Glucose sensing of individual pancreatic beta-cells involves transitions between steady-state and oscillatory cytoplasmic Ca2+.

Glucose stimulation of individual pancreatic beta-cells is associated with a rise of the cytoplasmic Ca2+ concentration ([Ca2+]i) manifested either as large amplitude oscillations (0.2-0.5/min) or as a sustained increase. Determinants for the transitions between the basal and the two stimulated states have now been studied using dual-wavelength fluorometric measurements on individual ob/ob mouse beta-cells loaded with the Ca2+ indicator Fura-2. The transition from the basal state to large amplitude oscillations was induced by raising the glucose concentration to 7 mM or above. The frequencies and shapes of the [Ca2+]i cycles remained largely unaffected when raising glucose as high as 40 mM. However, in some cells the oscillatory pattern was transformed into a sustained increase of [Ca2+]i at high glucose concentrations. Although the peak values for the oscillations exceeded the steady-state increase, the time average [Ca2+]i was higher during the latter phase. Both types of glucose-induced transitions were facilitated by the presence of 1-100 nM glucagon. Protein kinase C activation by 10 nM of the phorbol ester TPA resulted in a transformation of the glucose-induced oscillations into a sustained increase of [Ca2+]i but the levels reached were considerably lower than obtained with glucose alone. It is concluded that the glucose sensing of the individual beta-cell is based on sudden transitions between steady-state and oscillating cytoplasmic Ca2+. It is these transitions rather than alterations of the oscillatory characteristics which determine the average [Ca2+]i regulating insulin release.     

Glucose and tolbutamide trigger transients of Ca2+ in single pancreatic beta-cells exposed to tetraethylammonium.

Isolated pancreatic beta-cells respond to glucose stimulation with increase of the cytoplasmic Ca2+ concentration ([Ca2+]i) in terms of membrane-derived slow oscillations (0.2-0.5/min) with superimposed transient of intracellular origin. To evaluate under which conditions transients may result also from entry of extracellular Ca2+, the cytoplasmic concentration of the ion was measured with dual wavelength fluorometry and fura-2 in individual mouse beta-cells exposed to the K+ channel blocker tetraethylammonium (TEA). In the presence of 20 mM TEA, the beta-cells responded to closure of the KATP channels (increase of the glucose concentration to 11 mM or addition of 1 mM tolbutamide) with pronounced transients of [Ca2+]i. However, there were no transients when the beta-cells were depolarized by raising extracellular K+ to 30 mM in the presence of 20 mM TEA. The glucose-induced [Ca2+]i transients became more pronounced after thapsigargin inhibition of the endoplasmic reticulum Ca(2+)-ATPase. The tolbutamide-induced transients were amplified when promoting the entry of Ca2+ (rise of extracellular Ca2+ to 10 mM or addition of BAY K 8644), unaffected in the presence of thapsigargin and the Na+ channel blocker tetrodotoxin and slightly reduced by glucagon. Blockage of voltage-dependent Ca2+ channels with methoxyverapamil resulted in a prompt disappearance of the transients induced by glucose or tolbutamide. The observations indicate that closure of the KATP channels can precipitate pronounced transients of [Ca2+]i when other K+ conductances are suppressed.     

Propagation of cytoplasmic Ca2+ oscillations in clusters of pancreatic beta-cells exposed to glucose

Digital image analysis was employed for resolving the temporal and spatial variations of the cytoplasmic Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells loaded with the Ca(2+)-indicator Fura-2. Glucose-stimulated individual beta-cells exhibited large amplitude oscillations of [Ca2+]i with a mean frequency of 0.33 min-1. When Ca2+ diffusion was restricted by increasing the Ca2+ buffering capacity, the sugar-induced rise of [Ca2+]i preferentially affected the peripheral cytoplasm. When glucagon was present glucose also caused less prominent oscillations with about a 10-fold higher frequency superimposed on an elevated [Ca2+]i. In small clusters of 6-14 cells the average frequency of the large amplitude oscillations increased to 0.60 min-1. The clusters were found to contain micro-domains of electrically coupled cells with synchronized oscillations. After increasing the glucose concentration, adjacent domains became functionally coupled. The oscillations originated from different cells in the cluster. Also the fast glucagon-dependent oscillations were synchronized between cells and had different origins. The results indicate that coupling of beta-cells leads to an increased frequency of the large amplitude oscillations, and that the oscillatory characteristics are determined collectively among electrically coupled beta-cells rather than by particular pacemaker cells. In the light of these data it is necessary to reconsider the previous ideas that glucose-induced oscillations of membrane potential and [Ca2+]i require coupling between many beta-cells, and that the peak [Ca2+]i values reached during oscillations should increase with the size of the coupled cluster.     

