Ion interaction at the pore of Lc-type Ca2+ channel is sufficient to mediate depolarization-induced exocytosis

The coupling of voltage-gated Ca2+ channel (VGCC) to exocytotic proteins suggests a regulatory function for the channel in depolarization-evoked exocytosis. To explore this possibility we have examined catecholamine secretion in PC12 and chromaffin cells. We found that replacing Ca2+ with La3+ or other lanthanide ions supported exocytosis in divalent ion-free solution. Cd2+, nifedipine, or verapamil inhibited depolarization-evoked secretion in La3+, indicating specific binding of La3+ at the pore of L-type VGCC, probably at the poly-glutamate (EEEE) locus. Lanthanide efficacy was stringently dependent on ionic radius with La3+>Ce3+>Pr3+, consistent with a size-selective binding interface of trivalent cations at the channel pore. La3+ inward currents were not detected and the highly sensitive La3+/fura-2 imaging assay (approximately 1 pm) detected no La3+ entry, cytosolic La3+ build-up, or alterations in cytosolic Ca2. These results provide strong evidence that occupancy of the pore of the channel by an impermeable cation leads to a conformational change that is transmitted to the exocytotic machinery upstream of intracellular cation build-up (intracellular Ca2+ concentration). Our model allows for a tight temporal and spatial coupling between the excitatory stimulation event and vesicle fusion. It challenges the conventional view that intracellular Ca2+ ion build-up via VGCC permeation is required to trigger secretion and establishes the VGCC as a plausible Ca2+ sensor protein in the process of neuroendocrine secretion.     

Removal of Ca2+ channel beta3 subunit enhances Ca2+ oscillation frequency and insulin exocytosis

An oscillatory increase in pancreatic beta cell cytoplasmic free Ca2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel beta3 subunit in the molecular regulation of these [Ca2+]i oscillations has now been clarified by using beta3 subunit-deficient beta cells. beta3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca2+]i oscillation frequency in beta cells lacking the beta3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the beta3 subunit negatively modulated InsP3-induced Ca2+ release, which is not paralleled by any effect on the voltage-gated L type Ca2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the beta3 subunit in the beta cell may constitute the basis for a novel diabetes therapy.     

The C2A domain of synaptotagmin alters the kinetics of voltage-gated Ca2+ channels Ca(v)1.2 (Lc-type) and Ca(v)2.3 (R-type)

Biochemical and genetic studies implicate synaptotagmin (Syt 1) as a Ca2+ sensor for neuronal and neuroendocrine neurosecretion. Calcium binding to Syt 1 occurs through two cytoplasmic repeats termed the C2A and C2B domains. In addition, the C2A domain of Syt 1 has calcium-independent properties required for neurotransmitter release. For example, mutation of a polylysine motif (residues 189-192) reverses the inhibitory effect of injected recombinant Syt 1 C2A fragment on neurotransmitter release from PC12 cells. Here we examined the requirement of the C2A polylysine motif for Syt 1 interaction with the cardiac Cav1.2 (L-type) and the neuronal Cav2.3 (R-type) voltage-gated Ca2+ channels, two channels required for neurotransmission. We find that the C2A polylysine motif presents a critical interaction surface with Cav1.2 and Cav2.3 since truncated Syt 1 containing a mutated motif (Syt 1*1-264) was ineffective at modifying the channel kinetics. Mutating the polylysine motif also abolished C2A binding to Lc753-893, the cytosolic interacting domain of Syt 1 at Cav1.2 1 subunit. Syt 1 and Syt 1* harboring the mutation at the KKKK motif modified channel activation, while Syt 1* only partially reversed the syntaxin 1A effects on channel activity. This mutation would interfere with the assembly of Syt 1/channel/syntaxin into an exocytotic unit. The functional interaction of the C2A polylysine domain with Cav1.2 and Cav2.3 is consistent with tethering of the secretory vesicle to the Ca2+ channel. It indicates that calcium-independent properties of Syt 1 regulate voltage-gated Ca2+ channels and contribute to the molecular events underlying transmitter release.     

