Activation Of Voltage-gated Ca Channels Research Articles

Exendin-4 promotes retinal ganglion cell survival and function by inhibiting calcium channels in experimental diabetes

Progressive damage of retinal ganglion cells (RGCs) is observed in early diabetic retinopathy. Intracellular Ca2+ overload mediated by Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) is involved in neurodegeneration, whereas glucagon-like peptide-1 (GLP-1) provides neuroprotection. However, whether GLP-1 plays a neuroprotective role in diabetic retinas by modulating VGCCs remains unknown. We found that eye drops of exendin-4, a long-acting GLP-1 receptor (GLP-1R) agonist, prevented the increase of L-type Ca2+ current (ILCa) densities of RGCs induced by 4-week hyperglycemia and promoted RGC survival by suppressing L-type VGCC (L-VGCC) activity in streptozotocin-induced diabetic rats. Moreover, exendin-4-induced suppression of ILCa in RGCs may be mediated by a GLP-1R/Gs/cAMP-PKA/ryanodine/Ca2+/calmodulin/calcineurin/PP1 signaling pathway. Furthermore, exendin-4 functionally improved the light-evoked spiking ability of diabetic RGCs. These results suggest that GLP-1R activation enhances cAMP to PP1 signaling and that PP1 inactivates L-VGCCs by dephosphorylating them, thereby reducing Ca2+ influx, which could protect RGCs against excitotoxic Ca2+ overload.

Commiphora myrrha stimulates insulin secretion from β-cells through activation of atypical protein kinase C and mitogen-activated protein kinase

Ethnopharmacological relevanceAyurvedic medicine has been used in the treatment of diabetes mellitus for centuries. In Arabia and some areas of Africa, Commiphora myrrha (CM) has been extensively used as a plant-based remedy. We have previously shown that an aqueous CM resin solution directly stimulates insulin secretion from MIN6 cells, a mouse β-cell line, and isolated mouse and human islets. However, the signaling pathways involved in CM-induced insulin secretion are completely unknown. Insulin secretion is normally triggered by elevations in intracellular Ca2+ ([Ca2+]i) through voltage gated Ca2+ channels (VGCC) and activation of protein kinases. Protein and lipid kinases such as protein kinase A (PKA), Ca2+-calmodulin dependent protein kinase II (CaMKII), phosphoinositide 3-kinases (PI3Ks), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK), specifically extracellular signal-regulated kinases (ERK1/2), may be involved in receptor-operated insulin secretion. Therefore, we hypothesized that CM may induce insulin secretion by modulating the activity of VGCC and/or one or more of the above kinases. Aim of the studyTo investigate the possible molecular mechanism of action of CM-induced insulin secretion. The effects of aqueous CM resin extract on [Ca2+]i and protein kinase activation from β-cells were examined. MethodsThe effect of aqueous CM resin solution on [Ca2+]i was assessed using Ca2+ microfluorimetry. The involvement of VGCC in CM-induced insulin secretion was investigated using static and perifusion insulin secretion experiments in the presence of either EGTA, a Ca2+ chelator, or nifedipine, a blocker of VGCC. The involvement of kinase activation in the stimulatory effect of CM on insulin secretion was examined by using static and perifusion insulin secretion experiments in the presence of known pharmacological inhibitors and/or downregulation of specific kinases. The effects of CM on phosphorylation of PKCζ and ERK1/2 were also assessed using the Wes™ capillary-based protein electrophoresis. ResultsCa2+ microfluorimetry measurements showed that exposing MIN6 cells to CM (0.5–2 mg/mL) was not associated with changes in [Ca2+]i. Similarly, incubating MIN6 cells and mouse islets with EGTA and nifedipine, respectively, did not attenuate the insulin secretion induced by CM. However, incubating mouse and human islets with CM in the presence of staurosporine, a non-selective protein kinase inhibitor, completely blocked the effect of CM on insulin secretion. Exposing mouse islets to CM in the presence of H89, KN62 and LY294002, inhibitors of PKA, CaMKII and PI3K, respectively, did not reduce CM-induced insulin secretion. However, incubating mouse and human islets with CM in the presence of Ro 31–8220, a pan-PKC inhibitor, diminished insulin secretion stimulated by CM, whereas inhibiting the action of typical PKC (with Go6976) and PLCβ (with U73122) did not affect CM-stimulated insulin secretion. Similarly, downregulating typical and novel PKC by chronic exposure of mouse islets to phorbol 12-myristate 13-acetate (PMA) was also not associated with a decrease in the stimulatory effect of CM on insulin secretion. Interestingly, CM-induced insulin secretion from mouse islets was inhibited in the presence of the PKCζ inhibitor ZIP and a MAPK inhibitor PD 98059. In addition, Wes™ capillary-based protein electrophoresis indicated that expression of the phosphorylated forms of PKCζ and ERK1/2, a MAPK, was significantly increased following exposure of INS-1832/13 cells, a rat insulinoma cell line, to CM. ConclusionsOur data indicate that CM directly stimulates insulin secretion through activating known downstream effectors of insulin-stimulus secretion coupling. Indeed, the increase in insulin secretion seen with CM is independent of changes in [Ca2+]i and does not involve activation of VGCC. Instead, the CM stimulatory effect on insulin secretion is completely dependent on protein kinase activation. Our findings indicate that CM could induce insulin exocytosis by stimulating the phosphorylation and activation of PKCζ, which in turn phosphorylates and activates ERK1/2.

