Pulsatile Basal Insulin Secretion Is Driven by Glycolytic Oscillations.

In fasted and fed states, blood insulin levels are oscillatory. While this phenomenon is well studied at high glucose levels, comparatively little is known about its origin under basal conditions. We propose a possible mechanism for basal insulin oscillations based on oscillations in glycolysis, demonstrated using an established mathematical model. At high glucose, this is superseded by a calcium-dependent mechanism.

Deletion of Ia-2 and/or Ia-2ß in mice decreases insulin secretion by reducing the number of dense core vesicles.

AIMS/HYPOTHESIS: Islet antigen 2 (IA-2) and IA-2ß are dense core vesicle (DCV) transmembrane proteins and major autoantigens in type 1 diabetes. The present experiments were initiated to test the hypothesis that the knockout of the genes encoding these proteins impairs the secretion of insulin by reducing the number of DCV. METHODS: Insulin secretion, content and DCV number were evaluated in islets from single knockout (Ia-2 [also known as Ptprn] KO, Ia-2ß [also known as Ptprn2] KO) and double knockout (DKO) mice by a variety of techniques including electron and two-photon microscopy, membrane capacitance, Ca(2+) currents, DCV half-life, lysosome number and size and autophagy. RESULTS: Islets from single and DKO mice all showed a significant decrease in insulin content, insulin secretion and the number and half-life of DCV (p < 0.05 to 0.001). Exocytosis as evaluated by two-photon microscopy, membrane capacitance and Ca(2+) currents supports these findings. Electron microscopy of islets from KO mice revealed a marked increase (p < 0.05 to 0.001) in the number and size of lysosomes and enzymatic studies showed an increase in cathepsin D activity (p < 0.01). LC3 protein, an indicator of autophagy, also was increased in islets of KO compared with wild-type mice (p < 0.05 to 0.01) suggesting that autophagy might be involved in the deletion of DCV. CONCLUSIONS/INTERPRETATION: We conclude that the decrease in insulin content and secretion, resulting from the deletion of Ia-2 and/or Ia-2ß, is due to a decrease in the number of DCV.

An integrated instrument for rapidly deforming living cells using rapid pressure pulses and simultaneously monitoring applied strain in near real time.

Because many types of living cells are sensitive to applied strain, different in vitro models have been designed to elucidate the cellular and subcellular processes that respond to mechanical deformation at both the cell and tissue level. Our focus was to improve upon an already established strain system to make it capable of independently monitoring the deflection and applied pressure delivered to specific wells of a commercially available, deformable multiwell culture plate. To accomplish this, we devised a custom frame that was capable of mounting deformable 6 or 24 well plates, a pressurization system that could load wells within the plates, and a camera-based imaging system which was capable of capturing strain responses at a sufficiently high frame rate. The system used a user defined program constructed in Labview(®) to trigger plate pressurization while simultaneously allowing the deflection of the silicone elastomeric plate bottoms to be imaged in near real time. With this system, up to six wells could be pulsed simultaneously using compressed air or nitrogen. Digital image capture allowed near-real time monitoring of applied strain, strain rate, and the cell loading profiles. Although our ultimate goal is to determine how different strain rates applied to neurons modulates their intrinsic biochemical cascades, the same platform technology could be readily applied to other systems. Combining commercially available, deformable multiwell plates with a simple instrument having the monitoring capabilities described here should permit near real time calculations of stretch-induced membrane strain in multiple wells in real time for a wide variety of applications, including high throughput drug screening.

Niflumic acid-sensitive ion channels play an important role in the induction of glucose-stimulated insulin secretion by cyclic AMP in mice.

