Increase In Membrane Capacitance Research Articles

IR Laser-Induced Perturbations of the Voltage-Dependent Solute Carrier Protein SLC26a5

Alterations in membrane capacitance can arise from linear and nonlinear sources. For example, changes in membrane surface area or dielectric properties can modify capacitance linearly, whereas sensor residues of voltage-dependent proteins can modify capacitance nonlinearly. Here, we examined the effects of fast temperature jumps induced by an infrared (IR) laser in control and prestin (SLC26a5)-transfected human embryonic kidney (HEK) cells under whole-cell voltage clamp. Prestin’s voltage sensor imparts a characteristic bell-shaped, voltage-dependent nonlinear capacitance (NLC). Temperature jumps in control HEK cells cause a monophasic increase in membrane capacitance (Cm) regardless of holding voltage due to double-layer effects. Prestin-transfected HEK cells, however, additionally show a biphasic increase/decrease in Cm with a reversal potential corresponding to the voltage at peak NLC of prestin (Vh), attributable to a rapid temperature-following shift in Vh, with shift rates up to 14 V/s over the course of a 5 ms IR pulse. Treatment with salicylate, a known inhibitor of NLC, reestablishes control cell behavior. A simple kinetic model recapitulates our biophysical observations. These results verify a voltage-dependent protein’s ability to respond to fast temperature perturbations on a par with double-layer susceptibility. This likely arises from prestin’s unique ability to move sensor charge at kilohertz rates, which is required for the outer hair cells’ role as a cochlear amplifier.

Voltage-gated Ca2+ influx and mitochondrial Ca2+ initiate secretion from Aplysia neuroendocrine cells

Neuroendocrine secretion often requires prolonged voltage-gated Ca2+ entry; however, the ability of Ca2+ from intracellular stores, such as endoplasmic reticulum or mitochondria, to elicit secretion is less clear. We examined this using the bag cell neurons, which trigger ovulation in Aplysia by releasing egg-laying hormone (ELH) peptide. Secretion from cultured bag cell neurons was observed as an increase in plasma membrane capacitance following Ca2+ influx evoked by a 5-Hz, 1-min train of depolarizing steps under voltage-clamp. The response was similar for step durations of ⩾50ms, but fell off sharply with shorter stimuli. The capacitance change was attenuated by replacing external Ca2+ with Ba2+, blocking Ca2+ channels, buffering intracellular Ca2+ with EGTA, disrupting synaptic protein recycling, or genetic knock-down of ELH. Regarding intracellular stores, liberating mitochondrial Ca2+ with the protonophore, carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP), brought about an EGTA-sensitive elevation of capacitance. Conversely, no change was observed to Ca2+ released from the endoplasmic reticulum or acidic stores. Prior exposure to FCCP lessened the train-induced capacitance increase, suggesting overlap in the pool of releasable vesicles. Employing GTP-γ-S to interfere with endocytosis delayed recovery (presumed membrane retrieval) of the capacitance change following FCCP, but not the train. Finally, secretion was correlated with reproductive behavior, in that neurons isolated from animals engaged in egg-laying presented a greater train-induced capacitance elevation vs quiescent animals. The bag cell neuron capacitance increase is consistent with peptide secretion requiring high Ca2+, either from influx or stores, and may reflect the all-or-none nature of reproduction.

Complexin facilitates exocytosis and synchronizes vesicle release in two secretory model systems

Complexins (Cplxs) are small, SNARE-associated proteins believed to regulate fast, calcium-triggered exocytosis. However, studies have pointed to either an inhibitory and/or facilitatory role in exocytosis, and the role of Cplxs in synchronizing exocytosis is relatively unexplored. Here, we compare the function of two types of complexin, Cplx 1 and 2, in two model systems of calcium-dependent exocytosis. In mouse neuromuscular junctions (NMJs), we find that lack of Cplx 1 significantly reduces and desynchronizes calcium-triggered synaptic transmission; furthermore, high-frequency stimulation elicits synaptic facilitation, instead of normal synaptic depression, and the degree of facilitation is highly sensitive to the amount of cytoplasmic calcium buffering. In Cplx 2-null adrenal chromaffin cells, we also find decreased and desynchronized evoked release, and identify a significant reduction in the vesicle pool close to the calcium channels (immediately releasable pool, IRP). Viral transduction with either Cplx 1 or 2 rescues both the size of the evoked response and the synchronicity of release, and it restores the IRP size. Our findings in two model systems are mutually compatible and indicate a role of Cplx 1 and 2 in facilitating vesicle priming, and also lead to the new hypothesis that Cplxs may synchronize vesicle release by promoting coupling between secretory vesicles and calcium channels.

