ and shifted with changes of K+ concentrations in external solution, suggesting that this current is a K+-selective current. The KCNQ/M current selective blockers linopirdine (150 µM) and XE991 (5 µM) suppressed this current. The IC50 of XE991 blockade was 3.9 ìM. The KCNQ/M channel openers retigabine (10 µM) and flupirtine (10 µM) significantly increased the current and shifted its activation curve toward more negative potentials. E4031, a specific blocker of ERG K+ channels, did not significantly block this current. The results from immunocytochemistry suggest that MNCs express KCNQ2, KCNQ3, KCNQ4, and KCNQ5, but not KCNQ1. These data suggest that this osmosensitive current could be a KCNQ/M current. Studies using single unit extracellular recording in hypothalamic explants showed that 10 µM XE991 increased MNC firing rate and that 20 µM retigabine decreased firing rate or caused a cessation of firing. These data suggest that a KCNQ/M current contributes to the regulation of MNC firing. KCNQ/M channels play key roles in regulating neuronal excitability in many types of central neurons. Slow activation of this current during firing might suppress activity by hyperpolarizing the cells and thus contribute to a transition between fast continuous and burst firing.<p> Our studies will be beneficial to understand the mechanisms that control VP and OT in response to acute changes in osmolality and also the mechanisms underlying MNC adaptation during sustained dehydration.

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)- benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells and OC2 human oral cancer cells was unclear. This study explored whether m-3M3FBS changed basal [Ca2+]i levels in suspended MDCK and OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. m-3M3FBS at concentrations between 0.1-20 £gM increased [Ca2+]i in a concentration-dependent manner in MDCK cells, however in OC2 cells, m-3M3FBS at concentrations between 10-60 £gM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signals were reduced partly by removing extracellular Ca2+ in the two cell types. m-3M3FBS-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca2+-free medium, m-3M3FBS pretreatment abolished the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin, cyclopiazonic acid or 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with thapsigargin, cyclopiazonic acid or BHQ partly reduced m-3M3FBS-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter m-3M3FBS-induced [Ca2+]i rise. Collectively, in MDCK and OC2 cells, m-3M3FBS induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels and other unidentified Ca2+ channels. Additionally, 5-100 £gM of m-3M3FBS killed cells in a concentration-dependent manner in OC2 cells. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid (BAPTA). Propidium iodide staining data suggest that m-3M3FBS (20 or 50 £gM) induced apoptosis in a Ca2+-independent manner. We were also interested in knowing whether BAPTA suppressed cell death during oxidative stress in MDCK cells. BAPTA loading altered tBHP (tert-butyl hydroperoxide) and H2O2-induced cell death in a concentration-dependent manner. This suggests that the cell death induced by tBHP and H2O2 appears to be Ca2+-dependent in MDCK cells. The tBHP and H2O2-induced cell death was not suppressed by 2 £gM U73122 (PLC inhibitor), 50 £gM zVAD-fmk (caspase inhibitor), 2 £gM cyclosporin A (a potent inhibitor of the MPTP), 20 £gM PD98059 (ERK inhibitor) or 2 £gM SP600125 (JNK inhibitor). This suggests that the tBHP and H2O2-induced MDCK cells death was not via the PLC, MPTP, caspase, ERK or JNK pathways. Propidium iodide staining, caspase-3 activity assay and cell morphology data suggest that tBHP and H2O2-induced cell death was necrosis, not via apoptosis, and the cell death appears to be caspase-independent and Ca2+-dependent. The effect of the antidepressant paroxetine on [Ca2+]i in OC2 human oral cancer cells is unclear. This study also explored whether paroxetine changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Paroxetine at concentrations between 100-1000 £gM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 50% by removing extracellular Ca2+. Paroxetine-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and protein kinase C modulators. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished paroxetine¡Vinduced [Ca2+]i rise. Inhibition of PLC with U73122 did not alter paroxetine-induced [Ca2+]i rise. Paroxetine at 10-50 £gM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca2+ was chelated with BAPTA. Propidium iodide staining suggests that apoptosis played a role in the death. Collectively, in OC2 cells, paroxetine induced [Ca2+]i rise by causing PLC-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels in a manner regulated by protein kinase C and phospholipase A2. Paroxetine also induced cell death in a Ca2+-independent manner.