Nitric oxide-cyclic GMP system potentiates glucose-induced rise in cytosolic Ca2+ concentration in rat pancreatic beta-cells.

Involvement of nitric oxide (NO) in the regulation of insulin secretion from pancreatic beta-cells was investigated by measuring cytosolic Ca2+ concentration ([Ca2+]i) in isolated rat pancreatic beta-cells. At 7.0 mM glucose, L-arginine (0.1 mM) elevated [Ca2+]i in about 50% of the beta-cells examined. The response was partially inhibited by an NO synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMA; 0.1 mM), suggesting that part of the response was mediated by the production of NO from L-arginine. D-Arginine at higher concentrations (3 or 10 mM) also increased [Ca2+]i at 7.0 mM glucose; however, the response was not affected by L-NMA (0.1 mM). Similar [Ca2+]i elevation was produced by NO (10 nM) and sodium nitroprusside (SNP; 10 microM) at 7.0 mM glucose. The SNP-induced increase in [Ca2+]i was abolished by nicardipine (1 microM), suggesting that the [Ca2+]i response is mediated by Ca2+ influx through L-type voltage-operated Ca2+ channels. In the presence of oxyhemoglobin (1 microM), the [Ca2+]i elevation induced by NO (10 nM) was abolished. Neither degradation products of NO, NO2- nor NO3-, caused any changes in [Ca2+]i. 8-Bromo-cyclic GMP (8-Br-cGMP; 3 mM) and atrial natriuretic peptide (0.1 microM) elevated [Ca2+]i at 7.0 mM glucose. We conclude that NO, which is produced from L-arginine in pancreatic islets, facilitates glucose-induced [Ca2+]i increase via the elevation of cGMP in rat pancreatic beta-cells. NO-cGMP system may physiologically regulate insulin secretion from pancreatic beta-cells.     

Heterogeneous changes in [Ca2+]i induced by glucose, tolbutamide and K+ in single rat pancreatic B cells

The effects of glucose, tolbutamide and K+ on cytosolic free Ca2+ ([Ca2+]i) in single rat pancreatic B cells were examined using Fura-2 and dual wavelength microfluorimetry. At basal glucose concentration (2.8 mM), about half of the cells were found to display spontaneous Ca2+ oscillations. Glucose (greater than or equal to 11.1 mM), tolbutamide (greater than or equal to 50 microM) and K+ (50 mM) induced rises in [Ca2+]i that could be inhibited by the Ca2+ channel blocker D600. The pattern of response and the sensitivity to the secretagogues were characterized by a marked heterogeneity. The majority of the cells responded to glucose and tolbutamide by a progressive increase in [Ca2+]i onto which sinusoidal oscillations were superimposed. The periodicity of these oscillations was about 2.5/min. Occasionally, some cells displayed slow and major waves in Ca2+ levels (about 0.2/min). None of the cells responded to glucose by displaying an initial decrease in [Ca2+]i. Likewise, the sugar failed to decrease [Ca2+]i in the absence of extracellular Ca2+. The present study shows that, despite a large heterogeneity of the response, the majority of the pancreatic B cells respond to different secretagogues by displaying fast [Ca2+]i oscillations that are reminiscent of the bursts of electrical activity recorded in B cells.     

The role of Ca2+ in steroidogenesis in Leydig cells. Stimulation of intracellular free Ca2+ by lutropin (LH), luliberin (LHRH) agonist and cyclic AMP.