Noncompetitive, Voltage-Dependent NMDA Receptor Antagonism by Hydrophobic Anions

NMDA receptor (NMDAR) antagonists are dissociative anesthetics, drugs of abuse, and are of therapeutic interest in neurodegeneration and neuropsychiatric disease. Many well-known NMDAR antagonists are positively charged, voltage-dependent channel blockers. We recently showed that the hydrophobic anion dipicrylamine (DPA) negatively regulates GABA(A) receptor function by a mechanism indistinguishable from that of sulfated neurosteroids. Because sulfated neurosteroids also modulate NMDARs, here we examined the effects of DPA on NMDAR function. In rat hippocampal neurons DPA inhibited currents gated by 300 µM NMDA with an IC(50) of 2.3 µM. Neither onset nor offset of antagonism exhibited dependence on channel activation but exhibited a noncompetitive profile. DPA antagonism was independent of NMDAR subunit composition and was similar at extrasynaptic and total receptor populations. Surprisingly, similar to cationic channel blockers but unlike sulfated neurosteroids, DPA antagonism was voltage dependent. Onset and offset of DPA antagonism were nearly 10-fold faster than DPA-induced increases in membrane capacitance, suggesting that membrane interactions do not directly explain antagonism. Furthermore, voltage dependence did not derive from association of DPA with a site on NMDARs directly accessible to the outer membrane leaflet, assessed by DPA translocation experiments. Consistent with the expected lack of channel block, DPA antagonism did not interact with permeant ions. Therefore, we speculate that voltage dependence may arise from interactions of DPA with the inherent voltage dependence of channel gating. Overall, we conclude that DPA noncompetitively inhibits NMDA-induced current by a novel voltage-dependent mechanism and represents a new class of anionic NMDAR antagonists.

Bioimpedance of soft tissue under compression

In this paper compression-dependent bioimpedance measurements of porcine spleen tissue are presented. Using a Cole–Cole model, nonlinear compositional changes in extracellular and intracellular makeup; related to a loss of fluid from the tissue, are identified during compression. Bioimpedance measurements were made using a custom tetrapolar probe and bioimpedance circuitry. As the tissue is increasingly compressed up to 50%, both intracellular and extracellular resistances increase while bulk membrane capacitance decreases. Increasing compression to 80% results in an increase in intracellular resistance and bulk membrane capacitance while extracellular resistance decreases. Tissues compressed incrementally to 80% show a decreased extracellular resistance of 32%, an increased intracellular resistance of 107%, and an increased bulk membrane capacitance of 64% compared to their uncompressed values. Intracellular resistance exhibits double asymptotic curves when plotted against the peak tissue pressure during compression, possibly indicating two distinct phases of mechanical change in the tissue during compression. Based on these findings, differing theories as to what is happening at a cellular level during high tissue compression are discussed, including the possibility of cell rupture and mass exudation of cellular material.

Synapsins I and II Are Not Required for β-Cell Insulin Secretion: Granules Must Pool Their Own Weight

Synapsins I and II Are Not Required for β-Cell Insulin Secretion: Granules Must Pool Their Own Weight

Synapsins I and II Are Not Required for Insulin Secretion from Mouse Pancreatic β-cells