AIMS/HYPOTHESIS: We have previously reported that glucose-stimulated insulin secretion (GSIS) is induced by glucagon-like peptide-1 (GLP-1) in mice lacking ATP-sensitive K(+) (K(ATP)) channels (Kir6.2(-/-) mice [up-to-date symbol for Kir6.2 gene is Kcnj11]), in which glucose alone does not trigger insulin secretion. This study aimed to clarify the mechanism involved in the induction of GSIS by GLP-1. METHODS: Pancreas perfusion experiments were performed using wild-type (Kir6.2(+/+)) or Kir6.2(-/-) mice. Glucose concentrations were either changed abruptly from 2.8 to 16.7 mmol/l or increased stepwise (1.4 mmol/l per step) from 2.8 to 12.5 mmol/l. Electrophysiological experiments were performed using pancreatic beta cells isolated from Kir6.2(-/-) mice or clonal pancreatic beta cells (MIN6 cells) after pharmacologically inhibiting their K(ATP) channels with glibenclamide. RESULTS: The combination of cyclic AMP plus 16.7 mmol/l glucose evoked insulin secretion in Kir6.2(-/-) pancreases where glucose alone was ineffective as a secretagogue. The secretion was blocked by the application of niflumic acid. In K(ATP) channel-inactivated MIN6 cells, niflumic acid similarly inhibited the membrane depolarisation caused by cAMP plus glucose. Surprisingly, stepwise increases of glucose concentration triggered insulin secretion only in the presence of cAMP or GLP-1 in Kir6.2(+/+), as in Kir6.2(-/-) pancreases. CONCLUSIONS/INTERPRETATION: Niflumic acid-sensitive ion channels participate in the induction of GSIS by cyclic AMP in Kir6.2(-/-) beta cells. Cyclic AMP thus not only acts as a potentiator of insulin secretion, but appears to be permissive for GSIS via novel, niflumic acid-sensitive ion channels. This mechanism may be physiologically important for triggering insulin secretion when the plasma glucose concentration increases gradually rather than abruptly.

Calcium-activated K+ channels of mouse beta-cells are controlled by both store and cytoplasmic Ca2+: experimental and theoretical studies.

A novel calcium-dependent potassium current (K(slow)) that slowly activates in response to a simulated islet burst was identified recently in mouse pancreatic beta-cells (Göpel, S.O., T. Kanno, S. Barg, L. Eliasson, J. Galvanovskis, E. Renström, and P. Rorsman. 1999. J. Gen. Physiol. 114:759-769). K(slow) activation may help terminate the cyclic bursts of Ca(2+)-dependent action potentials that drive Ca(2+) influx and insulin secretion in beta-cells. Here, we report that when [Ca(2+)](i) handling was disrupted by blocking Ca(2+) uptake into the ER with two separate agents reported to block the sarco/endoplasmic calcium ATPase (SERCA), thapsigargin (1-5 microM) or insulin (200 nM), K(slow) was transiently potentiated and then inhibited. K(slow) amplitude could also be inhibited by increasing extracellular glucose concentration from 5 to 10 mM. The biphasic modulation of K(slow) by SERCA blockers could not be explained by a minimal mathematical model in which [Ca(2+)](i) is divided between two compartments, the cytosol and the ER, and K(slow) activation mirrors changes in cytosolic calcium induced by the burst protocol. However, the experimental findings were reproduced by a model in which K(slow) activation is mediated by a localized pool of [Ca(2+)] in a subspace located between the ER and the plasma membrane. In this model, the subspace [Ca(2+)] follows changes in cytosolic [Ca(2+)] but with a gradient that reflects Ca(2+) efflux from the ER. Slow modulation of this gradient as the ER empties and fills may enhance the role of K(slow) and [Ca(2+)] handling in influencing beta-cell electrical activity and insulin secretion.

Insulin activates ATP-sensitive K(+) channels in pancreatic beta-cells through a phosphatidylinositol 3-kinase-dependent pathway.

