Sulphonylureas do not increase insulin secretion by a mechanism other than a rise in cytoplasmic Ca2+ in pancreatic B-cells

The following sequence of events is thought to underlie the stimulation of insulin release by hypoglycaemic sulphonylureas. Interaction of the drugs with a high-affinity binding site (sulphonylurea receptor) in the B-cell membrane leads to closure of ATP-sensitive K+ channels, depolarization, opening of voltage-dependent Ca2+ channels, Ca2+ influx and rise in cytoplasmic [Ca2+]i. Recent experiments using permeabilized islet cells or measuring changes in B-cell membrane capacitance have suggested that sulphonylureas can increase insulin release by a mechanism independent of a change in [Ca2+]i. This provocative hypothesis was tested here with intact mouse islets. When B-cells were strongly depolarized by 60 mM K+, [Ca2+]i was increased and insulin secretion stimulated. Under these conditions, tolbutamide did not further increase [Ca2+]i or insulin release, whether it was applied before or after high K+, and whether the concentration of glucose was 3 or 15 mM. This contrasts with the ability of forskolin and phorbol 12-myristate 13-acetate (PMA) to increase release in the presence of high K+. Tolbutamide also failed to increase insulin release from islets depolarized with barium (substituted for extracellular Ca2+) or with arginine in the presence of high glucose. Glibenclamide and its non-sulphonylurea moiety meglitinide were also without effect on insulin release from already depolarized B-cells. In the absence of extracellular Ca2+, acetylcholine induced monophasic peaks of [Ca2+]i and insulin secretion which were both unaffected by tolbutamide. Insulin release from permeabilized islet cells was stimulated by raising free Ca2+ (between 0.1 and 23 microM). This effect was not affected by tolbutamide and inconsistently increased by glibenclamide. In conclusion, the present study does not support the proposal that hypoglycaemic sulphonylureas can increase insulin release even when they do not also raise [Ca2+]i in B-cells.     

Temporal and quantitative correlations between insulin secretion and stably elevated or oscillatory cytoplasmic Ca2+ in mouse pancreatic beta-cells

An increase in cytoplasmic Ca2+ in beta-cells is a key step in glucose-induced insulin secretion. However, whether changes in cytoplasmic free Ca2+ ([Ca2+]i) directly regulate secretion remains disputed. This question was addressed by investigating the temporal and quantitative relationships between [Ca2+]i and insulin secretion. Both events were measured simultaneously in single mouse islets loaded with fura-PE3 and perifused with a medium containing diazoxide (to prevent any effect of glucose on the membrane potential) and either 4.8 or 30 mmol/l K+. Continuous depolarization with 30 mmol/l K+ in the presence of 15 mmol/l glucose induced a sustained rise in [Ca2+]i and insulin release. No oscillations of secretion were detected even after mathematical analysis of the data (pulse, spectral and sample distribution analysis). In contrast, alternating between 30 and 4.8 mmol/l K+ (1 min/2 min or 2.5 min/5 min) triggered synchronous [Ca2+]i and insulin oscillations of regular amplitude in each islet. A good correlation was found between [Ca2+]i and insulin secretion, and it was independent of the presence or absence of oscillations. This quantitative correlation between [Ca2+]i and insulin secretion was confirmed by experiments in which extracellular Ca2+ was increased or decreased (0.1-2.5 mmol/l) stepwise in the presence of 30 mmol/l K+. This resulted in parallel stepwise increases or decreases in [Ca2+]i and insulin secretion. However, while the successive [Ca2+]i levels were unaffected by glucose, each plateau of secretion was much higher in 20 than in 3 mmol/l glucose. In conclusion, in our preparation of normal mouse islets, insulin secretion oscillates only when [Ca2+]i oscillates in beta-cells. This close temporal relationship between insulin secretion and [Ca2+]i changes attests of the regulatory role of Ca2+. There also exists a quantitative relationship that is markedly influenced by the concentration of glucose.     