The requirements of purified rat Leydig cells for intra- and extra-cellular Ca2+ during steroidogenesis stimulated by LH (lutropin), cyclic AMP analogues and LHRH (luliberin) agonist were investigated. The intracellular Ca2+ concentrations ([Ca2+]i) were measured by using the fluorescent Ca2+ chelator quin-2. The basal [Ca2+]i was found to be 89.4 +/- 16.6 nM (mean +/- S.D., n = 25). LH, 8-bromo cyclic AMP and dibutyryl cyclic AMP increased [Ca2+]i, by 300-500 nM at the highest concentrations of each stimulator, whereas LHRH agonist only increased [Ca2+]i by a maximum of approx. 60 nM. Low concentrations of LH (less than 1 pg/ml) and all concentrations of LHRH agonist increased testosterone without detectable changes in cyclic AMP. With amounts of LH greater than 1 pg/ml, parallel increases in cyclic AMP and [Ca2+]i occurred. The steroidogenic effect of the LHRH agonist was highly dependent on extracellular Ca2+ concentration ([Ca2+]e), whereas LH effects were only decreased by 35% when [Ca2+]e was lowered from 2.5 nM to 1.1 microM. No increase in [Ca2+]i occurred with the LHRH agonist in the low-[Ca2+]e medium, whereas LH (100 ng/ml) gave an increase of 52 nM. It is concluded that [Ca2+]i can be modulated in rat Leydig cells by LH via mechanisms that are both independent of and dependent on cyclic AMP, whereas LHRH-agonist action on [Ca2+]i is independent of cyclic AMP. The evidence obtained suggests that, at sub-maximal rates of testosterone production, Ca2+, rather than cyclic AMP, is the second messenger, whereas for maximum steroidogenesis both Ca2+- and cyclic-AMP-dependent pathways may be involved.     

Suppression of insulin release by galanin and somatostatin is mediated by a G-protein. An effect involving repolarization and reduction in cytoplasmic free Ca2+ concentration

The effects of galanin and somatostatin on insulin release, membrane potential, and cytoplasmic free Ca2+ concentration [( Ca2+]i) were investigated using beta-cells isolated from obese hyperglycemic mice. Whereas insulin release was measured in a column perifusion system, membrane potential and [Ca2+]i were measured with the fluorescent indicators bisoxonol (bis-(1,3-diethylthiobarbiturate)trimethineoxonol) and quin 2, in cell suspensions in a cuvette. Galanin (16 nM) and somatostatin (400 nM) suppressed glucose-stimulated insulin release in parallel to promoting repolarization and a reduction in [Ca2+]i. The reduction in [Ca2+]i comprised an initial nadir followed by a slow rise and the establishment of a new steady state level. The slow rise in [Ca2+]i was abolished by 50 microM D-600, a blocker of voltage-activated Ca2+ channels. Both peptides suppressed insulin release even when [Ca2+]i was raised by 25 mM K+. Under these conditions the inhibition of insulin release was partly reversed by an increase in the glucose concentration. Addition of 5 mM Ca2+ to a cell suspension, incubated in the presence of 20 mM glucose and either galanin, somatostatin, or the alpha 2-adrenergic agonist clonidine (10 nM), induced oscillations in [Ca2+]i, this effect disappearing subsequent to the addition of D-600. The effects of galanin, somatostatin, and clonidine on [Ca2+]i were abolished in beta-cells treated with pertussis toxin. In accordance with measurements of [Ca2+]i, treatment with pertussis toxin reversed the inhibitory effect of galanin on insulin release. The inhibitory action of galanin and somatostatin on insulin release is probably accounted for by not only a repolarization-induced reduction in [Ca2+]i and a decreased sensitivity of the secretory machinery to Ca2+, but also by a direct interaction with the exocytotic process. It is proposed that these effects are mediated by a pertussis toxin-sensitive GTP-binding protein.     

Role of voltage-dependent Na+ channels for rhythmic Ca2+ signalling in glucose-stimulated mouse pancreatic beta-cells.