Synapsins are a family of phosphoproteins that modulate the release of neurotransmitters from synaptic vesicles. The release of insulin from pancreatic β-cells has also been suggested to be regulated by synapsins. In this study, we have utilized a knock out mouse model with general disruptions of the synapsin I and II genes [synapsin double knockout (DKO)]. Stimulation with 20 mm glucose increased insulin secretion 9-fold in both wild-type (WT) and synapsin DKO islets, whereas secretion in the presence of 70 mm K(+) and 1 mm glucose was significantly enhanced in the synapsin DKO mice compared to WT. Exocytosis in single β-cells was investigated using patch clamp. The exocytotic response, measured by capacitance measurements and elicited by a depolarization protocol designed to visualize exocytosis of vesicles from the readily releasable pool and from the reserve pool, was of the same size in synapsin DKO and WT β-cells. The increase in membrane capacitance corresponding to readily releasable pool was approximately 50fF in both genotypes. We next investigated the voltage-dependent Ca(2+) influx. In both WT and synapsin DKO β-cells the Ca(2+) current peaked at 0 mV and measured peak current (I(p)) and net charge (Q) were of similar magnitude. Finally, ultrastructural data showed no variation in total number of granules (N(v)) or number of docked granules (N(s)) between the β-cells from synapsin DKO mice and WT control. We conclude that neither synapsin I nor synapsin II are directly involved in the regulation of glucose-stimulated insulin secretion and Ca(2)-dependent exocytosis in mouse pancreatic β-cells.

Novel standards in the measurement of rat insulin granules combining electron microscopy, high-content image analysis and in silico modelling.

Aims/hypothesisKnowledge of number, size and content of insulin secretory granules is pivotal for understanding the physiology of pancreatic beta cells. Here we re-evaluated key structural features of rat beta cells, including insulin granule size, number and distribution as well as cell size.MethodsElectron micrographs of rat beta cells fixed either chemically or by high-pressure freezing were compared using a high-content analysis approach. These data were used to develop three-dimensional in silico beta cell models, the slicing of which would reproduce the experimental datasets.ResultsAs previously reported, chemically fixed insulin secretory granules appeared as hollow spheres with a mean diameter of ∼350 nm. Remarkably, most granules fixed by high-pressure freezing lacked the characteristic halo between the dense core and the limiting membrane and were smaller than their chemically fixed counterparts. Based on our analyses, we conclude that the mean diameter of rat insulin secretory granules is 243 nm, corresponding to a surface area of 0.19 μm2. Rat beta cells have a mean volume of 763 μm3 and contain 5,000–6,000 granules.Conclusions/interpretationA major reason for the lower mean granule number/rat beta cell relative to previous accounts is a reduced estimation of the mean beta cell volume. These findings imply that each granule contains about twofold more insulin, while its exocytosis increases membrane capacitance about twofold less than assumed previously. Our integrated approach defines new standards for quantitative image analysis of beta cells and could be applied to other cellular systems.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-011-2438-4) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Rab27a Regulates Exocytosis of Large Dense-Core Granules in Mouse Chromaffin Cells

Rab-GTPases exert important roles in secretory vesicle cycles, with Rab3 and Rab27 participating in exocytotic throughput. While Rab3 and Rab27 share many effectors, some are unique, making it critical to identify specific functional roles. Studies on regulation of neurotransmitter release have focused principally on Rab3 and much less on Rab27. Among Rab-GTPases, Rab27 is unique in binding effectors in either the GTP- or GDP-bound state. We here compare secretory responses from ashen mice, which lack functional Rab27a protein, with strain-matched controls. Secretion was evaluated using measurements of membrane capacitance under whole-cell recording in isolated mouse chromaffin cells. UV flash uncaging of Ca2+ raised [Ca2+]i uniformly and induced catecholamine neurotransmitter release. RT-PCR demonstrated the presence of Rab27 in chromaffin cells, and immunoblotting further characterized the expressed isoform as Rab27a, not Rab27b. The exocytic burst (1s post-UV flash) of membrane capacitance increase (ΔCm) was fitted by a double exponential curve, from which the amount and fusion kinetics of granules from the readily- (RRP) and slowly-releasable pools (SRP) were extracted. Exocytic burst ΔCm was greater, and the size of both the RRP and SRP larger, in wild-type cells (RRP, 347 fF; SRP, 616 fF) than in Rab27a-/- cells (RRP, 157fF; SRP, 438 fF), while the kinetics of release from both pools remained unchanged. Expression of Rab27a in Rab27a-/- cells, using electroporation, rescued the wild-type phenotype. Rab 27a is therefore important for priming into the RRP and SRP. To assess dense-core vesicle docking at the plasma membrane, we analyzed granule distribution from E18 chromaffin cell EM micrographs. These data show that while Rab27a-/- cells possess fewer total granules, morphological docking of granules at the plasma membrane is unaffected. Further work is necessary to identify through which effector Rab27a is exerting its effects.

