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.     

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.     

Tetracaine stimulates insulin secretion from the pancreatic beta-cell by release of intracellular calcium

The role of intracellular calcium stores in stimulus-secretion coupling in the pancreatic beta-cell is largely unknown. We report here that tetracaine stimulates insulin secretion from collagenase-isolated mouse islets of Langerhans in the absence of glucose or extracellular calcium. We also found that the anesthetic evokes a dose-dependent rise of the intracellular free-calcium concentration ([Ca2+]i) in cultured rat and mouse beta-cells. The tetracaine-specific [Ca2+]i rise also occurs in the absence of glucose, or in beta-cells depolarized by exposure to a Ca(2+)-deficient medium (< 1 microM) or elevated [K+]o. Furthermore, tetracaine (> or = 300 microM) depolarized the beta-cell membrane in mouse pancreatic islets, but inhibited Ca2+ entry through voltage-gated Ca2+ channels in HIT cells, an insulin-secreting cell line. From these data we conclude that tetracaine-enhancement of insulin release occurs by mechanisms that are independent of Ca2+ entry across the cell membrane. The tetracaine-induced [Ca2+]i rise in cultured rat beta-cells and insulin secretion from mouse islets is insensitive to dantrolene (20 microM), a drug that inhibits Ca2+ release evoked by cholinergic agonists in the pancreatic beta-cell, and thapsigargin (3 microM), a blocker of the endoplasmic reticulum (ER) Ca2+ pump. We conclude that the Ca2+ required for tetracaine-potentiated insulin secretion is released from intracellular Ca2+ stores other than the ER. Furthermore, tetracaine-induced Ca2+ release was unaffected by the mitochondrial electron transfer inhibitors NaN3 and rotenone. Taken together, these data show that a calcium source other than the ER and mitochondria can affect beta-cell insulin secretion.     

Carbon monoxide stimulates insulin release and propagates Ca2+ signals between pancreatic beta-cells

A key question for understanding the mechanisms of pulsatile insulin release is how the underlying beta-cell oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i) are synchronized within and among the islets in the pancreas. Nitric oxide has been proposed to coordinate the activity of the beta-cells by precipitating transients of [Ca2+]i. Comparing ob/ob mice and lean controls, we have now studied the action of carbon monoxide (CO), another neurotransmitter with stimulatory effects on cGMP production. A strong immunoreactivity for the CO-producing constitutive heme oxygenase (HO-2) was found in ganglionic cells located in the periphery of the islets and in almost all islet endocrine cells. Islets from ob/ob mice had sixfold higher generation of CO (1 nmol.min-1.mg protein-1) than the lean controls. This is 100-fold the rate for their constitutive production of NO. Moreover, islets from ob/ob mice showed a threefold increase in HO-2 expression and expressed inducible HO (HO-1). The presence of an excessive islet production of CO in the ob/ob mouse had its counterpart in a pronounced suppression of the glucose-stimulated insulin release from islets exposed to the HO inhibitor Zn-protoporhyrin (10 microM) and in a 16 times higher frequency of [Ca2+]i transients in their beta-cells. Hemin (0.1 and 1.0 microM), the natural substrate for HO, promoted the appearance of [Ca2+]i transients, and 10 microM of the HO inhibitors Zn-protoporphyrin and Cr-mesoporphyrin had a suppressive action both on the firing of transients and their synchronization. It is concluded that the increased islet production of CO contributes to the hyperinsulinemia in ob/ob mice. In addition to serving as a positive modulator of glucose-stimulated insulin release, CO acts as a messenger propagating Ca2+ signals with coordinating effects on the beta-cell rhythmicity.     

Insulin-secreting INS-1 cells generate a novel type of poorly synchronized Ca2+ transients

Pancreatic beta-cells have an intrinsic oscillatory Ca2+ activity supposed to be synchronized among the islets by cytoplasmic Ca2+ transients elicited by nonadrenergic, noncholinergic (NANC) neurons. To improve the understanding of this process, the cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in two insulin-releasing cell lines using dual wavelength microfluorometry and the indicator fura-2. INS-1 cells but not RINm5F cells were found to generate transients of [Ca2+]i in the presence of the Ca2+ channel blocker methoxyverapamil. These transients differed from those occurring in native beta-cells persisting in the presence of thapsigargin or during prolonged exposure to ATP. Moreover, the [Ca2+]i transients were poorly synchronized whether or not the INS-1 cells had physical contact. If appearing in native beta-cells, the type of [Ca2+]i transients now observed may interfere with the coordination of the beta-cell rhythmicity evoked by NANC neurons.     