Gadolinium Differentially Affects Membrane Permeabilization and Ca2+ Influx Induced by Nanosecond Electric Pulses in Bovine Adrenal Chromaffin Cells

Our studies show that exposing isolated bovine adrenal chromaffin cells to a single 5 ns, 5 MV/m pulse (NEP) causes activation of voltage-gated Ca2+ channels (VGCCs) that in turn leads to Ca2+ influx and catecholamine release. The trigger for VGCC activation is hypothesized to be membrane depolarization caused by an instantaneous, NEP-elicited inward membrane current that is carried in part by Na+. Recent studies characterizing the properties of the NEP-elicited inward current by whole-cell patch clamp electrophysiology have shown that a large component of this unknown conductance displays properties that are consistent with those of ion channel proteins, in particular, TRP channels.

Simvastatin Inhibits L-Type Ca2+-Channel Activity Through Impairment of Mitochondrial Function.

Plasma membrane ion channels and mitochondrial electron transport complexes (mETC) are recognized "off targets" for certain drugs. Simvastatin is one such drug, a lipophilic statin used to treat hypercholesterolemia, but which is also associated with adverse effects like myopathy and increased risk of glucose intolerance. Such myopathy is thought to arise through adverse actions of simvastatin on skeletal muscle mETC and mitochondrial respiration. In this study, we investigated whether the glucose intolerance associated with simvastatin is also mediated via adverse effects on mETC in pancreatic beta-cells because mitochondrial respiration underlies insulin secretion from these cells, an effect in part mediated by promotion of Ca2+ influx via opening of voltage-gated Ca2+ channels (VGCCs). We used murine pancreatic beta-cells to investigate these ideas. Mitochondrial membrane potential, oxygen consumption, and ATP-sensitive-K+-channel activity were monitored as markers of mETC activity, respiration, and cellular ATP/ADP ratio respectively; Ca2+ channel activity and Ca2+ influx were also measured. In intact beta-cells, simvastatin inhibited oxidative respiration (IC50 approximately 3 µM) and mETC (1 < IC50 < 10 µM), effects expected to impair VGCC opening. Consistent with this idea simvastatin > 0.1 µM reversed activation of VGCCs by glucose but had no significant effect in the sugar's absence. The VGCC effects were mimicked by rotenone which also decreased respiration and ATP/ADP. This study demonstrates modulation of beta-cell VGCC activity by mitochondrial respiration and their sensitivity to mETC inhibitors. This reveals a novel outcome for the action of drugs like simvastatin for which mETC is an "off target".

Voltage-gated Ca2+ channel activity modulates smooth muscle cell calcium waves in hamster cremaster arterioles.

Cremaster muscle arteriolar smooth muscle cells (SMCs) display inositol 1,4,5-trisphosphate receptor-dependent Ca2+ waves that contribute to global myoplasmic Ca2+ concentration and myogenic tone. However, the contribution made by voltage-gated Ca2+ channels (VGCCs) to arteriolar SMC Ca2+ waves is unknown. We tested the hypothesis that VGCC activity modulates SMC Ca2+ waves in pressurized (80 cmH2O/59 mmHg, 34°C) hamster cremaster muscle arterioles loaded with Fluo-4 and imaged by confocal microscopy. Removal of extracellular Ca2+ dilated arterioles (32 ± 3 to 45 ± 3 μm, n = 15, P < 0.05) and inhibited the occurrence, amplitude, and frequency of Ca2+ waves ( n = 15, P < 0.05), indicating dependence of Ca2+ waves on Ca2+ influx. Blockade of VGCCs with nifedipine (1 μM) or diltiazem (10 μM) or deactivation of VGCCs by hyperpolarization of smooth muscle with the K+ channel agonist cromakalim (10 μM) produced similar inhibition of Ca2+ waves ( P < 0.05). Conversely, depolarization of SMCs with the K+ channel blocker tetraethylammonium (1 mM) constricted arterioles from 26 ± 3 to 14 ± 2 μm ( n = 11, P < 0.05) and increased wave occurrence (9 ± 3 to 16 ± 3 waves/SMC), amplitude (1.6 ± 0.07 to 1.9 ± 0.1), and frequency (0.5 ± 0.1 to 0.9 ± 0.2 Hz, n = 10, P < 0.05), effects that were blocked by nifedipine (1 μM, P < 0.05). Similarly, the VGCC agonist Bay K8644 (5 nM) constricted arterioles from 14 ± 1 to 8 ± 1 μm and increased wave occurrence (3 ± 1 to 10 ± 1 waves/SMC) and frequency (0.2 ± 0.1 to 0.6 ± 0.1 Hz, n = 6, P < 0.05), effects that were unaltered by ryanodine (50 μM, n = 6, P > 0.05). These data support the hypothesis that Ca2+ waves in arteriolar SMCs depend, in part, on the activity of VGCCs. NEW & NOTEWORTHY Arterioles that control blood flow to and within skeletal muscle depend on Ca2+ influx through voltage-gated Ca2+ channels and release of Ca2+ from internal stores through inositol 1,4,5-trisphosphate receptors in the form of Ca2+ waves to maintain pressure-induced smooth muscle tone.

Voltage Gated Calcium Channel Activation by Backpropagating Action Potentials Downregulates NMDAR Function.