Clinical dose of lidocaine destroys the cell membrane and induces both necrosis and apoptosis in an identified Lymnaea neuron

Although lidocaine-induced cell toxicity has been reported, its mechanism is unclear. Cell size, morphological change, and membrane resistance are related to homeostasis and damage to the cell membrane; however, the effects of lidocaine on these factors are unclear. Using an identified LPeD1 neuron from Lymnaea stagnalis, we sought to determine how lidocaine affects these factors and how lidocaine is related to damage of the cell membrane. Cell size and morphological form were measured by a micrograph and imaging analysis system. Membrane potential and survival rate were obtained by intracellular recording. Membrane resistance and capacitance were measured by whole-cell patch clamp. Phosphatidyl serine and nucleic acid were double stained and simultaneously measured by annexin V and propidium iodide. Lidocaine at a clinical dose (5-20 mM) induced morphological change (bulla and bleb) in the neuron and increased cell size in a concentration-dependent manner. Membrane potential was depolarized in a concentration-dependent manner. At perfusion of more than 5 mM lidocaine, the depolarized membrane potential was irreversible. Lidocaine decreased membrane resistance and increased membrane capacitance in a concentration-dependent manner. Both phosphatidyl serine and nucleic acid were stained under lidocaine exposure in a concentration-dependent manner. A clinical dose of lidocaine greater than 5 mM destroys the cell membrane and induces both necrosis and apoptosis in an identified Lymnaea neuron.

Compensatory Thrombopoietin Production from the Liver and Bone Marrow Stimulates Thrombopoiesis of Living Rat Megakaryocytes in Chronic Renal Failure

Background/Aims: Decreased thrombopoiesis has been ascribed a role in the pathogenesis of uremic bleeding in chronic renal failure (CRF). However, serum thrombopoietin (TPO) levels are usually elevated in CRF patients, suggesting increased thrombopoiesis. The aim of this study was to determine the thrombopoietic activity in CRF. Methods: Male Sprague-Dawley rats that underwent 5/6 nephrectomy were used as the model of CRF. Age-matched sham-operated rats were used as controls. Single megakaryocytes were isolated from the rat bone marrow, and their size distribution was examined. Megakaryocyte membrane invaginations were monitored by confocal imaging of di-8-ANEPPS staining, and patch clamp whole-cell recordings of membrane capacitance. TPO gene expression was assessed in various tissues. Results: Circulating platelet counts and the number of large megakaryocytes were increased in the bone marrow of CRF rats. Massive di-8-ANEPPS staining and increased membrane capacitance in large megakaryocytes demonstrated increased membrane invaginations. Unaffected Kv1.3-channel currents per cell surface area demonstrated unaltered channel densities. TPO transcription was decreased in the renal cortex but increased in the liver and bone marrow of CRF rats. Conclusion: Increased thrombopoiesis in CRF was thought to be a reactive mechanism to platelet dysfunction. Increased TPO production from the liver and bone marrow compensated for decreased production from damaged kidneys.

Biophysical Characteristics Reveal Neural Stem Cell Differentiation Potential

BackgroundDistinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers.Methodology/Principal FindingsWe used dielectrophoresis (DEP) to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates.Conclusions/SignificanceWe show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors.