Insulin is known to regulate pancreatic beta-cell function through the activation of cell surface insulin receptors, phosphorylation of insulin receptor substrate (IRS)-1 and -2, and activation of phosphatidylinositol (PI) 3-kinase. However, an acute effect of insulin in modulating beta-cell electrical activity and its underlying ionic currents has not been reported. Using the perforated patch clamp technique, we found that insulin (1-600 nmol/l) but not IGF-1 (100 nmol/l) reversibly hyperpolarized single mouse beta-cells and inhibited their electrical activity. The dose-response relationship for insulin yielded a maximal change (mean +/- SE) in membrane potential of -13.6 +/- 2.0 mV (P < 0.001) and a 50% effective dose of 25.9 +/- 0.1 nmol/l (n = 63). Exposing patched beta-cells within intact islets to 200 nmol/l insulin produced similar results, hyperpolarizing islets from -47.7 +/- 3.3 to -65.6 +/- 3.7 mV (P < 0.0001, n = 11). In single cells, insulin-induced hyperpolarization was associated with a threefold increase in whole-cell conductance from 0.6 +/- 0.1 to 1.7 +/- 0.2 nS (P < 0.001, n = 10) and a shift in the current reversal potential from -25.7 +/- 2.5 to -63.7 +/- 1.0 mV (P < 0.001 vs. control, n = 9; calculated K(+) equilibrium potential = -90 mV). The effects of insulin were reversed by tolbutamide, which decreased cell conductance to 0.5 +/- 0.1 nS and shifted the current reversal potential to -25.2 +/- 2.3 mV. Insulin-induced beta-cell hyperpolarization was sufficient to abolish intracellular calcium concentration ([Ca(2+)](i)) oscillations measured in pancreatic islets exposed to 10 mmol/l glucose. The application of 100 nmol/l wortmannin to inactivate PI 3-kinase, a key enzyme in insulin signaling, was found to reverse the effects of 100 nmol/l insulin. In cell-attached patches, single ATP-sensitive K(+) (K(ATP)) channels were activated by bath-applied insulin and subsequently inhibited by wortmannin. Our data thus demonstrate that insulin activates the K(ATP) channels of single mouse pancreatic beta-cells and islets, resulting in membrane hyperpolarization, an inhibition of electrical activity, and the abolition of [Ca(2+)](i) oscillations. We thus propose that locally released insulin might serve as a negative feedback signal within the islet under physiological conditions.

Chloride channels regulate HIT cell volume but cannot fully account for swelling-induced insulin secretion.

Insulin-secreting pancreatic islet beta-cells possess anion-permeable Cl- channels (I(Cl,islet)) that are swelling-activated, but the role of these channels in the cells is unclear. The Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and niflumic acid were evaluated for their ability to inhibit I(Cl,islet) in clonal beta-cells (HIT cells). Both drugs blocked the channel, but the blockade due to niflumic acid was less voltage-dependent than the blockade due to DIDS. HIT cell volume initially increased in hypotonic solution and was followed by a regulatory volume decrease (RVD). The addition of niflumic acid and, to a lesser extent, DIDS to the hypotonic solution potentiated swelling and blocked the RVD. In isotonic solution, niflumic acid produced swelling, suggesting that islet Cl- channels are activated under basal conditions. The channel blockers glyburide, gadolinium, or tetraethylammonium-Cl did not alter hypotonic-induced swelling or volume regulation. The Na/K/2Cl transport blocker furosemide produced cell shrinkage in isotonic solution and blocked cell swelling normally induced by hypotonic solution. Perifused HIT cells secreted insulin when challenged with hypotonic solutions. However, this could not be completely attributed to I(Cl,islet)-mediated depolarization, because secretion persisted even when Cl- channels were fully blocked. To test whether blocker-resistant secretion occurred via a distal pathway, distal secretion was isolated using 50 mmol/l potassium and diazoxide. Under these conditions, glucose-dependent secretion was blunted, but hypotonically induced secretion persisted, even with Cl- channel blockers present. These results suggest that beta-cell swelling stimulates insulin secretion primarily via a distal I(Cl,islet)-independent mechanism, as has been proposed for K(ATP)-independent glucose- and sulfonylurea-stimulated insulin secretion. Reverse transcriptase-polymerase chain reaction of HIT cell mRNA identified a CLC-3 transcript in HIT cells. In other systems, CLC-3 is believed to mediate swelling-induced outwardly rectifying Cl- channels. This suggests that the proximal effects of swelling to regulate cell volume may be mediated by CLC-3 or a closely related Cl- channel.

The phantom burster model for pancreatic beta-cells.