Cations residing at the selectivity filter of the voltage-gated Ca2+-channel modify fusion-pore kinetics

Strontium (Sr(2+)), Barium (Ba(2+)) and Lanthanum (La(3+)) can substitute for Ca(2+) in driving synaptic transmission during membrane depolarization. Ion recognition at the polyglutamate motif (EEEE), comprising the channel selectivity-filter, during voltage-driven transitions, controls the kinetics of the voltage-gated calcium channel (VGCC) and its interactions with the synaptic proteins. We tested the effect of different charge carriers on evoked-release, as a means of exploring the involvement of VGCC in the fusion pore configuration. Employing amperometry recordings in single bovine chromaffin cells we show that the size of the fusion pore, designated by the 'foot'-amplitude, was increased when Ba(2+) substituted for Ca(2+) and decreased, with La(3+). The fusion pore stability, indicated by 'foot'-width, was decreased in La(3+). Also, the mean open time of the fusion pore (tau(fp)) was significantly lower in Sr(2+) and La(3+) compared to Ba(2+) and Ca(2+). These cations when occupying the selectivity filter reduced the spike frequency in the order of Ca(2+) > Sr(2+) > Ba(2+) > La(3+), which is parallel to the reduction in total catecholamine release. The correlation between ion binding at the selectivity filter and fusion pore properties supports a model in which the Ca(2+) channel regulates secretion through a site at the selectivity filter, upstream to cation entry into the cell.     

Glucose increases extracellular [Ca2+] in rat insulinoma (INS-1E) pseudoislets as measured with Ca2+-sensitive microelectrodes

Secretory granules of pancreatic β-cells contain high concentrations of Ca2+ ions that are co-released with insulin in the extracellular milieu upon activation of exocytosis. As a consequence, an increase in the extracellular Ca2+ concentration ([Ca2+]ext) in the microenvironment immediately surrounding β-cells should be expected following the exocytotic event. Using Ca2+-selective microelectrodes we show here that both high glucose and non-nutrient insulinotropic agents elicit a reversible increase of [Ca2+]ext within rat insulinoma (INS-1E) β-cells pseudoislets. The glucose-induced increases in [Ca2+]ext are blocked by pretreatment with different Ca2+ channel blockers. Physiological agonists acting as positive or negative modulators of the insulin secretion and drugs known to intersect the secretory machinery at different levels also induce [Ca2+]ext changes as predicted on the basis of their described action on insulin secretion. Finally, the glucose-induced [Ca2+]ext increase is strongly inhibited after disruption of the actin web, indicating that the dynamic [Ca2+]ext changes recorded in INS-1E pseudoislets by Ca2+-selective microelectrodes occur mainly as a consequence of exocytosis of Ca2+-rich granules. In conclusion, our data directly demonstrate that the extracellular spaces surrounding β-cells constitute a restricted domain where Ca2+ is co-released during insulin exocytosis, creating the basis for an autocrine/paracrine cell-to-cell communication system via extracellular Ca2+ sensors.     

The molecular chaperone hsp70 interacts with the cytosolic II-III loop of the Cav2.3 E-type voltage-gated Ca2+ channel.

Multiple types of voltage-activated Ca2+ channels (T, L, N, P, Q, R type) coexist in excitable cells and participate in synaptic differentiation, secretion, transmitter release, and neuronal plasticity. Ca2+ ions entering cells trigger these events through their interaction with the ion channel itself or through Ca2+ binding to target proteins initiating signalling cascades at cytosolic loops of the ion conducting subunit (Cava1). These loops interact with target proteins in a Ca2+-dependent or independent manner. In Cav2.3-containing channels the cytosolic linker between domains II and III confers a novel Ca2+ sensitivity to E-type Ca2+ channels including phorbol ester sensitive signalling via protein kinase C (PKC) in Cav2.3 transfected HEK-293 cells. To understand Ca2+ and phorbol ester mediated activation of Cav2.3 Ca2+ channels, protein interaction partners of the II-III loop were identified. FLAG-tagged II-III - loop of human Cav2.3 was over-expressed in HEK 293 cells, and the molecular chaperone hsp70, which is known to interact with PKC, was identified as a novel functional interaction partner. Immunopurified II-III loop-protein of neuronal and endocrine Cav2.3 splice variants stimulate autophosphorylation of PKCa, leading to the suggestion that hsp70--binding to the II-III loop--may act as an adaptor for Ca2+ dependent targeting of PKC to E-type Ca2+ channels.     