Cytosolic Ca2+ gradients in pancreatic islet-cells stimulated by glucose and carbachol

Digital image analysis was employed to resolve the spatial differences in distribution of cytosolic free Ca2+ concentrations ([Ca2+]i) in mouse pancreatic islet-cells stimulated with glucose and carbachol. Using Indo-1 loaded mouse islet-cells, we have demonstrated that glucose induces steep spatial gradients of [Ca2+]i in isolated mouse islet-cells. Furthermore, the largest [Ca2+]i increase was always spatially restricted to a region just beneath the plasma membrane. Low concentrations of carbachol (0.6 microM) induced steep spatial gradients of [Ca2+]i which originated from the center of the cells. However, 10 microM carbachol increased [Ca2+]i to high levels collapsing the [Ca2+]i gradients in the center of the cells. Different patterns of [Ca2+]i oscillations were observed between dissociated pancreatic islet-cells and mouse pancreatic islets when challenged with 11 mM glucose. Under these conditions we could identify cells within the islet which oscillate with the same pattern as the whole islet. We postulate that "initiators" of insulin release, as glucose, induce greater [Ca2+]i increases at exocytotic sites than those induced by "potentiators", as carbachol.     

Glucose-stimulated elevation of cytoplasmic calcium is defective in the diabetic Chinese hamster islet B cell

To characterize insulin release and cytoplasmic free Ca2+ ([Ca2+]i) levels in the diabetic Chinese hamster islet B cell, islets from genetically normal (subline M) and diabetic (subline L) hamsters were collagenase isolated. Insulin release and glucose utilization (conversion of D-[5-(3H)]glucose to 3H2O) were measured in whole islets; [Ca2+]i levels were measured in single islet cells using fura-2. The Ca2+ channel agonist, 12 mmol/l perchlorate, ClO4-, increased the subnormal insulin response during 20 mmol/l glucose perifusion, but did not normalize it. Glucose utilization measured over a 2-h period was normal. Glucose induced an initial decrease and then a rise in [Ca2+]i in 85% of the normal (presumably B) cells. In diabetic cells, the [Ca2+]i response was delayed, subnormal and only observed in 23% of the cells. When perchlorate or another Ca2+ channel agonist, 10 mumol/l CGP 28392, was added with glucose, a larger proportion of the diabetic cells (61-67%) showed increased [Ca2+]i and the mean [Ca2+]i response was not different from normal. However, neither perchlorate nor CGP 28392 could normalize glucose-stimulated insulin release, and K(+)-induced insulin release was decreased in diabetic islets. The K(+)-induced [Ca2+]i rise was essentially normal in all the diabetic islet cells. Therefore, the diabetic hamster islet appears to metabolize glucose normally, but has a diminished insulin response to glucose and K+. The Ca2+ channel agonists markedly improve the subnormal [Ca2+]i response but not the insulin response. Glucose-induced elevation of [Ca2+]i and exocytosis appear defective in the diabetic Chinese hamster B cell.     

Pituitary adenylate cyclase-activating peptide, carbachol, and glucose stimulate insulin release in the absence of an increase in intracellular Ca2+

Insulin secretion from the pancreatic beta cell line HIT-T15 was examined under conditions in which the elevation of intracellular free Ca2+ concentration ([Ca2+]i) was inhibited by nitrendipine or diazoxide or by severe Ca2+ deprivation. Glucose-induced insulin release was completely abolished under these conditions. However, in the presence of 12-O-tetradecanoyl-phorbol-13-acetate or forskolin, 10 mM glucose significantly enhanced insulin release, even in the presence of 5 microM nitrendipine or 150 microM diazoxide. The [Ca2+]i was not increased under these conditions. Even under Ca(2+)-deprived conditions, achieved by 60-min preincubation in Ca(2+)-free buffer containing 1 mM ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), glucose in the complete absence of extracellular Ca2+ significantly enhanced insulin release when the cells were treated also with 12-O-tetradecanoylphorbol-13-acetate and forskolin. Because of these findings, additional studies were performed with pituitary adenylate cyclase-activating peptide (PACAP) and carbachol to see whether physiological stimulation via receptor activation could stimulate insulin release in the absence of a rise in [Ca2+]i. Under normal Ca(2+)-containing conditions, PACAP and carbachol stimulated insulin release and markedly potentiated glucose-stimulated release. In the presence of nitrendipine and thapsigargin, glucose failed to stimulate insulin release. Also, neither glucose in combination with PACAP nor glucose with carbachol was able to stimulate release. However, under the same conditions, the combination of glucose, PACAP, and carbachol did stimulate release while being unable to elevate [Ca2+]i. Thus, simultaneous activation of the beta cell by PACAP, carbachol, and glucose can stimulate insulin release even when [Ca2+]i is not elevated.     