On Depolarization-Evoked Exocytosis as a Function of Calcium Entry: Possibilities and Pitfalls

Secretion from many endocrine cells is a result of calcium-regulated exocytosis due to Ca2+ influx. Using the patch-clamp technique, voltage pulses can be applied to the cells to open Ca2+ channels, resulting in a measurable Ca2+ current, and evoke exocytosis, which can be seen as an increase in membrane capacitance. A common tool for evaluating the relation between Ca2+ influx and exocytosis is to plot the increase in capacitance (ΔCm) as a function of the integral of the measured Ca2+ current (Q). When depolarizations of different lengths are imposed, the rate of exocytosis is typically higher for shorter than for longer pulses, which has been suggested to result from depletion of a granule pool or from Ca2+ current inactivation. It is here demonstrated that ΔCm as a function of Q can reveal whether Ca2+ current inactivation masquerades as pool depletion. Moreover, it is shown that a convex, cooperativity-like, relation between ΔCm and Q surprisingly cannot occur as a result of cooperative effects, but can result from delays in the exocytotic process or in Ca2+ dynamics. An overview of expected ΔCm-versus-Q relations for a range of explicit situations is given, which should help in the interpretation of data of depolarization-evoked exocytosis in endocrine cells.

Glucose-dependent docking and SNARE protein-mediated exocytosis in mouse pancreatic alpha-cell

The function of alpha-cells in patients with type 2 diabetes is often disturbed; glucagon secretion is increased at hyperglycaemia, yet fails to respond to hypoglycaemia. A crucial mechanism behind the fine-tuned release of glucagon relies in the exocytotic machinery including SNARE proteins. Here, we aimed to investigate the temporal role of syntaxin 1A and SNAP-25 in mouse alpha-cell exocytosis. First, we used confocal imaging to investigate glucose dependency in the localisation of SNAP-25 and syntaxin 1A. SNAP-25 was mainly distributed in the plasma membrane at 2.8 mM glucose, whereas the syntaxin 1A distribution in the plasma membrane, as compared to the cytosolic fraction, was highest at 8.3 mM glucose. Furthermore, following inclusion of an antibody against SNAP-25 or syntaxin 1A, exocytosis evoked by a train of ten depolarisations and measured as an increase in membrane capacitance was reduced by ~50%. Closer inspection revealed a reduction in the refilling of granules from the reserve pool (RP), but also showed a decreased size of the readily releasable pool (RRP) by ~45%. Disparate from the situation in pancreatic beta-cells, the voltage-dependent Ca²⁺ current was not reduced, but the Ca²⁺ sensitivity of exocytosis decreased by the antibody against syntaxin 1A. Finally, ultrastructural analysis revealed that the number of docked granules was >2-fold higher at 16.7 mM than at 1 mM glucose. We conclude that syntaxin 1A and SNAP-25 are necessary for alpha-cell exocytosis and regulate fusion of granules belonging to both the RRP and RP without affecting the Ca²⁺ current.

Role of Calcium Independent Phospholipase A2 in Maintaining Mitochondrial Membrane Potential and Preventing Excessive Exocytosis in PC12 Cells

This study was carried out to elucidate the effects of calcium independent phospholipase A(2) (iPLA(2)) on mitochondrial function and exocytosis in neuroendocrine cells. iPLA(2) mRNA and protein were detected in cell lysates and mitochondria from PC12 cells. Treatment of cells with the iPLA(2) inhibitor, bromoenol lactone (BEL), resulted in reduction in the mitochondrial membrane potential. Increase in membrane capacitance and number of spikes at amperometry, indicating exocytosis, were detected from PC12 cells after treatment with BEL. The induced exocytosis was abolished by pre-incubation of cells with the antioxidant, glutathione monoethyl ester, spin-trap/free radical scavenger, PBN, or inhibitors of the mitochondrial permeability transition pore, cyclosporine A and bongkrekic acid. These findings indicate that inhibition of iPLA(2) results in excessive exocytosis through increased oxidative damage (or failure to repair such damage) and defects in mitochondrial function. A similar process may occur in neurons with mutations in iPLA(2), leading to neuronal injury.