Significance of Na/Ca exchange for Ca2+ buffering and electrical activity in mouse pancreatic beta-cells

We have combined the patch-clamp technique with microfluorimetry of the cytoplasmic Ca2+ concentration ([Ca2+]i) to characterize Na/Ca exchange in mouse beta-cells and to determine its importance for [Ca2+]i buffering and shaping of glucose-induced electrical activity. The exchanger contributes to Ca2+ removal at [Ca2+]i above 1 microM, where it accounts for >35% of the total removal rate. At lower [Ca2+]i, thapsigargin-sensitive Ca2+-ATPases constitute a major (70% at 0.8 microM [Ca2+]i) mechanism for Ca2+ removal. The beta-cell Na/Ca exchanger is electrogenic and has a stoichiometry of three Na+ for one Ca2+. The current arising from its operation reverses at approximately -20 mV (current inward at more negative voltages), has a conductance of 53 pS/pF (14 microM [Ca2+]i), and is abolished by removal of external Na+ or by intracellularly applied XIP (exchange inhibitory peptide). Inhibition of the exchanger results in shortening (50%) of the bursts of action potentials of glucose-stimulated beta-cells in intact islets and a slight (5 mV) hyperpolarization. Mathematical simulations suggest that the stimulatory action of glucose on beta-cell electrical activity may be accounted for in part by glucose-induced reduction of the cytoplasmic Na+ concentration with resultant activation of the exchanger.     

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.     

Delayed rectifying and calcium-activated K+ channels and their significance for action potential repolarization in mouse pancreatic beta-cells

The contribution of Ca2(+)-activated and delayed rectifying K+ channels to the voltage-dependent outward current involved in spike repolarization in mouse pancreatic beta-cells (Rorsman, P., and G. Trube. 1986. J. Physiol. 374:531-550) was assessed using patch-clamp techniques. A Ca2(+)-dependent component could be identified by its rapid inactivation and sensitivity to the Ca2+ channel blocker Cd2+. This current showed the same voltage dependence as the voltage-activated (Cd2(+)-sensitive) Ca2+ current and contributed 10-20% to the total beta-cell delayed outward current. The single-channel events underlying the Ca2(+)-activated component were investigated in cell-attached patches. Increase of [Ca2+]i invariably induced a dramatic increase in the open state probability of a Ca2(+)-activated K+ channel. This channel had a single-channel conductance of 70 pS [( K+]o = 5.6 mM). The Ca2(+)-independent outward current (constituting greater than 80% of the total) reflected the activation of an 8 pS [( K+]o = 5.6 mM; [K+]i = 155 mM) K+ channel. This channel was the only type observed to be associated with action potentials in cell-attached patches. It is suggested that in mouse beta-cells spike repolarization results mainly from the opening of the 8-pS delayed rectifying K+ channel.     

Role of single-channel stochastic noise on bursting clusters of pancreatic beta-cells.

To study why pancreatic beta-cells prefer to burst as a multi-cellular complex, we have formulated a stochastic model for bursting clusters of excitable cells. Our model incorporated a delayed rectifier K+ channel, a fast voltage-gated Ca2+ channel, and a slow Cai-blockable Ca2+ channel. The fraction of ATP-sensitive K+ channels that may still be active in the bursting regime was included in the model as a leak current. We then developed an efficient method for simulating an ionic current component of an excitable cell that contains several thousands of channels opening simultaneously under unclamped voltage. Single channel open-close stochastic events were incorporated into the model by use of binomially distributed random numbers. Our simulations revealed that in an isolated beta-cell [Ca2+]i oscillates with a small amplitude about a low [Ca2+]i. However, in a large cluster of tightly coupled cells, stable bursts develop, and [Ca2+]i oscillates with a larger amplitude about a higher [Ca2+]i. This may explain why single beta-cells do not burst and also do not release insulin.     