Glucose decreases Na+,K+-ATPase activity in pancreatic beta-cells. An effect mediated via Ca2+-independent phospholipase A2 and protein kinase C-dependent phosphorylation of the alpha-subunit

In the pancreatic beta-cell, glucose-induced membrane depolarization promotes opening of voltage-gated L-type Ca2+ channels, an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i), and exocytosis of insulin. Inhibition of Na+,K+-ATPase activity by ouabain leads to beta-cell membrane depolarization and Ca2+ influx. Because glucose-induced beta-cell membrane depolarization cannot be attributed solely to closure of ATP-regulated K+ channels, we investigated whether glucose regulates other transport proteins, such as the Na+,K+-ATPase. Glucose inhibited Na+,K+-ATPase activity in single pancreatic islets and intact beta-cells. This effect was reversible and required glucose metabolism. The inhibitory action of glucose was blocked by pretreatment of the islets with a selective inhibitor of a Ca2+-independent phospholipase A2. Arachidonic acid, the hydrolytic product of this phospholipase A2, also inhibited Na+, K+-ATPase activity. This effect, like that of glucose, was blocked by nordihydroguaiaretic acid, a selective inhibitor of the lipooxygenase metabolic pathway, but not by inhibitors of the cyclooxygenase or cytochrome P450-monooxygenase pathways. The lipooxygenase product 12(S)-HETE (12-S-hydroxyeicosatetranoic acid) inhibited Na+,K+-ATPase activity, and this effect, as well as that of glucose, was blocked by bisindolylmaleimide, a specific protein kinase C inhibitor. Moreover, glucose increased the state of alpha-subunit phosphorylation by a protein kinase C-dependent process. These results demonstrate that glucose inhibits Na+, K+-ATPase activity in beta-cells by activating a distinct intracellular signaling network. Inhibition of Na+,K+-ATPase activity may thus be part of the mechanisms whereby glucose promotes membrane depolarization, an increase in [Ca2+]i, and thereby insulin secretion in the pancreatic beta-cell.     

Cytoplasmic Ca2+ oscillations in pancreatic beta-cells

In the last 15 years it has been a growing interest in the cyclic variations of circulating insulin [46]. After the suggestion that this phenomenon may be due to oscillations of the beta-cell membrane potential [8,39], it was demonstrated that [Ca2+]i oscillates in the glucose-stimulated beta-cell with a similar frequency to that of pulsatile insulin release. The present review describes four types of [Ca2+]i oscillations in the pancreatic beta-cell. The slow sinusoidal oscillations, referred to as type-a, are those which most closely correspond to pulsatile insulin release. Although not affecting the properties of the type-a oscillations in individual beta-cells, the concentration of glucose is a determinant for their generation and further transformation into a sustained increase. Accordingly, cytoplasmic Ca2+ is regulated by sudden transitions between oscillatory and steady-state levels at threshold concentrations of glucose, which are characteristic for the individual beta-cell. This behaviour explains the observation of a gradual recruitment of previously non-secreting cells with increase of the extracellular glucose concentration [44]. However, it still remains to be elucidated how the sudden transitions between these three states translate into the co-ordinated slow oscillations of [Ca2+]i in the intact islet. Cyclic variations of circulating insulin require a synchronization of the [Ca2+]i cycles also among the islets in the pancreas. It is still an open question by which means the millions of islets communicate mutually to establish a pattern of pulsatile insulin release from the whole pancreas. The discovery that the beta-cell is not only the functional unit for insulin synthesis but also generates the [Ca2+]i oscillations required for pulsatile insulin release has both physiological and clinical implications. The fact that minor damage to the beta-cells prevents the type-a oscillations with maintenance of a glucose response in terms of raised [Ca2+]i reinforces previous arguments [54] that loss of insulin oscillations is an early indicator of type-2 diabetes. Further analyses of the [Ca2+]i oscillations in the beta-cells should include not only the mechanisms for their generation and subsequent propagation within or among the islets but also how modulation of their frequency affects the insulin sensitivity of various target cells. The latter approach may be important in the attempts to maintain normoglycemia under conditions minimizing the vascular effects of insulin supposed to precipitate hypertonia and atherosclerosis [70,71,77].     