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice

Insulin is secreted from pancreatic beta cells in response to an elevation of cytoplasmic Ca(2+) resulting from enhanced Ca(2+) influx through voltage-gated Ca(2+) channels. Mouse beta cells express several types of Ca(2+) channel (L-, R- and possibly P/Q-type). beta cell-selective ablation of the gene encoding the L-type Ca(2+) channel subtype Ca(v)1.2 (betaCa(v)1.2(-/-) mouse) decreased the whole-cell Ca(2+) current by only approximately 45%, but almost abolished first-phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca(2+) handling and glucose-induced electrical activity. However, high-resolution capacitance measurements of exocytosis in single beta cells revealed that the loss of first-phase insulin secretion in the betaCa(v)1.2(-/-) mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Ca(v)1.2 channel. Thus, the conduit of Ca(2+) entry determines the ability of the cation to elicit secretion.     

Functional specialization of presynaptic Cav2.3 Ca2+ channels

Ca2+ influx into presynaptic terminals via voltage-dependent Ca2+ channels triggers fast neurotransmitter release as well as different forms of synaptic plasticity. Using electrophysiological and genetic techniques we demonstrate that presynaptic Ca2+ entry through Cav2.3 subunits contributes to the induction of mossy fiber LTP and posttetanic potentiation by brief trains of presynaptic action potentials while they do not play a role in fast synaptic transmission, paired-pulse facilitation, or frequency facilitation. This functional specialization is most likely achieved by a localization remote from the release machinery and by a Cav2.3 channel-dependent facilitation of presynaptic Ca2+ influx. Thus, the presence of Cav2.3 channels boosts the accumulation of presynaptic Ca2+ triggering presynaptic LTP and posttetanic potentiation without affecting the low release probability that is a prerequisite for the enormous plasticity displayed by mossy fiber synapses.     

Glycerotoxin stimulates neurotransmitter release from N-type Ca2+ channel expressing neurons

Glycerotoxin (GLTx) is capable of stimulating neurotransmitter release at the frog neuromuscular junction by directly interacting with N-type Ca2+ (Cav2.2) channels. Here we have utilized GLTx as a tool to investigate the functionality of Cav2.2 channels in various mammalian neuronal preparations. We first adapted a fluorescent-based high-throughput assay to monitor glutamate release from rat cortical synaptosomes. GLTx potently stimulates glutamate secretion and Ca2+ influx in synaptosomes with an EC50 of 50 pm. Both these effects were prevented using selective Cav2.2 channel blockers suggesting the functional involvement of Cav2.2 channels in mediating glutamate release in this system. We further show that both Cav2.1 (P/Q-type) and Cav2.2 channels contribute equally to depolarization-induced glutamate release. We then investigated the functionality of Cav2.2 channels at the neonatal rat neuromuscular junction. GLTx enhances both spontaneous and evoked neurotransmitter release causing a significant increase in the frequency of postsynaptic action potentials. These effects were blocked by specific Cav2.2 channel blockers demonstrating that either GLTx or its derivatives could be used to selectively enhance the neurotransmitter release from Cav2.2-expressing mammalian neurons.     

Chronic exposure to beta-hydroxybutyrate inhibits glucose-induced insulin release from pancreatic islets by decreasing NADH contents

To investigate the effects of chronic exposure to ketone bodies on glucose-induced insulin secretion, we evaluated insulin release, intracellular Ca2+ and metabolism, and Ca2+ efficacy of the exocytotic system in rat pancreatic islets. Fifteen-hour exposure to 5 mM d-beta-hydroxybutyrate (HB) reduced high glucose-induced insulin secretion and augmented basal insulin secretion. Augmentation of basal release was derived from promoting the Ca2+-independent and ATP-independent component of insulin release, which was suppressed by the GDP analog. Chronic exposure to HB affected mostly the second phase of glucose-induced biphasic secretion. Dynamic experiments showed that insulin release and NAD(P)H fluorescence were lower, although the intracellular Ca2+ concentration ([Ca2+](i)) was not affected 10 min after exposure to high glucose. Additionally, [Ca2+](i) efficacy in exocytotic system at clamped concentrations of ATP was not affected. NADH content, ATP content, and ATP-to-ADP ratio in the HB-cultured islets in the presence of high glucose were lower, whereas glucose utilization and oxidation were not affected. Mitochondrial ATP production shows that the respiratory chain downstream of complex II is not affected by chronic exposure to HB, and that the decrease in ATP production is due to decreased NADH content in the mitochondrial matrix. Chronic exposure to HB suppresses glucose-induced insulin secretion by lowering the ATP level, at least partly by inhibiting ATP production by reducing the supply of NADH to the respiratory chain. Glucose-induced insulin release in the presence of aminooxyacetate was not reduced, which implies that chronic exposure to HB affects the malate/aspartate shuttle and thus reduces NADH supply to mitochondria.     