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.     

Inhibition of calpain blocks pancreatic beta-cell spreading and insulin secretion

In addition to promoting insulin secretion, an increase in cytosolic Ca(2+) triggered by glucose has been shown to be crucial for spreading of beta-cells attached on extracellular matrix (804G matrix). Calpains are Ca(2+)-dependent cysteine proteases involved in an extended spectrum of cellular responses, including cytoskeletal rearrangements and vesicular trafficking. The present work aimed to assess whether calpain is also implicated in the process of Ca(2+)-induced insulin secretion and spreading of rat pancreatic beta-cells. The results indicate calpain dependency of beta-cell spreading on 804G matrix. Indeed, treatment with three distinct calpain inhibitors (N-Ac-Leu-Leu-norleucinal, calpeptin, and ethyl(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butyl-carbamoyl]-2-ox-iranecarboxylate) inhibited cell spreading induced by glucose and KCl, whereas cell attachment was not significantly modified. Calpain inhibitors also suppressed glucose- and KCl-stimulated insulin secretion without affecting insulin synthesis. Washing the inhibitor out of the cell culture restored spreading on 804G matrix and insulin secretory response after 24 h. In addition, incubation with calpeptin did not affect insulin secretory response to mastoparan that acts on exocytosis downstream of intracellular calcium [Ca(2+)]i. Finally, calpeptin was shown to affect the [Ca(2+)]i response to glucose but not to KCl. In summary, the results show that inhibition of calpain blocks spreading and insulin secretion of primary pancreatic beta-cells. It is therefore suggested that calpain could be a mediator of Ca(2+)-induced-insulin secretion and beta-cell spreading.     

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.     

High dose of lipoxin A4 induces apoptosis in rat renal interstitial fibroblasts

Studies have implicated that lipoxinA4 (LXA4) inhibited nuclear factor-kappaB (NF-kappaB), Akt/PKB and PI 3-kinase activity and proliferation of glomerular mesangial cells. It is speculated that LXA4 might serve as pro-apoptotic factor. Rat renal interstitial fibroblasts (NRK-49F cells) were exposed to LXA4 in 5% FCS for 24 h. LXA4 at 0.1 and 1 microM induced 9.83% and 33.82% apoptosis of the cells, respectively, upregulated the expression of calpain 10 and Smac, the levels of [Ca2+]i and activity of caspase-3, and downregulated the activity of STAT3 and threonine phosphorylated Akt1. Transfection of calpain 10 or Smac antisense oligodeoxynucleotide into the cells inhibited the LXA4-induced apoptosis, activity of caspase-3. Pretreatment of the cells with calcium inhibitor SK&F96365 inhibited the LXA4-induced apoptosis, levels of [Ca2+]i, expression of calpain 10 and Smac. In conclusion, LXA4 at high concentrations induced apoptosis of renal interstitial fibroblasts via [Ca2+]i-dependent upregulation of calpain 10 and Smac expression.     

Phorbol ester-stimulated insulin secretion by RINm5F insulinoma cells is linked with membrane depolarization and an increase in cytosolic free Ca2+ concentration

In studying the regulation of insulin secretion by phorbol esters, we examined their effects on the cytosolic free Ca2+ concentration ([Ca2+]i), using the Ca2+ indicator fura-2 in the rat insulin-secreting beta-cell line RINm5F. [Ca2+]i was measured in parallel with the rate of insulin release. 50 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), which may act via protein kinase C, stimulated insulin release and caused an increase in [Ca2+]i. Ca2+-free conditions eliminated the increase in [Ca2+]i and resulted in a reduced stimulation of insulin release by TPA. The Ca2+ channel blocker nitrendipine (300 nM) inhibited both the increase in [Ca2+]i and the increased rate of insulin secretion. Another phorbol ester, 4 beta-phorbol 12,13-didecanoate, which activates protein kinase C, also induced an increase in [Ca2+]i and in the rate of insulin release, while 4 alpha-phorbol 12,13-didecanoate, which fails to stimulate protein kinase C, was without effect. Further studies with bis-oxonol as an indicator of membrane potential showed that TPA depolarized the beta-cell plasma membrane. From these results, it is concluded that TPA depolarizes the plasma membrane, induces the opening of Ca2+ channels in the RINm5F beta-cell plasma membrane, increases [Ca2+]i, and results in insulin secretion. The action of TPA was next compared with that of a depolarizing concentration of KC1 (25 mM), which stimulates insulin secretion simply by opening Ca2+ channels. TPA consistently elicited less depolarization, a smaller rise of [Ca2+]i, but a greater release of insulin than KC1. Therefore an additional action of TPA is suggested, which potentiates the action of the elevated [Ca2+]i on insulin secretion.     