The majority of excitatory synapses are located on dendritic spines of cortical glutamatergic neurons. In spines, compartmentalized Ca2+ signals transduce electrical activity into specific long-term biochemical and structural changes. Action potentials (APs) propagate back into the dendritic tree and activate voltage gated Ca2+ channels (VGCCs). For spines, this global mode of spine Ca2+ signaling is a direct biochemical feedback of suprathreshold neuronal activity. We previously demonstrated that backpropagating action potentials (bAPs) result in long-term enhancement of spine VGCCs. This activity-dependent VGCC plasticity results in a large interspine variability of VGCC Ca2+ influx. Here, we investigate how spine VGCCs affect glutamatergic synaptic transmission. We combined electrophysiology, two-photon Ca2+ imaging and two-photon glutamate uncaging in acute brain slices from rats. T- and R-type VGCCs were the dominant depolarization-associated Ca2+conductances in dendritic spines of excitatory layer 2 neurons and do not affect synaptic excitatory postsynaptic potentials (EPSPs) measured at the soma. Using two-photon glutamate uncaging, we compared the properties of glutamatergic synapses of single spines that express different levels of VGCCs. While VGCCs contributed to EPSP mediated Ca2+ influx, the amount of EPSP mediated Ca2+ influx is not determined by spine VGCC expression. On a longer timescale, the activation of VGCCs by bAP bursts results in downregulation of spine NMDAR function.

Novel Ca2+-dependent mechanisms regulate spontaneous release at excitatory synapses onto CA1 pyramidal cells.

Although long thought to simply be a source of synaptic noise, spontaneous, action potential-independent release of neurotransmitter from presynaptic terminals has multiple roles in synaptic function. We explored whether and to what extent the two predominantly proposed mechanisms for explaining spontaneous release, stochastic activation of voltage-gated Ca2+ channels (VGCCs) or activation of Ca2+-sensing receptors (CaSRs) by extracellular Ca2+, played a role in the sensitivity of spontaneous release to the level of extracellular Ca2+ concentration at excitatory synapses at CA1 pyramidal cells of the adult male mouse hippocampus. Blocking VGCCs with Cd2+ had no effect on spontaneous release, ruling out stochastic activation of VGCCs. Although divalent cation agonists of CaSRs, Co2+ and Mg2+, dramatically enhanced miniature excitatory postsynaptic current (mEPSC) frequency, potent positive and negative allosteric modulators of CaSRs had no effect. Moreover, immunoblot analysis of hippocampal lysates failed to detect CaSR expression, ruling out the CaSR. Instead, the increase in mEPSC frequency induced by Co2+ and Mg2+ was mimicked by lowering postsynaptic Ca2+ levels with BAPTA. Together, our results suggest that a reduction in intracellular Ca2+ may trigger a homeostatic-like compensatory response that upregulates spontaneous transmission at excitatory synapses onto CA1 pyramidal cells in the adult hippocampus. NEW & NOTEWORTHY We show that the predominant theories for explaining the regulation of spontaneous, action potential-independent synaptic release do not explain the sensitivity of this type of synaptic transmission to external Ca2+ concentration at excitatory synapses onto hippocampal CA1 pyramidal cells. In addition, our data indicate that intracellular Ca2+ levels in CA1 pyramidal cells regulate spontaneous release, suggesting that excitatory synapses onto CA1 pyramidal cells may express a novel, rapid form of homeostatic plasticity.

Bud extracts from Salix caprea L. inhibit voltage gated calcium channels and catecholamines secretion in mouse chromaffin cells

BackgroundSalix caprea L. is an ornamental plant with prominent antioxidant activity. In the last decades Salix caprea bud extracts (SCBEs) have been used for the treatment of oxidative stress related disorders. PurposeA large part of cellular functions depends on the amount of intracellular Ca2+ concentration which in turn is mainly determined by Ca2+ ions movements across plasma membrane as well as by Ca2+ released from the stores. For better evaluating the mechanism of action of SCBEs, we focused on the effect of SCBEs on voltage gated Ca2+ channels (VGCCs) functioning and related catecholamines secretion in mouse chromaffin cells (MCCs). These latter are neuroendocrine cells that share a wide variety of functions with neurons. They are particularly interesting for studying the relationship between VGCCs activation and catecholamines secretion both in control and under stressful conditions. Study design and methodsWe focused on the effect of SCBEs on VGCCs being these latter considered one of the main pathway of Ca2+ influx across plasma membrane. Ca2+ currents and capacitance changes were measured in patch clamp experiments performed in voltage clamp configuration. ResultsWe show that SCBEs inhibited VGCCs in a dose dependent manner. On average, the saturating concentration of SCBEs (SCBEsmax) is able to block 36% of the maximum Ca2+ current amplitude (ICa) without selectivity for L (ICa, L) or non-L type (ICa, non-L) Ca2+ channels. Furthermore, ICa inhibition is not followed by alteration of VGCCs gating kinetics, but is responsible for a marked decrease of Ca2+ dependent catecholamines secretion. ConclusionWe conclude that the ability of SCBEs to inhibit VGCCs function, known to be potentiated during oxidative stress, could contribute to the already known antioxidant properties of Salix caprea L. We finally suggest that the inhibitory effect of SCBEs on catecholamines secretion may contribute to treat stress dependent cellular dysfunctions.