The effects of the antidepressant mirtazapine on cytosolic Ca2+ concentrations ([Ca2+]i) in human prostate cancer PC3 cells and human osteosarcoma MG63 cells were measured by Ca2+-sensitive fluorescent probe fura-2. In Ca2+-containing medium, mirtazapine induced [Ca2+]i rises in a concentration-dependent manner in both PC3 and MG63 cells. Removal of extracellular Ca2+ inhibited the mirtazapine-induced Ca2+ signal. In Ca2+-free medium, thapsigargin (an inhibitor of the endoplasmic reticulum Ca2+-ATPase pump) induced [Ca2+]i rises by passively depleting the endoplasmic reticulum Ca2+ store, after which the increasing effect of mirtazapine (1.5 mM) on [Ca2+]i was reduced. Conversely, pretreatment with mirtazapine decreased thapsigargin-induced [Ca2+]i rises in PC3 and MG63 cells. When PC3 cells were pretreated with U73122, a phospholipase C inhibitor, mirtazapine-induced [Ca2+]i rises were inhibited by 47%. But in MG63 cells, 2 mM U73122 did not change mirtazapine-induced [Ca2+]i rises. These finding suggest that mirtazapine-induced [Ca2+]i rises were caused both by extracellular Ca2+ influx and intracellular depletion of the endoplasmic reticulum Ca2+ stores. Furthermore, the mechanism of mirtazapine-induced Ca2+ release may be different between PC3 and MG63 cells. Additionally, cell proliferation assays suggest that overnight incubation with higher concentrations of mirtazapine decreased cell viability in a concentration-dependent manner.

Depression is a physiological disorder that may be treated by increasing the body¡¦s amount of one or a few of the following neurotransmitters: serotonin, dopamine and norepinephrine. Although there are seven distinct classes of antidepressants, selective serotonin reuptake inhibitors (SSRIs) and tetracyclic antidepressants are widely prescribed and generally regarded as the first-line drugs in the treatment of depression. However, many physiological roles of some SSRIs appear to be dissociated with the inhibition of serotonin reuptake. For instance, paroxetine, a member of SSRIs and maprotiline, a member of tetracyclic antidepressant, have been shown to induce apoptosis or to prevent other agents from inducing apoptosis in several cell lines. Thus the effects of these two drugs on the apoptosis are still controversial. The aim of this study is to investigate the molecular mechanisms of paroxetine and maprotiline in induction of cell death in human osteosarcoma and murine neuroblastoma cells. First, WST-1 reduction assays and propidium iodide-staining assays were used to determine cell viability and apoptosis in the presence of paroxetine and maprotiline. Then immunoblotting was used to measure the activity of apoptotic markers caspase-3 and mitogen-activated protein kinases (MAPKs) to survey the apoptotic pathways induced by these two antidepressants. The experimental results may be helpful to understand the pharmacological and toxicological effects of these two antidepressants in cells from important organs. Results showed that paroxetine caused apoptosis via the activation of caspase-3 in cultured human osteosarcoma cells (MG63). Although paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine was also found to induce [Ca2+]i increases but pretreatment with BAPTA/AM, a Ca2+ chelator, prevented paroxetine-induced [Ca2+]i increases, and thus did not protect cells from death. These results suggest that paroxetine caused Ca2+-independent apoptosis via the activation of p38 MAPK-associated caspase-3 in MG63 cells. Maprotiline was also found to induce apoptosis through increased caspase-3 activation in murine neuroblastoma Neuro-2a cells. Induction of JNK phosphorylation contributed to the activation of caspase-3 resulting in maprotiline-induced Neuro-2a cell apoptosis. Thus, it appears that maprotiline induced apoptosis via JNK/caspase-3-dependent signaling pathways. Blockage of activation of ERK was found to increase the activation of caspase-3 leading to an enhancement of maprotiline-induced apoptosis. These data suggest that ERK was a survival signal to oppose maprotiline-caused apoptotic effect in Neuro-2a cells. Thus the activation of caspase-3 by maprotiline appears to depend on the activation of JNK and the inactivation of ERK. [Ca2+]i measurement in the presence of maprotiline showed that the antidepressant induced [Ca2+]i increases. Interestingly, pretreatment with BAPTA/AM could suppress maprotiline-induced ERK phosphorylation, enhance caspase-3 activation and increase maprotiline-induced apoptosis. In conclusion, this study demonstrates that maprotiline induced apoptosis in murine neuroblastoma cells through activation of JNK-associated caspase-3 pathways. Maprotiline also evoked an anti-apoptotic response that was both Ca2+- and ERK-dependent. This thesis contains some published data in the journal of Toxicology and Applied Pharmacology and some data were submitted in the journal of Toxicology Letters.