The putative role of voltage-dependent Na+ channels for glucose induction of rhythmic Ca2+ signalling was studied in mouse pancreatic beta-cells with the use of the Ca2+ indicator fura-2. A rise in glucose from 3 to 11 mM resulted in slow oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i). These oscillations, as well as superimposed transients seen during forskolin-induced elevation of cAMP, remained unaffected in the presence of the Na+ channel blocker tetrodotoxin. During exposure to 1-10 microM veratridine, which facilitates the opening of voltage-dependent Na+ channels, the slow oscillations were replaced by repetitive and pronounced [Ca2+]i transients arising from the basal level. The effects of veratridine were reversed by tetrodotoxin. The veratridine-induced [Ca2+]i transients were critically dependent on the influx of Ca2+ and persisted after thapsigargin inhibition of the endoplasmic reticulum Ca2+-ATPase. Both tolbutamide and ketoisocaproate mimicked the action of glucose in promoting [Ca2+]i transients in the presence of veratridine. It is suggested that activation of voltage-dependent Na+ channels is a useful approach for amplifying Ca2+ signals for insulin release.     

Microfluorometric analysis of Cl- permeability and its relation to oscillatory Ca2+ signalling in glucose-stimulated pancreatic beta-cells

The cytoplasmic concentrations of Cl-([Cl-]i) and Ca2+ ([Ca2+]i) were measured with the fluorescent indicators N-(ethoxycarbonylmethyl)-6-methoxyquinilinum bromide (MQAE) and fura-2 in pancreatic beta-cells isolated from ob/ob mice. Steady-state [Cl-]i in unstimulated beta-cells was 34 mM, which is higher than expected from a passive distribution. Increase of the glucose concentration from 3 to 20 mM resulted in an accelerated entry of Cl- into beta-cells depleted of this ion. The exposure to 20 mM glucose did not affect steady-state [Cl-]i either in the absence or presence of furosemide inhibition of Na+, K+, 2 Cl- co-transport. Glucose-induced oscillations of [Ca2+]i were transformed into sustained elevation in the presence of 4,4' diisothiocyanato-dihydrostilbene-2,2'-disulfonic acid (H2DIDS). A similar effect was noted when replacing 25% of extracellular Cl- with the more easily permeating anions SCN-, I-, NO3- or Br-. It is concluded that glucose stimulation of the beta-cells is coupled to an increase in their Cl- permeability and that the oscillatory Ca2+ signalling is critically dependent on transmembrane Cl- fluxes.     

Evidence of cyclic AMP-independent action of glucagon on calcium mobilization in rat hepatocytes

Glucagon increases the cytoplasmic free calcium concentration as measured by aequorin bioluminescence. It has been proposed by Wakelam et al. (Nature 323 (1986) 68-71) that low concentrations of glucagon mobilize calcium from an intracellular pool by causing polyphosphoinositide breakdown. To identify whether cyclic AMP mediates changes in the cytoplasmic free calcium concentration ([Ca2+]c) induced by glucagon, the effects of forskolin and exogenous cyclic AMP on [Ca2+]c were compared with that of glucagon in aequorin-loaded hepatocytes. Although the magnitudes of the [Ca2+]c responses to 250 microM forskolin and 1 mM 8-bromo cyclic AMP were identical to that of 5 nM glucagon, these two agents induced a more prolonged elevation of [Ca2+]c. Glucagon-induced elevation of [Ca2+]c was accompanied by a smaller increase in cyclic AMP than that induced by forskolin. When the cyclic AMP response to glucagon was potentiated by an inhibitor of phosphodiesterase, 3-isobutyl-1-methylxanthine, the glucagon-induced increase in [Ca2+]c was not affected. Conversely, when the cyclic AMP response to glucagon was reduced by pretreatment of the cells with angiotensin II, glucagon-induced changes in [Ca2+]c were rather enhanced. Furthermore, vasopressin potentiated glucagon-induced changes in [Ca2+]c despite the reduction of the cyclic AMP response to glucagon. In the presence of 1 microM extracellular calcium, angiotensin II did not enhance glucagon-induced changes in [Ca2+]c. These results suggest that at least part of the action of 5 nM glucagon on calcium mobilization is independent of cyclic AMP.     