Functional coupling of diverse voltage-gated Ca(2+) channels underlies high fidelity of fast dendritic Ca(2+) signals during burst firing.

In neurons, the Ca(2+) signal associated with the dendritic back-propagating action potential codes a chemical message to the different dendritic sites, playing a crucial role in electrical signalling, synaptic transmission and synaptic plasticity. The study of the underlying Ca(2+) current, mediated by different types of voltage-gated Ca(2+) channels, cannot be achieved by using the patch clamp technique. In this article, we used a recently developed cutting-edge optical technique to investigate the physiological behaviour of local Ca(2+) currents along the apical dendrite of CA1 hippocampal pyramidal neurons. We directly measure, for the first time, the synergistic activation and deactivation of the diverse dendritic voltage-gated Ca(2+) channels operating during bursts of back-propagating action potentials to precisely control the Ca(2+) signal. We demonstrate that the Ca(2+) loss via high-voltage-activated channels is compensated by the Ca(2+) entry via the other channels translating in high fidelity of Ca(2+) signalling. In CA1 hippocampal pyramidal neurons, the dendritic Ca(2+) signal associated with somatic firing represents a fundamental activation code for several proteins. This signal, mediated by voltage-gated Ca(2+) channels (VGCCs), varies along the dendrites. In this study, using a recent optical technique based on the low-affinity indicator Oregon Green 488 BAPTA-5N, we analysed how activation and deactivation of VGCCs produced by back-propagating action potentials (bAPs) along the apical dendrite shape the Ca(2+) signal at different locations in CA1 hippocampal pyramidal neurons of the mouse. We measured, at multiple dendritic sites, the Ca(2+) transients and the changes in membrane potential associated with bAPs at 50μs temporal resolution and we estimated the kinetics of the Ca(2+) current. We found that during somatic bursts, the bAPs decrease in amplitude along the apical dendrite but the amplitude of the associated Ca(2+) signal in the initial 200μm dendritic segment does not change. Using a detailed pharmacological analysis, we demonstrate that this effect is due to the perfect compensation of the loss of Ca(2+) via high-voltage-activated (HVA) VGCCs by a larger Ca(2+) component via low-voltage-activated (LVA) VGCCs, revealing a mechanism coupling the two VGCC families of K(+) channels. More distally, where the bAP does not activate HVA-VGCCs, the Ca(2+) signal is variable during the burst. Thus, we demonstrate that HVA- and LVA-VGCCs operate synergistically to stabilise Ca(2+) signals associated with bAPs in the most proximal 200μm dendritic segment.

Comprehensive behavioral analysis of voltage-gated calcium channel beta-anchoring and -regulatory protein knockout mice.

Calcium (Ca2+) influx through voltage-gated Ca2+ channels (VGCCs) induces numerous intracellular events such as neuronal excitability, neurotransmitter release, synaptic plasticity, and gene regulation. It has been shown that genes related to Ca2+ signaling, such as the CACNA1C, CACNB2, and CACNA1I genes that encode VGCC subunits, are associated with schizophrenia and other psychiatric disorders. Recently, VGCC beta-anchoring and -regulatory protein (BARP) was identified as a novel regulator of VGCC activity via the interaction of VGCC β subunits. To examine the role of the BARP in higher brain functions, we generated BARP knockout (KO) mice and conducted a comprehensive battery of behavioral tests. BARP KO mice exhibited greatly reduced locomotor activity, as evidenced by decreased vertical activity, stereotypic counts in the open field test, and activity level in the home cage, and longer latency to complete a session in spontaneous T-maze alteration test, which reached “study-wide significance.” Acoustic startle response was also reduced in the mutants. Interestingly, they showed multiple behavioral phenotypes that are seemingly opposite to those seen in the mouse models of schizophrenia and its related disorders, including increased working memory, flexibility, prepulse inhibition, and social interaction, and decreased locomotor activity, though many of these phenotypes are statistically weak and require further replications. These results demonstrate that BARP is involved in the regulation of locomotor activity and, possibly, emotionality. The possibility was also suggested that BARP KO mice may serve as a unique tool for investigating the pathogenesis/pathophysiology of schizophrenia and related disorders. Further evaluation of the molecular and physiological phenotypes of the mutant mice would provide new insights into the role of BARP in higher brain functions.

Ca 2+ Influx Through Vascular Smooth Muscle Cell Voltage‐Gated Ca 2+ Channels Increases Endothelial Cell Ca 2+ to Evoke Vasodilation

Global increases in vascular smooth muscle cell (SMC) intracellular Ca2+ typically lead to the formation of Ca2+-dependent cross-bridges between actin and myosin causing SMC contraction and vasoconstriction. We now show in skeletal muscle arterioles that Ca2+ influx through SMC voltage-gated Ca2+ channels (VGCCs) can activate a vasodilator signaling pathway in adjacent endothelial cells (ECs), so forming a bidirectional, voltage-dependent circuit between the SMCs and ECs, which provides negative feedback to supress vasoconstriction. Rat isolated cremaster arterioles from male Wistar rats were cannulated, pressurized and loaded with Oregon Green® 488 BAPTA-1 to image [Ca2+]i changes in either ECs and/or SMCs using laser scanning confocal microscopy. Direct activation of SMC VGCCs with BayK8644, or depolarization with raised K+, 4-AP or the α1-adrenoceptor agonist phenylephrine (PE), each indirectly stimulated local and propagating cell-wide Ca2+ events in the adjacent ECs. In freshly isolated EC tubes, which lack influence from SMCs, direct and indirect activators of VGCCs did not alter EC [Ca2+]i. These data demonstrate a SMC-EC circuit that links VGCC-dependent EC Ca2+ activity to feedback regulation of vascular tone. Selective block of EC Ca2+-activated K+ (KCa) channels markedly enhanced contraction to both direct (BayK8644) or indirect (PE) activators of VGCCs. Our data clearly demonstrate a link between global increases in SMC Ca2+ with a secondary increase in EC Ca2+ and activation of EC KCa channels, to provide EC-dependent feedback to supress vasoconstriction. This work was supported by the British Heart Foundation and the Wellcome Trust.