Requirement for aralar and its Ca2+-binding sites in Ca2+ signal transduction in mitochondria from INS-1 clonal beta-cells

Aralar, the mitochondrial aspartate-glutamate carrier present in beta-cells, is a component of the malate-aspartate NADH shuttle (MAS). MAS is activated by Ca2+ in mitochondria from tissues with aralar as the only AGC isoform with an S0.5 of approximately 300 nm. We have studied the role of aralar and its Ca2+-binding EF-hand motifs in glucose-induced mitochondrial NAD(P)H generation by two-photon microscopy imaging in INS-1 beta-cells lacking aralar or expressing aralar mutants blocked for Ca2+ binding. Aralar knock-down in INS-1 beta-cell lines resulted in undetectable levels of aralar protein and complete loss of MAS activity in isolated mitochondria and in a 25% decrease in glucose-stimulated insulin secretion. MAS activity in mitochondria from INS-1 cells was activated 2-3-fold by extramitochondrial Ca2+, whereas aralar mutants were Ca2+ insensitive. In Ca2+-free medium, glucose-induced increases in mitochondrial NAD(P)H were small (1.3-fold) and unchanged regardless of the lack of aralar. In the presence of 1.5 mm Ca2+, glucose induced robust increases in mitochondrial NAD(P)H (approximately 2-fold) in cell lines with wild-type or mutant aralar. There was a approximately 20% reduction in NAD(P)H response in cells lacking aralar, illustrating the importance of MAS in glucose action. When small Ca2+ signals that resulted in extremely small mitochondrial Ca2+ transients were induced in the presence of glucose, the rise in mitochondrial NAD(P)H was maintained in cells with wild-type aralar but was reduced by approximately 50% in cells lacking or expressing mutant aralar. These results indicate that 1) glucose-induced activation of MAS requires Ca2+ potentiation; 2) Ca2+ activation of MAS represents a larger fraction of glucose-induced mitochondrial NAD(P)H production under conditions where suboptimal Ca2+ signals are associated with a glucose challenge (50 versus 20%, respectively); 3) inactivation of EF-hand motifs in aralar prevents activation of MAS by small Ca2+ signals. The results suggest a possible role for aralar and MAS in priming the beta-cell by Ca2+-mobilizing neurotransmitter or hormones.     

Cations Residing at the Selectivity Filter of the Voltage-Gated Ca2+-Channel Modify Fusion-Pore Kinetics

Strontium (Sr²⁺), Barium (Ba²⁺), and Lanthanum (La³⁺) can substitute for Ca²⁺ in driving synaptic transmission during membrane depolarization. Ion recognition at the polyglutamate motif (EEEE), comprising the channel selectivity-filter, during voltage-driven transitions, controls the kinetics of the voltage-gated calcium channel (VGCC) and its interactions with the synaptic proteins. We tested the effect of different charge carriers on evoked-release, as a means of exploring the involvement of VGCC in the fusion pore configuration. Employing amperometry recordings in single bovine chromaffin cells we show that the size of the fusion pore, designated by the 'foot'-amplitude, was increased when Ba²⁺ substituted for Ca²⁺ and decreased, with La³⁺. The fusion pore stability, indicated by 'foot'-width, was decreased in La³⁺. Also, the mean open time of the fusion pore (tfp) was significantly lower in Sr²⁺ and La³⁺ compared to Ba²⁺ and Ca²⁺. These cations when occupying the selectivity filter reduced the spike frequency in the order of Ca²⁺ > Sr²⁺ > Ba²⁺ > La³⁺, which is parallel to the reduction in total catecholamine release. The correlation between ion binding at the selectivity filter and fusion pore properties supports a model in which the Ca²⁺ channel regulates secretion through a site at the selectivity filter, upstream to cation entry into the cell.     

Formation of an endophilin-Ca2+ channel complex is critical for clathrin-mediated synaptic vesicle endocytosis

A tight balance between synaptic vesicle exocytosis and endocytosis is fundamental to maintaining synaptic structure and function. Calcium influx through voltage-gated Ca2+ channels is crucial in regulating synaptic vesicle exocytosis. However, much less is known about how Ca2+ regulates vesicle endocytosis or how the endocytic machinery becomes enriched at the nerve terminal. We report here a direct interaction between voltage-gated Ca2+ channels and endophilin, a key regulator of clathrin-mediated synaptic vesicle endocytosis. Formation of the endophlin-Ca2+ channel complex is Ca2+ dependent. The primary Ca2+ binding domain resides within endophilin and regulates both endophilin-Ca2+ channel and endophilin-dynamin complexes. Introduction into hippocampal neurons of a dominant-negative endophilin construct, which constitutively binds to Ca2+ channels, significantly reduces endocytosis-mediated uptake of FM 4-64 dye without abolishing exocytosis. These results suggest an important role for Ca2+ channels in coordinating synaptic vesicle recycling by directly coupling to both exocytotic and endocytic machineries.     