Pancreatic beta-cells exhibit bursting oscillations with a wide range of periods. Whereas periods in isolated cells are generally either a few seconds or a few minutes, in intact islets of Langerhans they are intermediate (10-60 s). We develop a mathematical model for beta-cell electrical activity capable of generating this wide range of bursting oscillations. Unlike previous models, bursting is driven by the interaction of two slow processes, one with a relatively small time constant (1-5 s) and the other with a much larger time constant (1-2 min). Bursting on the intermediate time scale is generated without need for a slow process having an intermediate time constant, hence phantom bursting. The model suggests that isolated cells exhibiting a fast pattern may nonetheless possess slower processes that can be brought out by injecting suitable exogenous currents. Guided by this, we devise an experimental protocol using the dynamic clamp technique that reliably elicits islet-like, medium period oscillations from isolated cells. Finally, we show that strong electrical coupling between a fast burster and a slow burster can produce synchronized medium bursting, suggesting that islets may be composed of cells that are intrinsically either fast or slow, with few or none that are intrinsically medium.

Localized calcium influx in pancreatic beta-cells: its significance for Ca2+-dependent insulin secretion from the islets of Langerhans.

Ca2+ influx through voltage-dependent Ca2+ channels plays a crucial role in stimulus-secretion coupling in pancreatic islet beta-cells. Molecular and physiologic studies have identified multiple Ca2+ channel subtypes in rodent islets and insulin-secreting cell lines. The differential targeting of Ca2+ channel subtypes to the vicinity of the insulin secretory apparatus is likely to account for their selective coupling to glucose-dependent insulin secretion. In this article, I review these studies. In addition, I discuss temporal and spatial aspects of Ca2+ signaling in beta-cells, the former involving the oscillatory activation of Ca2+ channels during glucose-induced electrical bursting, and the latter involving [Ca2+]i elevation in restricted microscopic "domains," as well as direct interactions between Ca2+ channels and secretory SNARE proteins. Finally, I review the evidence supporting a possible role for Ca2+ release from the endoplasmic reticulum in glucose-dependent insulin secretion, and evidence to support the existence of novel Ca2+ entry pathways. I also show that the beta-cell has an elaborate and complex set of [Ca2+]i signaling mechanisms that are capable of generating diverse and extremely precise [Ca2+]i patterns. These signals, in turn, are exquisitely coupled in space and time to the beta-cell secretory machinery to produce the precise minute-to-minute control of insulin secretion necessary for body energy homeostasis.

Cannabinoid receptors can activate and inhibit G protein-coupled inwardly rectifying potassium channels in a xenopus oocyte expression system.

In this study, we focused on the pharmacological characterization of cannabinoid receptor coupling to G protein-gated inwardly rectifying potassium (GIRK) channels. Cannabinoids were tested on Xenopus laevis oocytes coexpressing the CB(1) receptor and GIRK1 and GIRK4 channels (CB(1)/GIRK1/4) or the CB(2) receptor and GIRK1/4 channels (CB(2)/GIRK1/4). WIN 55,212-2 enhanced currents carried by GIRK channels in the CB(1)/GIRK1/4 and CB(2)/GIRK1/4 system; however, the CB(2) receptor did not couple efficiently to GIRK1/4 channels. In the CB(1)/GIRK1/4 system, WIN 55,212-2 was the most efficacious compound tested. CP 55,940 and anandamide acted as partial agonists. The rank order of potency was CP 55,940 > WIN 55,212-2 = anandamide. The CB(1)-selective antagonist SR141716A alone acted as a inverse agonist by inhibiting GIRK currents in oocytes expressing CB(1)/GIRK1/4, suggesting the CB(1) receptor is constitutively activated. A conserved aspartate residue, which was previously shown to be critical for G protein coupling in cannabinoid receptors, was mutated (to asparagine, D163N) and analyzed. Oocytes coexpressing CB(1)/GIRK1/4 or D163N/GIRK1/4 were compared. The potency of WIN 55, 212-2 at the mutant receptor was similar to wild type, but its efficacy was substantially reduced. CP 55,940 did not elicit currents in oocytes expressing D163N/GIRK1/4. In summary, it appears the CB(1) and CB(2) receptors couple differently to GIRK1/4 channels. In the CB(1)/GIRK1/4 system, cannabinoids evaluated demonstrated the ability to enhance or inhibit GIRK currents. Furthermore, a conserved aspartate residue in the CB(1) receptor is required for normal communication with GIRK channels in oocytes demonstrating the interaction between receptor and channels is G protein dependent.