Defective autophagy in vascular smooth muscle cells alters contractility and Ca²⁺ homeostasis in mice.

Autophagy is an evolutionary preserved process that prevents the accumulation of unwanted cytosolic material through the formation of autophagosomes. Although autophagy has been extensively studied to understand its function in normal physiology, the role of vascular smooth muscle (SM) cell (VSMC) autophagy in Ca(2+) mobilization and contraction remains poorly understood. Recent evidence shows that autophagy is involved in controlling contractile function and Ca(2+) homeostasis in certain cell types. Therefore, autophagy might also regulate contractile capacity and Ca(2+)-mobilizing pathways in VSMCs. Contractility (organ chambers) and Ca(2+) homeostasis (myograph) were investigated in aortic segments of 3.5-mo-old mice containing a SM cell-specific deletion of autophagy-related 7 (Atg7; Atg7(fl/fl) SM22α-Cre(+) mice) and in segments of corresponding control mice (Atg7(+/+) SM22α-Cre(+)). Our results indicate that voltage-gated Ca(2+) channels (VGCCs) of Atg7(fl/fl) SM22α-Cre(+) VSMCs were more sensitive to depolarization, independent of changes in resting membrane potential. Contractions elicited with K(+) (50 mM) or the VGCC agonist BAY K8644 (100 nM) were significantly higher due to increased VGCC expression and activity. Interestingly, the sarcoplasmic reticulum of Atg7(fl/fl) SM22α-Cre(+) VSMCs was enlarged, which, combined with increased sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 expression and higher store-operated Ca(2+) entry, promoted inositol 1,4,5-trisphosphate-mediated contractions of Atg7(fl/fl) SM22α-Cre(+) segments and maximized the Ca(2+) storing capacity of the sarcoplasmic reticulum. Moreover, decreased plasma membrane Ca(2+)-ATPase expression in Atg7(fl/fl) SM22α-Cre(+) VSMCs hampered Ca(2+) extrusion to the extracellular environment. Overall, our study indicates that defective autophagy in VSMCs leads to an imbalance between Ca(2+) release/influx and Ca(2+) reuptake/extrusion, resulting in higher basal Ca(2+) concentrations and significant effects on vascular reactivity.

Functional Coupling between ANO1 and TRPV1 Channels in Sensory Neurons

Ca2+ activated Cl- channel ANO1 (TMEM16A) is one of the most recently discovered pro-algesic ion channels expressed in peripheral sensory neurons. In these neurons ANO1 is specifically activated by Ca2+ release from intracellular stores in response to inflammatory mediators such as bradykinin (BK). In addition, ANO1 is also activated by noxious heat in the temperature range that is similar to that of the heat sensor TRPV1. As TRPV1 is permeable to Ca2+, there is an intriguing possibility that both channels can function in a coordinated fashion thus increasing the dynamic range of response to noxious heat. In order to investigate activation of ANO1 in nociceptors by intracellular Ca2+ signals we developed a single-cell imaging approach that allows simultaneous monitoring of Cl- channel activity and intracellular Ca2+ concentration in cultured dorsal root ganglion (DRG) neurons using simultaneous imaging of halide-sensitive H148Q/I152L EYFP mutant and fura-2. We investigated activation of Ca2+-activated anion conductance in DRG neurons in response to i) Ca2+ release from the IP3-sensitive intracellular stores induced by BK; ii) Ca2+ influx through the TRPV1 channels activated by capsaicin (CAP) and iii) Ca2+ influx via the voltage-gated Ca2+ channels (VGCC) induced by depolarization with extracellular solution containing 50 mM KCl (HK). Both BK and CAP produced large Ca2+ transients and caused significant quenching of YFP fluorescence. The onset of the YFP response to CAP was delayed as compared to the response to BK, possibly reflecting a shallower Ca2+ transient. Consistent with previous finding, activation of VGCC was least efficacious in activating CaCC and in approximately two thirds of neurons the YFP quenching was indistinguishable from the baseline rundown even despite strong Ca2+ transients produced by HK. Our results suggest that ANO1 in DRG neurons is preferentially coupled to colocalised Ca2+ sources.

Controlling insulin secretion: an exciting TASK.