Relation of exocytotic release of gamma-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the gamma-aminobutyric acid (GABA) carrier, NNC-711, (1-[(2-diphenylmethylene)amino]oxyethyl)- 1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride, blocks the Ca(2+)-independent release of [3H]GABA from rat brain synaptosomes induced by 50 mM K+ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca(2+)-dependent release of [3H]GABA in the total absence of carrier-mediated release. Reversal of the Na+/Ca2+ exchanger was used to increase the intracellular free Ca2+ concentration ([Ca2+]i) to test whether an increase in [Ca2+]i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca2+]i may rise to values above 400 nM, as a result of Na+/Ca2+ exchange, without inducing release of [3H]GABA, but subsequent K+ depolarization immediately induced [3H]GABA release. Thus, a rise of only a few nanomolar Ca2+ in the cytoplasm induced by 50 mM K+ depolarization, after loading the synaptosomes with Ca2+ by Na+/Ca2+ exchange, induced exocytotic [3H]GABA release, whereas the rise in cytoplasmic [Ca2+] caused by reversal of the Na+/Ca2+ exchanger was insufficient to induce exocytosis, although the value for [Ca2+]i attained was higher than that required for exocytosis induced by K+ depolarization. The voltage-dependent Ca2+ entry due to K+ depolarization, after maximal Ca2+ loading of the synaptosomes by Na+/Ca2+ exchange, and the consequent [3H]GABA release could be blocked by 50 microM verapamil. Although preloading the synaptosomes with Ca2+ by Na+/Ca2+ exchange did not cause [3H]GABA release under any conditions studied, the rise in cytoplasmic [Ca2+] due to Na+/Ca2+ exchange increased the sensitivity to external Ca2+ of the exocytotic release of [3H]GABA induced by subsequent K+ depolarization. Thus, our results show that the vesicular release of [3H]GABA is rather insensitive to bulk cytoplasmic [Ca2+] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca2+ entry through Ca2+ channels near the active zones.     

Insulin exocytosis and glucose-mediated increase in cytoplasmic free Ca2+ concentration in the pancreatic beta-cell are independent of cyclic ADP-ribose

Stimulation of pancreatic beta-cells by glucose gives rise to an increase in the cytoplasmic free calcium concentration ([Ca2+]i) and exocytosis of insulin. Cyclic adenosine 5'-diphosphate ribose (cADPR), a metabolite of beta-NAD+, has been reported to increase [Ca2+]i in pancreatic beta-cells by releasing Ca2+ from inositol 1,4,5-trisphosphate-insensitive intracellular stores. In the present study, we have examined the role of cADPR in glucose-mediated increases in [Ca2+]i and insulin exocytosis. Dispersed ob/ob mouse beta-cell aggregates were either pressure microinjected with fura-2 salt or loaded with fura-2 acetoxymethyl ester, and [Ca2+]i was monitored by microfluorimetry. Microinjection of beta-NAD+ into fura-2-loaded beta-cells did not increase [Ca2+]i nor did it alter the cells' subsequent [Ca2+]i response to glucose. Cells microinjected with the cADPR antagonist 8NH2-cADPR increased [Ca2+]i in response to glucose equally well as those injected with cADPR. Finally, the ability of cADPR to promote exocytosis of insulin in electropermeabilized beta-cells was investigated. cADPR on its own did not increase insulin secretion nor did it potentiate Ca2+-induced insulin secretion. We conclude that cADPR neither plays a significant role in glucose-mediated increases in [Ca2+]i nor interacts directly with the molecular mechanisms regulating exocytosis of insulin in normal pancreatic beta-cells.     

The presence of Human Papillomavirus (HPV) in microinvasive in situ spinocellular carcinoma of the oral cavity. Preliminary report