Enhancement of AMPA-mediated current after traumatic injury in cortical neurons.

Overactivation of ionotropic glutamate receptors has been implicated in the pathophysiology of traumatic brain injury. Using an in vitro cell injury model, we examined the effects of stretch-induced traumatic injury on the AMPA subtype of ionotropic glutamate receptors in cultured neonatal cortical neurons. Recordings made using the whole-cell patch-clamp technique revealed that a subpopulation of injured neurons exhibited an increased current in response to AMPA. The current-voltage relationship of these injured neurons showed an increased slope conductance but no change in reversal potential compared with uninjured neurons. Additionally, the EC(50) values of uninjured and injured neurons were nearly identical. Thus, current potentiation was not caused by changes in the voltage-dependence, ion selectivity, or apparent agonist affinity of the AMPA channel. AMPA-elicited current could also be fully inhibited by the application of selective AMPA receptor antagonists, thereby excluding the possibility that current potentiation in injured neurons was caused by the activation of other, nondesensitizing receptors. The difference in current densities between control and injured neurons was abolished when AMPA receptor desensitization was inhibited by the coapplication of AMPA and cyclothiazide or by the use of kainate as an agonist, suggesting that mechanical injury alters AMPA receptor desensitization. Reduction of AMPA receptor desensitization after brain injury would be expected to further exacerbate the effects of increased postinjury extracellular glutamate and contribute to trauma-related cell loss and dysfunctional synaptic information processing.

Modulation of the bursting properties of single mouse pancreatic beta-cells by artificial conductances.

Glucose triggers bursting activity in pancreatic islets, which mediates the Ca2+ uptake that triggers insulin secretion. Aside from the channel mechanism responsible for bursting, which remains unsettled, it is not clear whether bursting is an endogenous property of individual beta-cells or requires an electrically coupled islet. While many workers report stochastic firing or quasibursting in single cells, a few reports describe single-cell bursts much longer (minutes) than those of islets (15-60 s). We studied the behavior of single cells systematically to help resolve this issue. Perforated patch recordings were made from single mouse beta-cells or hamster insulinoma tumor cells in current clamp at 30-35 degrees C, using standard K+-rich pipette solution and external solutions containing 11.1 mM glucose. Dynamic clamp was used to apply artificial KATP and Ca2+ channel conductances to cells in current clamp to assess the role of Ca2+ and KATP channels in single cell firing. The electrical activity we observed in mouse beta-cells was heterogeneous, with three basic patterns encountered: 1) repetitive fast spiking; 2) fast spikes superimposed on brief (<5 s) plateaus; or 3) periodic plateaus of longer duration (10-20 s) with small spikes. Pattern 2 was most similar to islet bursting but was significantly faster. Burst plateaus lasting on the order of minutes were only observed when recordings were made from cell clusters. Adding gCa to cells increased the depolarizing drive of bursting and lengthened the plateaus, whereas adding gKATP hyperpolarized the cells and lengthened the silent phases. Adding gCa and gKATP together did not cancel out their individual effects but could induce robust bursts that resembled those of islets, and with increased period. These added currents had no slow components, indicating that the mechanisms of physiological bursting are likely to be endogenous to single beta-cells. It is unlikely that the fast bursting (class 2) was due to oscillations in gKATP because it persisted in 100 microM tolbutamide. The ability of small exogenous currents to modify beta-cell firing patterns supports the hypothesis that single cells contain the necessary mechanisms for bursting but often fail to exhibit this behavior because of heterogeneity of cell parameters.