Deficiency in RNA editing enzyme ADAR2 impairs regulated exocytosis

Mammalian RNA editing catalyzed by adenosine deaminases acting on RNA (ADARs) ADAR1 and ADAR2 plays pivotal roles in the brain through functional modifications of neurotransmitter receptors and ion channels. We have demonstrated previously that RNA editing by ADAR2 is regulated metabolically in pancreatic β cells. To investigate the cellular functions of ADAR2 in professional secretory cells, we studied the effects of ADAR2 knockdown on regulated exocytosis. Selective knockdown of ADAR2 expression markedly impaired glucose-stimulated insulin secretion in the rat insulinoma INS-1 cells and primary pancreatic islets and significantly diminished KCl-stimulated secretion of exogenous human growth hormone or endogenous chromogranin B protein in the rat adrenal pheochromocytoma PC12 cells. Notably, restored overexpression of catalytically active but not editing-deficient mutant ADAR2 could rescue the impairment in stimulated secretion from ADAR2 knockdown cells. Moreover, ADAR2 suppression significantly attenuated Ca(2+)-evoked membrane capacitance increases and appreciably reduced the number of membrane-docked insulin granules in INS-1 cells. Interestingly, the secretory defects resulting from ADAR2 deficiency were coupled to decreased expression of Munc18-1 and synaptotagmin-7, two key molecules in the regulation of vesicle exocytosis. Thus, these findings reveal an important aspect of ADAR2 actions in regulated exocytosis, implicating RNA editing in the control of cellular secretory machinery.

Inhibition of calcineurin phosphatase promotes exocytosis of renin from juxtaglomerular cells

To examine the role of the calcium/calmodulin-dependent phosphatase calcineurin in regulation of renin release, we assayed exocytosis using whole-cell patch clamp of single juxtaglomerular cells in culture. The calcineurin inhibitor, cyclosporine A (CsA), significantly increased juxtaglomerular cell membrane capacitance, an index of cell surface area and an established measure of exocytosis in single-cell assays. This effect was mimicked by intracellular delivery of a calcineurin inhibitory peptide, the calcium chelator ethylene glycol tetraacetic acid (EGTA), or the calmodulin inhibitor W-13. Simultaneous exposure to EGTA and CsA had no additive effect. The protein kinase A (PKA) blocker RpcAMPs had no effect on the CsA-induced increase in membrane capacitance. Intra- and extracellular application of tacrolimus did not alter membrane capacitance. A calmodulin antagonist (calmidazolium) and CsA, but not tacrolimus, significantly stimulated renin release from cultured juxtaglomerular cells. Juxtaglomerular cells expressed the calcineurin isoforms A-beta and A-gamma but not A-alpha. Plasma renin concentrations (PRCs) were not different in wild-type, calcineurin A-alpha, or A-beta knockout mice but increased after CsA treatment of the A-alpha knockout, while renin mRNA was suppressed. We conclude that calcineurin and calcium/calmodulin suppress exocytosis of renin from juxtaglomerular cells independent of PKA.

Calcineurin Suppression of Alpha7 nAChR Currents in Xenopus Oocytes

As part of our study of mechanisms underlying upregulation of neuronal nicotinic acetylcholine receptors (nAChRs) in brain, due to chronic nicotine exposure, we are characterizing signaling pathways that influence the magnitude of α7 nAChR currents in Xenopus oocytes. In previous studies of Kv1.1 channels in oocytes, we found that calcineurin (CaN) and protein tyrosine kinases (PTKs) mediated calcium-dependent suppression of Kv1.1 currents, through both endocytosis of channels as well as effects on channel gating. Thus, we hypothesized that these molecules would similarly regulate nAChR currents. We first determined that 100 uM bath-applied genistein (an inhibitor of many PTKs) enhanced murine peak α7 nAChR currents in oocytes, by a factor of 2-3, as reported by others (Cho et al., 2005, J. Neuroscience). We also measured an increase in membrane capacitance (Cm), as expected if genistein's potentiation of peak currents was due to increased addition of channels to the plasma membrane via exocytosis. Conversely, injection of vanadate ion (VO43- ) into oocytes expressing α7 nAChRs produced a dramatic reduction (∼90%) in peak currents, but no clear decrease in Cm. Exposure of oocytes expressing α7 nAChRs to 10 uM cyclosporine A (CsA), a potent and specific inhibitor of CaN, increased the magnitude of α7 currents by a factor of ∼2, and also increased Cm. The facilitatory effect of CsA upon α7 current was blocked by 100 uM H-7, a non-specific inhibitor of serine/threonine PKs. The combination of genistein and CsA produced a greater enhancement of the α7 current than either agent alone; their combined effect was approximately additive. Our results suggest that CaN activity may provide a readout of the intracellular calcium concentration, due to recent activity of α7 nAChRs, and thereby negatively regulate the number of α7 nAChRs expressed on the plasma membrane.