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.     

Effect of intracellular delivery of energy metabolites on intracellular Ca2+ in mouse islets of Langerhans

Regulation of glucose-induced oscillations in intracellular Ca2+ concentration ([Ca2+]i) was investigated by using a novel technique, electroporation from an electrolyte-filled capillary, to deliver energy metabolites to the intracellular compartment of mouse islets. Intracellular application of ATP resulted in a nifedipine-sensitive increase in [Ca2+]i, consistent with a KATP-channel dependent mechanism of Ca2+ influx. [Ca2+]i in islets exposed to 10 mM glucose oscillated with a period of approximately 3 min, often superimposed with faster oscillations. Electroporation of ATP blocked all types of oscillations and elevated [Ca2+]i while delivery of ADP had no effect on oscillations. Intracellular delivery of glucose-6-phosphate or fructose-1,6-bisphosphate tended to transform slow oscillations to fast oscillations. These results demonstrate that modulation of ATP concentrations and glycolytic flux are important in development of slow oscillations.     

Video imaging of cytosolic Ca2+ in pancreatic beta-cells stimulated by glucose, carbachol, and ATP.

In order to define the differences in the distribution of cytosolic free Ca2+ ([Ca2+]i) in pancreatic beta-cells stimulated with the fuel secretagogue glucose or the Ca(2+)-mobilizing agents carbachol and ATP, we applied digital video imaging to beta-cells loaded with fura-2.83% of the cells responded to glucose with an increase in [Ca2+]i after a latency of 117 +/- 24 s (mean +/- S.E., 85 cells). Of these cells, 16% showed slow wave oscillations (frequency 0.35/min). In order to assess the relationship between membrane potential and the distribution of the [Ca2+]i rise, digital image analysis and perforated patch-clamp methods were applied simultaneously. The system used allowed sufficient temporal resolution to visualize a subplasmalemmal Ca2+ transient due to a single glucose-induced action potential. Glucose could also elicit a slow depolarization which did not cause Ca2+ influx until the appearance of the first of a train of action potentials. [Ca2+]i rose progressively during spike firing. Inhibition of Ca2+ influx by EGTA abolished the glucose-induced rise in [Ca2+]i. In contrast, the peak amplitude of the [Ca2+]i response to carbachol was not significantly different in normal or in Ca(2+)-deprived medium. Occasionally, the increase of the [Ca2+]i rise was polarized to one area of the cell different from the subplasmalemmal rise caused by glucose. The amplitude of the response and the number of responding cells were significantly increased when carbachol was applied after the addition of high glucose (11.2 mM). ATP also raised [Ca2+]i and promoted both Ca2+ mobilization and Ca2+ influx. The intracellular distribution of [Ca2+]i was homogeneous during the onset of the response. A polarity in the [Ca2+]i distribution could be detected either in the descending phase of the peak or in subsequent peaks during [Ca2+]i oscillations caused by ATP. In the absence of extracellular Ca2+, the sequential application of ATP and carbachol revealed that carbachol was still able to raise [Ca2+]i after exhaustion of the ATP response. This may be due to desensitization to the former agonist, since the response occurred in the same area of the cell. These results reveal subtle differences in [Ca2+]i distribution following membrane depolarization with glucose or the application of Ca(2+)-mobilizing agonists.     