<p>Background and aim: The calcium ion (Ca2+) is an important ion involved in intracellular signalling. An increase in the free intracellular calcium concentration ([Ca2+]i) is essential for triggering insulin secretion from pancreatic beta cells. Beta cell death or disturbed insulin secretion are key factors in the pathogenesis of type 1 and type 2 diabetes respectively. A number of Ca2+ channels located on the plasma membrane or on the endoplasmic reticulum (ER) mediate Ca2+ increase in beta cells. Among the plasma membrane Ca2+ channels, members of the Transient Receptor Potential (TRP) family are currently of great interest. Transient Receptor Potential Vanilloid subtype 1 (TRPV1) is one of the 28 members of the TRP family. This ion channel is activated by heat and pungent chemicals like capsaicin. The main aim of this study was to investigate if functional TRPV1 channels are present in insulin secreting cells. Further more we examined if TRP channels could be studied by using microfluorometry in single cells. A third objective was to investigate if members of the TRP family could be identified by western blot.</p><p>Methods: We used S5 cells, a highly differentiated rat insulinoma cell line, as a model of beta cells. A ratiometric fluorescence technique was used for measurement of [Ca2+]i concentration from single Fura-2 loaded cells. [Ca2+]i was measured continuously using microscope based fluorometry with the time resolution of 1 Hz. For western blot we used proteins extracted from S5 cells and human islets. The blots were probed with antibodies directed against both the N-terminal and the C-terminal end of the protein.</p><p>Results: Capsaicin, an activator of TRPV1, increased [Ca2+]i in a dose-dependent manner with a half maximal effective concentration (EC50) ~ 100 nM. In nominally Ca2+ free buffer the capsaicin-induced [Ca2+]i increase was completely lost, while the intracellular depots of Ca2+ were not emptied as shown by administration of carbachol. The capsaicin-induced [Ca2+]i increase was completely blocked by capsazepine, an antagonist of TRPV1. An increase in temperature in the range of 43 – 49 °C increased [Ca2+]i, whereas temperatures < 42 °C did not. In nominally Ca2+ free medium the response to heat was reduced. Subsequent administration of carbachol showed that intracellular depots of Ca2+ were not emptied. Ruthenium red, an antagonist of TRPV1, also reduced the heat induced [Ca2+]i response. Another heat-sensitive, Ca2+ permeable protein Transient Receptor Potential Melastatin-like subtype 2 (TRPM2) was detected in S5 cells and human islets by western blot. The 171 kDa band represents the full length TRPM2 and is clearly visible in human islets, while the 95 KDa band represents the truncated form of TRPM2 and is more prominent in S5 cells.</p><p>Interpretation and conclusions: Microscope based fluorometry is a powerful method for studying ion channels of the TRP family in single living cells. We found that pancreatic beta cells express functional TRPV1 channels that were activated by capsaicin and heat. TRPV1 channels of beta cells are located on the plasma membrane and not on the ER. TRP channel proteins can also be detected by the western blot technique. The ease of studying TRP channels by microfluorometry and our demonstration of functionalTRPV1 channels in beta cells paves the way for studying the role of these channels in insulin secretion and in the pathogenesis of diabetes.</p>