Cystic Fibrosis Transmembrane Conductance Regulator in Mouse Pancreatic Beta-Cells

Cystic fibrosis (CF) is a monogenic autosomal recessive disease caused by mutation in the cystic fibrosis gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR), which is an ion-channel that conducts negatively charged chloride ions. Cystic fibrosis related diabetes (CFRD) is the leading complication of CF and exocrine pancreatic dysfunction affects ∼85% of patients. Recently, it was discovered that rat α-cells and β-cells in the islets of Langerhans express both CFTR mRNA and protein. The aim of this study was to investigate the presence of active CFTR channels in pancreatic β-cells and if these influence insulin secretion. For this purpose we have used the patch-clamp technique and capacitance measurements on single mouse β-cells and insulin secretion measurements using RIA. First we measured the presence of CFTR in β-cells using the patch-clamp technique. A membrane conductance of 0.05 ± 0.01 nS/pF (n=10) and 1.05 ± 0.22 nS/pF at negative and positive potentials, respectively, was activated by the cAMP-increasing agent forskolin. The conductance was significantly reduced (P<0.001) and the current almost totally inhibited in the presence of 10 μM of the CFTR-antagonist, CFTRinh-172. Glucose-stimulated and cAMP-amplified insulin secretion measured on islets was not reduced using this concentration although there was a tendency towards reduction. Moreover, exocytosis elicited by a train of ten membrane depolarisations and measured as an increase in membrane capacitance on single β-cells was significantly reduced by 70 ± 10% (P<0.01, n=9) in the presence of 10 μM CFTRinh-172. We conclude that active CFTR is present in mouse pancreatic β-cells and that it has a crucial role in exocytosis of secretory granules in the β-cells.

Enhancement of glucagon secretion in mouse and human pancreatic alpha cells by protein kinase C (PKC) involves intracellular trafficking of PKCα and PKCδ

Protein kinase C (PKC) regulates exocytosis in various secretory cells. Here we studied intracellular translocation of the PKC isoenzymes PKCalpha and PKCdelta, and investigated how activation of PKC influences glucagon secretion in mouse and human pancreatic alpha cells. Glucagon release from intact islets was measured in static incubations, and the amounts released were determined by RIA. Exocytosis was monitored as increases in membrane capacitance using the patch-clamp technique. The expression of genes encoding PKC isoforms was analysed by real-time PCR. Intracellular PKC distribution was assessed by confocal microscopy. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated glucagon secretion from mouse and human islets about fivefold (p < 0.01). This stimulation was abolished by the PKC inhibitor bisindolylmaleimide (BIM). Whereas PMA potentiated exocytosis more than threefold (p < 0.001), BIM inhibited alpha cell exocytosis by 60% (p < 0.05). In mouse islets, the PKC isoenzymes, PKCalpha and PKCbeta1, were highly abundant, while in human islets PKCeta, PKCepsilon and PKCzeta were the dominant variants. PMA stimulation of human alpha cells correlated with the translocation of PKCalpha and PKCdelta from the cytosol to the cell periphery. In the mouse alpha cells, PKCdelta was similarly affected by PMA, whereas PKCalpha was already present at the cell membrane in the absence of PMA. This association of PKCalpha in alpha cells was principally dependent on Ca(2+) influx through the L-type Ca(2+) channel. PKC activation augments glucagon secretion in mouse and human alpha cells. This effect involves translocation of PKCalpha and PKCdelta to the plasma membrane, culminating in increased Ca(2+)-dependent exocytosis. In addition, we demonstrated that PKCalpha translocation and exocytosis exhibit differential Ca(2+) channel dependence.