Neurotransmitters and their receptors in the islets of Langerhans of the pancreas: what messages do acetylcholine, glutamate, and GABA transmit?

Although neurotransmitters are present in pancreatic islets of Langerhans and can be shown to alter hormone secretion, their precise physiological roles in islet function and their cellular mechanisms of action are unclear. Recent research has identified specific neurotransmitter receptor isoforms in islets that may be important physiologically, because selective receptor agonists activate islet ion channels, modify intracellular [Ca2+], and affect secretion. This article focuses on the putative roles of acetylcholine, glutamate, and GABA in islet function. It has been hypothesized that acetylcholine potentiates insulin secretion by either promoting Ca release from cellular stores, activating a store depletion-activated channel, or activating a novel Na channel. GABA and glutamate, in contrast, have been proposed to mediate a novel paracrine signaling pathway whereby alpha- and beta-cells communicate within the islet. The evidence supporting these hypotheses will be critically evaluated.

Effects of intracellular calcium on GABAA receptors in mouse cortical neurons.

Using the patch-clamp technique, we studied the effect of intracellular Ca2+ on Cl- current gated by type A gamma-aminobutyric acid receptors (GABAA) in mouse cortical neurons. When the rapid Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was in the pipette solution, the GABA-activated Cl- current amplitude decreased over time to 49 +/- 7% of control. In contrast, equimolar replacement of BAPTA with ethylenebis(oxonitrilo)tetraacetate (EGTA) caused a 60 +/- 10% increase in GABA current. An increased intracellular Ca2+ concentration caused a transient augmentation of the GABA current. This effect of Ca2+ was concentration dependent (10 nM to 34 muM). Ca2+ increased the amplitude of the current by enhancing the maximal response to GABA rather than by changing the affinity of the receptor to GABA (EC50 = 5 +/- 0.4 muM vs. 7 +/- 0.3 muM). Both calmodulin (CaM) and a CaM kinase II inhibitor (200 muM) blocked the potentiating effect of Ca2+ suggesting that it was mediated by activation of CaM kinase II. We found that regulation of GABAA receptors by intracellular Ca2+ in cortical neurons has important physiological implications since the potentiating effect of increasing the intracellular Ca2+ on responses to GABA was mimicked by activating excitatory receptors with 100 muM N-methyl-D-aspartate (NMDA). These findings suggest that modulation of GABAA receptor activity by glutamate may be brought about via changes in intracellular Ca2+.

Inhibition of the electrogenic Na pump underlies delayed depolarization of cortical neurons after mechanical injury or glutamate.

We previously characterized the electrophysiological response of cortical neurons to a brief sublethal stretch-injury using an in vitro model of traumatic brain injury. This model revealed that cortical neurons undergo a stretch-induced delayed depolarization (SIDD) of their resting membrane potential (RMP) which is approximately 10 mV in magnitude. SIDD is dependent on N-methyl-D-aspartate (NMDA) receptor activation, neuronal firing, and extracellular calcium for its induction but not its maintenance. SIDD was maximal 1 h after the insult and required incubation at 37 degrees C. The present study examined the mechanism mediating SIDD and its relation to glutamate receptor activation. The Na pump inhibitor ouabain was used to assess the contribution of the Na pump to the RMP of control and stretched neurons using whole cell patch-clamp techniques. The nitric oxide (NO) synthase inhibitor N omega-nitro-L-arginine and a polyethylene glycol conjugate of superoxide dismutase were used to assess whether NO or superoxide anion, respectively, were involved in the induction of SIDD. Neurons were exposed to exogenous glutamate in the absence of cell stretch to determine whether glutamate alone can mimic SIDD. We report that SIDD is mediated by Na pump inhibition and is likely to result from reduced energy levels since the RMP of neurons dialyzed with a pipette solution containing 5 mM ATP were identical to controls. NO, but not superoxide anion, also may contribute to SIDD. A 3-min exposure to 10 microM glutamate produced a SIDD-like depolarization also associated with Na pump inhibition. The results suggest that Na pump inhibition secondary to alterations in cellular energetics underlies SIDD. Na pump inhibition due to glutamate exposure may contribute to traumatic brain injury or neurodegenerative diseases linked to glutamate receptor activation.