The -cells of the pancreatic islets of Langerhans secrete insulin in response to metabolic fuels and neurohormonal inputs after a meal. The proper regulation of insulin secretion is critical for the maintenance of glucose homeostasis. An absolute or relative impairment of insulin secretion in the face of insulin resistance underlies the pathophysiology of type 2 diabetes. Accordingly, some of the key antidiabetes agents promote increased insulin secretion from islets, including the newer incretin-based therapeutics and the sulfonylurea class of drugs. The consensus mechanism for glucose-stimulated insulin secretion was elucidated almost 30 years ago and has remained largely unchanged since. A glucose-dependent increase in the intracellular ATP to ADP ratio closes ATP-sensitive potassium (KATP) ion channels (1, 2), themselves the target of the sulfonylurea drugs (3). This results in depolarization of the -cell membrane and the firing of action potentials, which allows the activation of voltage-gated Ca channels and a rise in intracellular Ca that triggers insulin release (4). It is well-recognized, however, that a diverse array of additional ion channels contribute to -cell excitability, and only recently have we begun to understand the roles and molecular identities of the many key channels that shape the electrical response in these cells. Although the outward flux of K mediated by KATP channels dominates the electrical conductance of the -cell membrane in the resting condition,holding themembranepotential of these cells near the equilibrium potential for K ( 70 mV), closure of KATP channels upon glucose stimulation is not in itself sufficient to depolarize the cell. For the -cell to depolarize, an inward depolarizing leak current is required to push the membrane potential toward a more positive equilibrium (or plateau, Figure 1) sufficient to allow the activation of the voltage-gated Na , Ca , and K channels that mediate regenerative action potential firing (5). This depolarizing force is likely mediated by channels that mainly allow the inward leak of Na (and possibly Ca ) such as the transient receptor potential (TRP) family of channels (6) and/or the muscarinic-activated nonselective Na -leak channel (NALNC) (7). Additional currents, such as those mediated by the smallconductance Ca -sensitive K (SK) channels (8) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (9), contribute to the oscillatory nature of the electrical activity that characterizes -cell electrical function. Although required for membrane depolarization after glucose stimulation, these inward leak currents pose a problem of their own: the equilibrium potential for Na and Ca is much too positive ( 50 mV) to allow the rhythmic opening and closing of voltage-dependent ion channels required for action potential firing. What this means is that upon KATP channel closure in response to elevated glucose or sulfonylurea, the inward leak currents could depolarize the -cell too much, unless a counterbalancing outward leak is also present. In the present issue of Endocrinology, Dadi et al (10) present evidence to suggest that this key outward leak current is mediated by TASK-1, a member of the 2-poredomain K (K2P) channel family shown to be expressed at the mRNA level in human -cells only very recently (11, 12) and suggested previously as a target of inhaled anesthetics (13) and for the treatment of multiple sclerosis (14), T cell-mediated autoimmunity (15), and cardiac arrhythmia (16) among other conditions. Using cell-type–specific knockout mice and a selective TASK-1 inhibitor, the authors demonstrate that this channel mediates a back-

Abstract 488: Arteriolar Smooth Muscle Calcium Waves Depend on Calcium Influx through Voltage-gated Calcium Channels

Smooth muscle cells (SMCs) in arterioles from striated muscle display IP 3 receptor-dependent Ca 2+ waves that contribute to global myoplasmic Ca 2+ concentration and myogenic tone. However, the contribution of voltage-gated Ca 2+ channels (VGCC) to these arteriolar Ca 2+ signals is unknown. We tested the hypothesis that Ca 2+ waves depend on Ca 2+ influx through VGCC in cremaster muscle arterioles loaded with Fluo-4 and imaged by confocal microscopy. At rest, with vessels pressurized to 80 cm H 2 O in 2 mM Ca 2+ , arteriolar diameter was 28 ± 2 μm (n = 5), and SMCs displayed Ca 2+ waves with frequency (FREQ) = 0.21 ± 0.06 Hz, occurrence (OCC) = 3.5 ± 1.0 waves/SMC and amplitude (AMP) = 1.7 ± 0.1 F/Fo. Removal of extracellular Ca 2+ dilated the arterioles to 39 ± 1 μm, and inhibited Ca 2+ waves (FREQ = 0.1 ± 0.03, OCC = 1.7 ± 0.5 waves/SMC and AMP = 1.4 ± 0.06 F/Fo; p &lt; 0.05 vs. rest) indicating that Ca 2+ waves depended, in part, on influx of extracellular Ca 2+ . Similarly, the VGCC antagonist, nifedipine (1 μM), dilated the arterioles to 34 ± 1.3 μm and also inhibited Ca 2+ waves (FREQ = 0.07 ± 0.02 Hz, OCC = 1.1 ± 0.5 waves/SMC, AMP = 1.4 ± 0.05 F/Fo; p &lt; 0.05 vs. rest). Hyperpolarization of SMCs with the K + channel agonist, cromakalim (10 μM), dilated arterioles from 49 ± 3 to 59 ± 4 μm (n = 4, p &lt; 0.05) and also reduced Ca 2+ wave FREQ (0.1 ± 0.04 to 0.03 ± 0.003 Hz), OCC (1.7 ± 0.04 to 0.5 ± 0.05 waves/SMC) and AMP (1.5 ± 0.04 to 1.2 ± 0.004 F/Fo) (p &lt; 0.05). Conversely, depolarization of SMCs with the BK Ca channel blocker, TEA (1 mM), constricted arterioles from 28 ± 2 to 16 ± 1 μm (n = 5, p &lt; 0.05) and increased wave FREQ (0.2 ± 0.1 to 0.5 ± 0.1 Hz, p &lt; 0.05) and OCC (4 ± 1 to 8 ± 2 waves/SMC, p &lt; 0.05), effects blocked by nifedipine (1μM) (p &lt; 0.05). Similarly, in arterioles pressurized to 20 cm H 2 O to eliminate myogenic tone and reduce basal VGCC activity, application of the VGCC agonist, BayK 8644 (5 nM) constricted the arterioles from 14 ± 1 to 8 ± 1 μm and increased wave FREQ (0.2 ± 0.1 to 0.6 ± 0.1 Hz) and OCC (3 ± 1 to 10 ± 1 waves/SMC) (n = 6; p &lt; 0.05), effects that were independent of ryanodine receptors, as Ca 2+ waves were unaffected by ryanodine (50 μM) in the absence or presence of BayK 8644 (n = 6; p &gt; 0.05). These data support the hypothesis that Ca 2+ waves in arteriolar SMCs depend, in part, on Ca 2+ influx through VGCC.