Induction and activity of NO synthase in bone-marrow-derived macrophages are independent of Ca2+.

The aim of the present study was to analyse whether an increase in the intracellular free Ca2+ concentration ([Ca2+]i) plays a role as a signal mediating synthesis of nitric oxide (NO) in bone-marrow-derived macrophages, either by stimulating induction of NO synthase or by regulating the activity of the enzyme. Therefore we compared the effects of various synthetic analogues of bacterial lipopeptide and of lipopolysaccharide (LPS) on NO production (assessed as nitrite formation during an incubation for 24 h) and on [Ca2+]i [measured with the fluorescent probe indo-1 (1-[2-amino-5-(6-carboxyindol-2-yl)phenoxy]-2- 2-(2'-amino-5'-methylphenoxy)ethane-NNN'N'-tetra-acetic acid)]. Strongly dissociating effects were evoked on nitrite formation and on [Ca2+]i by the stimuli. LPS was preferentially effective on nitrite formation, whereas the Ca2+ ionophore ionomycin and AlF3 induced increases only in [Ca2+]i. The lipopeptides N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)-(2RS)- propyl]-(R)-cysteinylalanylglycine, N-palmitoyl-(S)-[2,3-bis(palmitoyloxy)- (2RS)-propyl]-(R)-cysteinylseryl-lysyl-lysyl-lysine and (S)-(1,2- dicarboxyhexadecyl)ethyl-N-palmitoylcysteinylseryl-lysyl-lys yl-lysine stimulated both parameters, but the maximal effects on nitrite formation and the shape of the dose-response curves did not parallel the effects on [Ca2+]i. Reduction of extracellular Ca2+ with EGTA significantly inhibited increases in [Ca2+]i, but did not change nitrite formation. Furthermore, NO synthesis in the cytosolic fraction of stimulated macrophages was not affected by Ca2+ over the concentration range 10 nM-2 microM. We conclude that increases in [Ca2+]i are not required for NO production in bone-marrow-derived macrophages. Thus the cellular regulation of NO production strikingly differs from that in the vascular endothelium, brain and adrenal gland.     

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.     

Alterations of extracellular calcium elicit selective modes of cell death and protease activation in SH-SY5Y human neuroblastoma cells

The role of intracellular Ca2+ homeostasis in mechanisms of neuronal cell death and cysteine protease activation was investigated in SH-SY5Y human neuroblastoma cells. Cells were incubated in 2 mM EGTA to lower intracellular Ca2+ or 5 mM CaCl2 to raise it. Cell death and activation of calpain and caspase-3 were measured. Both EGTA and excess CaCl2 elicited cell death. EGTA induced DNA laddering and an increase in caspase-3-like, but not calpain, activity. Pan-caspase inhibitors protected against EGTA-, but not CaCl2-, induced cell death. Conversely, excess Ca2+ elicited necrosis and activated calpain but not caspase-3. Calpain inhibitors did not preserve cell viability. Ca2+ was the death-mediating factor, because restoration of extracellular Ca2+ protected against cell death induced by EGTA and blockade of Ca2+ channels by Ni2+ protected against that induced by high Ca2+. We conclude that the EGTA treatment lowered intracellular Ca2+ and elicited caspase-3-like protease activity, which led to apoptosis. Conversely, excess extracellular Ca2+ entered Ca2+ channels and increased intracellular Ca2+ leading to calpain activation and necrosis. The mode of cell death and protease activation in response to changing Ca2+ were selective and mutually exclusive, demonstrating that these are useful models to individually investigate apoptosis and necrosis.