Evidence for cAMP-dependent protein kinase in mediating the parathyroid hormone-stimulated rise in cytosolic free calcium in rabbit connecting tubules

We investigated cellular mechanisms mediating the parathyroid hormone (PTH)-induced increase in cytosolic free Ca2+ concentration ([Ca2+]i) in isolated perfused rabbit connecting tubules. Prior and/or concomitant exposure to 0.5 mM of N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-8), a cyclic nucleotide-dependent protein kinase inhibitor, abolished the rise in [Ca2+]i produced by 0.1 nM PTH in five connecting tubules and suppressed it by approximately 50% in another five. In the latter, there was a delayed onset in the rise of [Ca2+]i. Such responses contrasted to the prompt increase in [Ca2+]i in PTH-stimulated control tubules. However, when H-8 was withdrawn, [Ca2+]i rose within minutes to reach a plateau value similar to the uninhibited response to PTH in controls, indicating rapidly reversible inhibition by H-8. In an otherwise identical protocol, 0.5 mM H-8 also reversibly suppressed the rise in [Ca2+]i induced by 0.175 mM 8-Br-cAMP. In contrast to the stimulatory effect of 8-Br-cAMP on [Ca2+]i, 1 mM 8-Br-cGMP caused no increase. At a concentration of 0.4 mM, the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS), a well-characterized cAMP-dependent protein kinase inhibitor, totally abolished the rise in [Ca2+]i caused by 0.1 nM PTH. We conclude that a cAMP-dependent protein kinase plays an important role in the PTH-stimulated rise in [Ca2+]i in the rabbit connecting tubule. Since the increase in [Ca2+]i was shown previously to depend on extracellular Ca2+, we propose that cAMP-dependent protein phosphorylation is important in mediating PTH-stimulated Ca2+ fluxes across plasma membranes of connecting tubule cells.     

Single pancreatic beta-cells from normal rats exhibit an initial decrease and subsequent increase in cytosolic free Ca2+ in response to glucose.

Since it was reported that glucose stimulation initially lowers as well as subsequently raises the cytosolic free calcium concentration [( Ca2+]i) in pancreatic islet cells from hyperglycemic ob/ob mice, it has been argued whether the lowering of [Ca2+]i is physiological or artifactual. In the present study, [Ca2+]i in single pancreatic beta-cells from normal rats was measured by Fura-2 microfluorometry. Following elevation of the glucose concentration from 2.8 mM (basal) to 16.7 mM, a bimodal change in [Ca2+]i, an initial decrease and subsequent increase, was demonstrated. When the basal glucose concentration was raised to 5.6 mM, the stimulation with 16.7 mM glucose also induced the decrease in [Ca2+]i in the majority of the cells, though the amplitude of the decrease was reduced. An elevation of the glucose concentration from 2.8 to 5.6 mM induced the decrease in [Ca2+]i but not usually the increase in [Ca2+]i. Removal of extracellular Ca2+ eliminated the increase in [Ca2+]i without affecting the decrease in [Ca2+]i. Thus, the decrease and increase in [Ca2+]i were clearly dissociated under certain conditions. In contrast, mannoheptulose (an inhibitor of glucose metabolism) inhibited both the decrease and increase in [Ca2+]i. These results demonstrate that the glucose-induced bimodal change in [Ca2+]i is a physiological response of islet beta-cells, and that the decrease and increase in [Ca2+]i are generated by mutually-independent mechanisms which are operated through glucose metabolism by islet beta-cells.     

Glucose induces temperature-dependent oscillations of cytoplasmic Ca2+ in single pancreatic beta-cells related to their electrical activity

Glucose induces large amplitude oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells. The effects of temperature on these oscillations were examined by monitoring [Ca2+]i continuously in single beta-cells from ob/ob-mice using dual wavelength microfluorometry. The oscillations of [Ca2+]i disappeared when the temperature was increased above 42 degrees C and were reversibly inhibited below 30 degrees C. However, cooling did not prevent a glucose response in terms of the average rise of [Ca2+]i. Since patch clamp studies of single beta-cells have indicated a random occurrence of glucose-induced action potentials at room temperature, it was important to explore how the sugar affected the electrical activity at 37 degrees C. Using the cell-attached configuration of the patch clamp technique for such analyses, the action potentials were found to occur in bursts with durations similar to the large amplitude oscillations of [Ca2+]i.     