Reduction of voltage-dependent Mg2+ blockade of NMDA current in mechanically injured neurons.

Activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors is implicated in the pathophysiology of traumatic brain injury. Here, the effects of mechanical injury on the voltage-dependent magnesium (Mg2+) block of NMDA currents in cultured rat cortical neurons were examined. Stretch-induced injury was found to reduce the Mg2+ blockade, resulting in significantly larger ionic currents and increases in intracellular free calcium (Ca2+) concentration after NMDA stimulation of injured neurons. The Mg2+ blockade was partially restored by increased extracellular Mg2+ concentration or by pretreatment with the protein kinase C inhibitor calphostin C. These findings could account for the secondary pathological changes associated with traumatic brain injury.

Temperature modulates the Ca2+ current of HIT-T15 and mouse pancreatic beta-cells.

The effects of raising temperature on the Ca2+ currents of insulin-secreting HIT and mouse pancreatic beta-cells were studied. Currents were measured in 3 mM Ca2+ containing solutions using standard whole-cell techniques. Increasing temperature from 22 degrees C to 35 degrees C increased peak Ca2+ current amplitude, percent (fast) inactivation and decreased the time-to-peak of the current. Ca2+ currents in HIT and mouse beta-cells responded in the same manner to an imposed physiological burstwave with test-pulses: (i) application of the burstwave inactivated the test-pulse Ca2+ current at both high and low temperatures; (ii) Ca2+ current inactivation leveled off during the plateau phase at 20-22 degrees C whereas there was an apparent continual decay at 33-35 degrees C; and (iii) recovery from inactivation occurred during the interburst period at both temperatures. Application of a physiological burstwave without test-pulses to mouse beta-cells before and after addition of 0.2 mM Cd2+ resulted in a Ca2+ difference current. This current activated during the hyperpolarized interburst phase, activated, inactivated and deactivated rapidly and continually during the plateau phase, and recovered from inactivation during the interburst. Although raising temperature strongly modified HIT and mouse beta-cells Ca2+ current, our work suggests that other channels, in addition to Ca2+ channels, are likely to be involved in the control of islet bursts, particularly at different temperatures. In addition, the effect of temperature on islet cell Ca2+ current may be partly responsible for the well-known temperature dependence of glucose-dependent secretion.

New mechanisms for sulfonylurea control of insulin secretion.

Oral antidiabetic sulfonylureas like tolbutamide and glyburide have been used to treat patients with noninsulin dependent diabetes mellitus. These agents lower blood glucose by stimulating insulin secretion from the pancreatic islets of Langerhans. A major component of this stimulation is sulfonylurea-mediated closure of the ATP-inhibited potassium channels (K(ATP) channels) of islet ß-cells. Closure of these channels leads to cell depolarization, calcium uptake, and insulin exocytosis. Progress leading up to the recent cloning of the high-affinity sulfonylurea receptor and reconstitution of the K(ATP) channel is reviewed in this article together with new data showing that sulfonylureas may control secretion by activating a novel chloride ion channel, inhibiting an islet Na/K/ATPase or via distal stimulation of granule exocytosis by a kinase C dependent mechanism.

Mechanical perturbation of cultured cortical neurons reveals a stretch-induced delayed depolarization.

1. An in vitro cellular model of injury was used to elucidate mechanisms contributing to traumatic brain injury (TBI). Neonatal rat cortical neurons cultured on a flexible silastic membrane were stretched rapidly and reversibly by a 50-ms pulse of pressurized air. 2. Sublethal cell stretch depolarized neuronal resting membrane potential by approximately 10 mV but only if cells were incubated for 1 h after injury. Stretch-induced delayed depolarization (or SIDD) returned to baseline values within 24 h. 3. SIDD was dependent on the degree of cell stretch and required neuronal firing, calcium entry, and N-methyl-D-aspartate receptor activation for its induction but not its maintainance. 4. Similarities between SIDD and TBI suggest that SIDD may play a role in brain injury.