A new signalling pathway for parallel fibre presynaptic type 4 metabotropic glutamate receptors (mGluR4) in the rat cerebellar cortex

In the rodent cerebellum, pharmacological activation of mGluR4 acutely depresses excitatory synaptic transmission at parallel fibre–Purkinje cell synapses. This depression involves the inhibition of presynaptic calcium (Ca2+) influx that ultimately controls glutamate release. In this study, we investigate the molecular basis of mGluR4-mediated inhibition of presynaptic Ca2+ transients. Our results demonstrate that the mGluR4 effect does not depend on selective inhibition of a specific type of presynaptic voltage-gated Ca2+ channel, but rather involves modulation of all classes of Ca2+ channels present in the presynaptic terminals. In addition, this inhibitory effect does not involve the activation of G protein-activated inwardly rectifying potassium channels, TEA-sensitive potassium channels or two-pore-domain potassium channels. Furthermore, this inhibition does not require pertussis toxin-sensitive G proteins, and is independent of any effect on adenylyl cyclases, protein kinase A, mitogen-activated protein kinases or phosphoinositol-3 kinase activity. Interestingly we found that mGluR4 inhibition of presynaptic Ca2+ influx employs a newly defined signalling pathway, notably that involving the activation of phospholipase C and ultimately protein kinase C.

1645 DIFFERENTIAL EFFECTS OF BOTULINUM NEUROTOXIN-A (BONT/A) ON BLADDER CONTRACTILE RESPONSES TO ACTIVATION OF EFFERENT NERVES, SMOOTH MUSCLES AND AFFERENT NERVES IN RATS

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Neurogenic Voiding Dysfunction1 Apr 20121645 DIFFERENTIAL EFFECTS OF BOTULINUM NEUROTOXIN-A (BONT/A) ON BLADDER CONTRACTILE RESPONSES TO ACTIVATION OF EFFERENT NERVES, SMOOTH MUSCLES AND AFFERENT NERVES IN RATS Ryosuke Takahashi, Takakazu Yunoki, Seiji Naito, and Naoki Yoshimura Ryosuke TakahashiRyosuke Takahashi Pittsburgh, PA More articles by this author , Takakazu YunokiTakakazu Yunoki Fukuoka, Japan More articles by this author , Seiji NaitoSeiji Naito Fukuoka, Japan More articles by this author , and Naoki YoshimuraNaoki Yoshimura Pittsburgh, PA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1477AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES BoNT/A, an effective therapeutic agent for overactive bladder, reportedly has inhibitory effects not only on acetylcholine exocytosis from efferent nerve terminals, but also on transmitter release from afferent nerve terminals. We also showed that BoNT/A inhibits voltage-gated Ca2+ channels (VGCC) in rat and human detrusor smooth muscles using patch-clamp techniques (AUA 2009). We therefore compared the effects of BoNT/A on rat bladder smooth muscle contractions induced by activation of efferent nerves, afferent nerves and VGCC on smooth muscle to examine the relative importance of these three inhibitory mechanisms. METHODS Bladder strips without urothelium were obtained from female SD rats. These strips were incubated for 3 h at 37°C in Krebs solution with different concentrations of BoNT/A (0.3-100 nM) and tension development was measured in an organ bath. To examine the effect of BoNT/A on neurotransmitter release from efferent nerve terminals and afferent nerve terminals and on VGCC of smooth muscle, changes in electrical field stimulation (EFS: 20V; 0.5-64 Hz), 1 μM capsaicin and 70 mM KCl-induced contractions were evaluated, respectively. In addition, the contractile response to carbachol (CCh) was also examined. RESULTS The significant inhibitory effect of BoNT/A on KCl-induced contractions was observed at a concentration of 3nM or higher, and the maximal inhibition at 100 nM was 30% compared to control strips. EFS-induced contractions were significantly inhibited by the incubation with BoNT/A of 10 nM or higher, and the maximal inhibition at 100 nM was 70% compared to control strips. The pretreatment of 1μM atropine plus 10μM αβMet-ATP inhibited EFS-induced contractions to the same level as in BoNT/A treatment. Capsaicin-induced contractions were inhibited by 30% with overnight incubation of 100 nM BoTN/A, but not with 3 h incubation. The contractile responses to CCh were not altered by the incubation with BoNT/A. CONCLUSIONS The order of inhibitory potency of BoNT/A is efferent nerve terminals > L-type VGCC on smooth muscles > afferent nerve terminals. In addition, BoNT/A can inhibit both acetylcholine and ATP release from efferent nerve terminals. Thus, BoNT/A has multiple inhibitory mechanisms affecting bladder function, which would enhance the efficacy of BoNT/A treatment for overactive bladder. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e665 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ryosuke Takahashi Pittsburgh, PA More articles by this author Takakazu Yunoki Fukuoka, Japan More articles by this author Seiji Naito Fukuoka, Japan More articles by this author Naoki Yoshimura Pittsburgh, PA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Metabotropic Regulation of RhoA/Rho-Associated Kinase by L-type Ca 2+ Channels