The Ca2+ dynamics of isolated mouse beta-cells and islets: implications for mathematical models

[Ca(2+)](i) and electrical activity were compared in isolated beta-cells and islets using standard techniques. In islets, raising glucose caused a decrease in [Ca(2+)](i) followed by a plateau and then fast (2-3 min(-1)), slow (0.2-0.8 min(-1)), or a mixture of fast and slow [Ca(2+)](i) oscillations. In beta-cells, glucose transiently decreased and then increased [Ca(2+)](i), but no islet-like oscillations occurred. Simultaneous recordings of [Ca(2+)](i) and electrical activity suggested that differences in [Ca(2+)](i) signaling are due to differences in islet versus beta-cell electrical activity. Whereas islets exhibited bursts of spikes on medium/slow plateaus, isolated beta-cells were depolarized and exhibited spiking, fast-bursting, or spikeless plateaus. These electrical patterns in turn produced distinct [Ca(2+)](i) patterns. Thus, although isolated beta-cells display several key features of islets, their oscillations were faster and more irregular. beta-cells could display islet-like [Ca(2+)](i) oscillations if their electrical activity was converted to a slower islet-like pattern using dynamic clamp. Islet and beta-cell [Ca(2+)](i) changes followed membrane potential, suggesting that electrical activity is mainly responsible for the [Ca(2+)] dynamics of beta-cells and islets. A recent model consisting of two slow feedback processes and passive endoplasmic reticulum Ca(2+) release was able to account for islet [Ca(2+)](i) responses to glucose, islet oscillations, and conversion of single cell to islet-like [Ca(2+)](i) oscillations. With minimal parameter variation, the model could also account for the diverse behaviors of isolated beta-cells, suggesting that these behaviors reflect natural cell heterogeneity. These results support our recent model and point to the important role of beta-cell electrical events in controlling [Ca(2+)](i) over diverse time scales in islets.     

Direct measurements of increased free cytoplasmic Ca2+ in mouse pancreatic beta-cells following stimulation by hypoglycemic sulfonylureas

The effects of the hypoglycemic sulfonylureas tolbutamide and glibenclamide on free cytoplasmic Ca2+, [Ca2+]i, were compared with that of a depolarizing concentration of K+ in dispersed and cultured pancreatic beta-cells from ob/ob mice. [Ca2+]i was measured with the fluorescent Ca2+-indicator quin2. The basal level corresponded to 150 nM and increased to 600 nM after exposure to 30.9 mM K+. The corresponding levels after stimulation with 1 microM glibenclamide and 100 microM tolbutamide were 390 and 270 nM respectively. K+ depolarization increased [Ca2+]i more rapidly than either of the sulfonylureas. It is suggested that the increased [Ca2+]i obtained after stimulation by sulfonylureas is due to depolarization of the beta-cells with subsequent entry of Ca2+ through voltage-dependent channels.     

Glucose-induced increase in cytoplasmic pH in pancreatic beta-cells is mediated by Na+/H+ exchange, an effect not dependent on protein kinase C.

Glucose-induced changes in cytoplasmic pH (pHi) were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Glucose, at concentrations above 3-5 mM, depolarized the beta-cell and increased pHi, cytoplasmic free Ca2+ ([Ca2+]i), and insulin release. This increase in pHi was dependent on the presence of extracellular Na+ and was inhibited by 5-(N-ethyl-N-isopropyl) amiloride, a blocker of Na+/H+ exchange. Stimulation of protein kinase C with phorbol ester also induced an alkalinization. However, when protein kinase C activity was down-regulated, glucose stimulation still induced alkalinization. At 20 mM glucose, 10 mM NH4Cl induced a marked rise in pHi, paralleled by repolarization, inhibition of electrical activity, and decreases in both [Ca2+]i and insulin release. Reduction in [Ca2+]i was prevented by 200 microM tolbutamide, but not by 10 mM tetraethylammonium. At 4 mM glucose, NH4Cl induced a transient increase in insulin release, without changing [Ca2+]i. Exposure of beta-cells to 10 mM sodium acetate caused a persistent decrease in pHi, an effect paralleled by a small transient increase in [Ca2+]i. Acidification per se did not change the beta-cell sensitivity to glucose, not excluding that the activity of the ATP-regulated K+ channels may be modulated by changes in pHi.