Caldendrin, a neuron-specific modulator of Cav/1.2 (L-type) Ca2+ channels

EF-hand Ca2+-binding proteins such as calmodulin and CaBP1 have emerged as important regulatory subunits of voltage-gated Ca2+ channels. Here, we show that caldendrin, a variant of CaBP1 enriched in the brain, interacts with and distinctly modulates Cav1.2 (L-type) voltage-gated Ca2+ channels relative to other Ca2+-binding proteins. Caldendrin binds to the C-terminal IQ-domain of the pore-forming alpha1-subunit of Cav1.2 (alpha(1)1.2) and competitively displaces calmodulin and CaBP1 from this site. Compared with CaBP1, caldendrin causes a more modest suppression of Ca2+-dependent inactivation of Cav1.2 through a different subset of molecular determinants. Caldendrin does not bind to the N-terminal domain of alpha11.2, a site that is critical for functional interactions of the channel with CaBP1. Deletion of the N-terminal domain inhibits CaBP1, but spares caldendrin modulation of Cav1.2 inactivation. In contrast, mutations of the IQ-domain abolish physical and functional interactions of caldendrin and Cav1.2, but do not prevent channel modulation by CaBP1. Using antibodies specific for caldendrin and Cav1.2, we show that caldendrin coimmunoprecipitates with Cav1.2 from the brain and colocalizes with Cav1.2 in somatodendritic puncta of cortical neurons in culture. Our findings reveal functional diversity within related Ca2+-binding proteins, which may enhance the specificity of Ca2+ signaling by Cav1.2 channels in different cellular contexts.     

Properties of voltage-gated Ca2+ currents measured from mouse pancreatic beta-cells in situ

We used the single-microelectrode voltage-clamp technique to record ionic currents from pancreatic beta-cells within intact mouse islets of Langerhans at 37 degrees C, the typical preparation for studies of glucose-induced "bursting" electrical activity. Cells were impaled with intracellular microelectrodes, and voltage pulses were applied in the presence of tetraethylammonium. Under these conditions, a voltage-dependent Ca2+ current (I(Cav)), containing L-type and non-L-type components, was observed. The current measured in situ was larger than that measured in single cells with whole-cell patch clamping, particularly at membrane potentials corresponding to the action potentials of beta-cell electrical activity. The temperature dependence of I(Cav) was not sufficient to account for the difference in size of the currents recorded with the two methods. During prolonged pulses, the voltage-dependent Ca2+ current measured in situ displayed both rapid and slow components of inactivation. The rapid component was Ca2+-dependent and was inhibited by the membrane-permeable Ca2+ chelator, BAPTA-AM. The effect of BAPTA-AM on beta-cell electrical activity then demonstrated that Ca2+-dependent inactivation of I(Cav) contributes to action potential repolarization and to control of burst frequency. Our results demonstrate the utility of voltage clamping beta-cells in situ for determining the roles of ion channels in electrical activity and insulin secretion.     

Neuropeptide W in the rat pancreas: potentiation of glucose-induced insulin release and Ca2+ influx through L-type Ca2+ channels in beta-cells and localization in islets

Neuropeptide W (NPW) is a regulatory peptide that acts via two subtypes of G protein-coupled receptors, GPR7 and GPR8. Evidence has been provided that NPW is involved in the central regulation of energy homeostasis and feeding behavior. In this study, we examined the effects of NPW on insulin release and localization of NPW in the rat pancreas. NPW (10-100 nM) significantly increased insulin release in the presence of 8.3 mM, but not 2.8 mM, glucose in the isolated rat islets. By fura-2 microfluorometry, NPW (1-100 nM) concentration-dependently increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) at 8.3 mM glucose in rat single beta-cells. The NPW-induced [Ca(2+)](i) increase was abolished under external Ca(2+)-free conditions and by an L-type Ca(2+) channel blocker nifedipine (10 microM). RT-PCR analysis revealed that mRNA for NPW was expressed in the rat pancreas and hypothalamus. Double immunohistochemical analysis showed that NPW-immunoreactivity was found in islets and co-localized with insulin-containing beta-cells, but not glucagon-containing alpha-cells and somatostatin-containing delta-cells. These results suggest that NPW could serve as a local modulator of glucose-induced insulin release in rat islets. NPW directly activates beta-cells to enhance Ca(2+) influx through voltage-dependent L-type Ca(2+) channels and potentiates glucose-induced insulin release.     