Background: Sustained vascular smooth muscle contraction is mediated by extracellular Ca 2+ influx through L-type voltage-gated Ca 2+ channels (VGCC) and RhoA/Rho-associated kinase (ROCK)-dependent Ca 2+ sensitization of the contractile machinery. VGCC activation can also trigger an ion-independent metabotropic pathway that involves G-protein/phospholipase C activation, inositol 1,4,5-trisphosphate synthesis, and Ca 2+ release from the sarcoplasmic reticulum (calcium channel-induced Ca 2+ release). We have studied the functional role of calcium channel-induced Ca 2+ release and the inter-relations between Ca 2+ channel and RhoA/ROCK activation. Methods and Results: We have used normal and genetically modified animals to study single myocyte electrophysiology and fluorimetry as well as cytosolic Ca 2+ and diameter in intact arteries. These analyses were complemented with measurement of tension and RhoA activity in normal and reversibly permeabilized arterial rings. We have found that, unexpectedly, L-type Ca 2+ channel activation and subsequent metabotropic Ca 2+ release from sarcoplasmic reticulum participate in depolarization-evoked RhoA/ROCK activity and sustained arterial contraction. We show that these phenomena do not depend on the change in the membrane potential itself, or the mere release of Ca 2+ from the sarcoplasmic reticulum, but they require the simultaneous activation of VGCC and the downstream metabotropic pathway with concomitant Ca 2+ release. During protracted depolarizations, refilling of the stores by a residual extracellular Ca 2+ influx through VGCC helps maintaining RhoA activity and sustained arterial contraction. Conclusions: These findings reveal that calcium channel-induced Ca 2+ release has a major role in tonic vascular smooth muscle contractility because it links membrane depolarization and Ca 2+ channel activation with metabotropic Ca 2+ release and sensitization (RhoA/ROCK stimulation).

GLP-2 potentiates L-type Ca2+ channel activity associated with stimulated glucose uptake in hippocampal neurons

Glucagon-like peptide-2 (GLP-2) is a neuropeptide secreted from endocrine cells in the gut and neurons in the brain. GLP-2 stimulates intestinal crypt cell proliferation and mucosal blood flow while decreasing gastric emptying and gut motility. However, a GLP-2-mediated signaling network has not been fully established in primary cells. Since the GLP-2 receptor mRNA and protein were highly expressed in the mouse hippocampus, we further characterized that human (125)I-labeled GLP-2(1-33) specifically bound to cultured hippocampal neurons with K(d) = 0.48 nM, and GLP-2 acutely induced subcellular translocalization of the early gene c-Fos. Using the whole cell patch clamp, we recorded barium currents (I(Ba)) flowing through voltage-gated Ca(2+) channels (VGCC) in those neurons in the presence of GLP-2 with and without inhibitors. We showed that GLP-2 (20 nM) enhanced the whole cell I(Ba) mediated by L-type VGCC that was defined using an L-type Ca(2+) channel blocker (nifedipine, 10 microM). Moreover, GLP-2-potentiation of L-type VGCC was abolished in neurons pretreated with a PKA inhibitor (PKI(14-22), 1 microM). Finally, using a fluorescent nonmetabolized glucose analog (6-NBDG) tracing imaging, we showed that glucose was taken up directly by cultured neurons. GLP-2 increased 2-deoxy-d-[(3)H]glucose uptake that was dependent upon dosage, activation of PKA, and potentiation of L-type VGCC. We conclude that GLP-2 potentiates L-type VGCC activity through activating PKA signaling, partially stimulating glucose uptake by primary cultured hippocampal neurons. The potentiation of L-type VGCC may be physiologically relevant to GLP-2-induced neuroendocrine modulation of neurotransmitter release and hormone secretion.

Biphasic Synaptic Ca Influx Arising from Compartmentalized Electrical Signals in Dendritic Spines

Author SummaryThe vast majority of excitatory synapses in the mammalian central nervous system are made onto dendritic spines, small (< 1 fL) membranous structures stippled along the dendrite. The head of each spine is separated from its parent dendrite by a thin neck – a morphological feature that intuitively suggests it might function to limit the transmission of electrical and biochemical signals. Unfortunately, the extremely small size of spines has made direct measurements of their electrical properties difficult and, therefore, the functional implications of electrical compartmentalization have remained elusive. In this study, we use spatiotemporally controlled stimulation to generate calcium signals within the spine head and/or neighboring dendrite. By comparing these measurements we demonstrate that spines create specialized electrical signaling compartments, which has at least two functional consequences. First, synaptic stimulation, but not similar dendritic depolarization, can trigger the activation of voltage-gated calcium channels within the spine. Second, voltage changes in the spine head arising from compartmentalization shape the time course of synaptically evoked calcium influx such that it is biphasic. Thus, the electrical compartmentalization provided by spines allows for multiple modes of calcium signaling at excitatory synapses.