Ca2+ signaling and exocytosis in pituitary corticotropes

The secretion of adrenocorticotrophin (ACTH) from corticotropes is a key component in the endocrine response to stress. The resting potential of corticotropes is set by the basal activities of TWIK-related K(+) (TREK)-1 channel. Corticotrophin-releasing hormone (CRH), the major ACTH secretagogue, closes the background TREK-1 channels via the cAMP-dependent pathway, resulting in depolarization and a sustained rise in cytosolic [Ca(2+)] ([Ca(2+)](i)). By contrast, arginine vasopressin and norepinephrine evoke Ca(2+) release from the inositol trisphosphate (IP(3))-sensitive store, resulting in the activation of small conductance Ca(2+)-activated K(+) channels and hyperpolarization. Following [Ca(2+)](i) rise, cytosolic Ca(2+) is taken into the mitochondria via the uniporter. Mitochondrial inhibition slows the decay of the Ca(2+) signal and enhances the depolarization-triggered exocytotic response. Both voltage-gated Ca(2+) channel activation and intracellular Ca(2+) release generate spatial Ca(2+) gradients near the exocytic sites such that the local [Ca(2+)] is ~3-fold higher than the average [Ca(2+)](i). The stimulation of mitochondrial metabolism during the agonist-induced Ca(2+) signal and the robust endocytosis following stimulated exocytosis enable corticotropes to maintain sustained secretion during the diurnal ACTH surge. Arachidonic acid (AA) which is generated during CRH stimulation activates TREK-1 channels and causes hyperpolarization. Thus, corticotropes may regulate ACTH release via an autocrine feedback mechanism.     

Calmodulin inhibits inositol trisphosphate-induced Ca2+ mobilization from the endoplasmic reticulum of islets

IP3-induced Ca2+ release from the endoplasmic reticulum (ER) of islets is believed to be a key intracellular event in glucose-induced insulin secretion. Calmodulin was shown to increase ATP-dependent Ca2+ steady-state and inhibit by 57.2% IP3-induced Ca2+ mobilization from the ER. Conversely, the calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide (W-7), induced Ca2+ release from the ER. The combination of W-7 (100 microM) and IP3 (10 microM), resulted in a greater release of Ca2+ from the ER than either W-7 or IP3 alone. W-7 was shown not to affect the structural integrity of the ER. Our results suggest that IP3-induced Ca2+ release from the ER is regulated by a calmodulin-dependent process.     

Intracellular Ca2+ mobilization by arachidonic acid. Comparison with myo-inositol 1,4,5-trisphosphate in isolated pancreatic islets

Previous studies have demonstrated that myo-inositol 1,4,5-trisphosphate (IP3) mobilizes Ca2+ from the endoplasmic reticulum (ER) of digitonin-permeabilized islets and that an increase in intracellular free Ca2+ stimulates insulin release. Furthermore, glucose stimulates arachidonic acid metabolism in islets. In digitonin-permeabilized islets, exogenous arachidonic acid at concentrations between 1.25 to 10 microM elicited significant Ca2+ release from the ER at a free Ca2+ concentration of 0.1 microM. Arachidonic acid-induced Ca2+ release was not due to the metabolites of arachidonic acid. Arachidonic acid induced a rapid release of Ca2+ within 2 min. Comparison of arachidonic acid-induced Ca2+ release with IP3-induced Ca2+ release revealed a similar molar potency of arachidonic acid and IP3. The combination of both arachidonic acid and IP3 resulted in a greater effect on Ca2+ mobilization from the ER than either compound alone. The mass of endogenous arachidonic acid released by islets incubated with 28 mM glucose was measured by mass spectrometric methods and was found to be sufficient to achieve arachidonic acid concentrations equal to or exceeding those required to induce release of Ca2+ sequestered in the ER. These observations indicate that glucose-induced arachidonic acid release could participate in glucose-induced Ca2+ mobilization and insulin secretion by pancreatic islets, possibly in cooperation with IP3.