To determine whether functional Ca2+ channels are present in vestibular dark cells, changes in intracellular Ca2+ concentration ([Ca2+]i) due to K+ applications were measured using the Ca(2+)-sensitive dye (fura-2) and patchclamp whole-cell recordings were made in dark cells isolated from the ampullae of the semicircular canal of the guinea pig. Exchange of the external solution with a buffer medium containing a high K+ concentration (80 mM K+ or 150 mM K+) caused a concentration-dependent increase in [Ca2+]i in vestibular dark cells. Application of 1 microM nifedipine as a Ca2+ channel antagonist completely blocked the increase in [Ca2+]i. Further treatment with 10 microM BAY K 8644 as a Ca2+ channel agonist caused an increase in [Ca2+]i. In the patch-clamp whole-cell recordings a 1-s depolarizing pulse given into the dark cell in the presence of a high barium concentration (50 mM Ba2+) induced an inward current. In determining the current-voltage relationship, a current was detected at a potential that depolarized at-50 mV and was maximal at +10 mV. This inward current was completely blocked by 1 mM La3+ as a Ca2+ channel antagonist. These findings suggest the presence of voltage-dependent Ca2+ channels in dark cells, which have a presumed function in the regulation of [Ca2+]i in the vestibular endolymph.     

Activation of stimulus-secretion coupling in pancreatic beta-cells by specific products of glucose metabolism. Evidence for privileged signaling by glycolysis

The energy requirements of most cells supplied with glucose are fulfilled by glycolytic and oxidative metabolism, yielding ATP. In pancreatic beta-cells, a rise in cytosolic ATP is also a critical signaling event, coupling closure of ATP-sensitive K+ channels (KATP) to insulin secretion via depolarization-driven increases in intracellular Ca2+ ([Ca2+]i). We report that glycolytic but not Krebs cycle metabolism of glucose is critically involved in this signaling process. While inhibitors of glycolysis suppressed glucose-stimulated insulin secretion, blockers of pyruvate transport or Krebs cycle enzymes were without effect. While pyruvate was metabolized in islets to the same extent as glucose, it produced no stimulation of insulin secretion and did not block KATP. A membrane-permeant analog, methyl pyruvate, however, produced a block of KATP, a sustained rise in [Ca2+]i, and an increase in insulin secretion 6-fold the magnitude of that induced by glucose. These results indicate that ATP derived from mitochondrial pyruvate metabolism does not substantially contribute to the regulation of KATP responses to a glucose challenge, supporting the notion of subcompartmentation of ATP within the beta-cell. Supranormal stimulation of the Krebs cycle by methyl pyruvate can, however, overwhelm intracellular partitioning of ATP and thereby drive insulin secretion.     

Effect of cilostazol, a phosphodiesterase type III inhibitor, on histamine-induced increase in [Ca2+]i and force in middle cerebral artery of the rabbit

1. The effect of cilostazol, an inhibitor of phosphodiesterase type III (PDE III), on the contraction induced by histamine was studied by making simultaneous measurements of isometric force and the intracellular concentration of Ca2+ ([Ca2+]i) in endothelium-denuded muscle strips from the peripheral part of the middle cerebral artery of the rabbit. 2. High K+ (80 mM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force. Cilostazol (10 microM) did not modify the resting [Ca2+]i, but it did significantly decrease the tonic contraction induced by high K+ without a corresponding change in the [Ca2+]i response. 3. Histamine (3 microM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force. Cilostazol (3 and 10 microM) significantly reduced both the phasic and tonic increases in [Ca2+]i and force induced by histamine, in a concentration-dependent manner. 4. Rp-adenosine-3':5'-cyclic monophosphorothioate (Rp-cAMPS, 0.1 mM), a PDE-resistant inhibitor of protein kinase A (and as such a cyclic AMP antagonist), did not modify the increases in [Ca2+]i and force induced by histamine alone, but it did significantly decrease the cilostazol-induced inhibition of the histamine-induced responses. 5. In Ca2+-free solution containing 2 mM EGTA, both histamine (3 microM) and caffeine (10 mM) transiently increased [Ca2+]i and force. Cilostazol (1-10 microM) (i) significantly reduced the increases in [Ca2+]i and force induced by histamine, and (ii) significantly reduced the increase in force but not the increase in [Ca2+]i induced by caffeine. 6. In ryanodine-treated strips, which had functionally lost the histamine-sensitive Ca2+ storage sites, histamine (3 microM) slowly increased [Ca2+]i and force. Cilostazol (3 and 10 microM) lowered the resting [Ca2+]i, but did not modify the histamine-induced increase in [Ca2+]i, suggesting that functional Ca2+ storage sites are required for the cilostazol-induced inhibition of histamine-induced Ca2+ mobilization. 7. The [Ca2+]i-force relationship was obtained in ryanodine-treated strips by applying ascending concentrations of Ca2+ (0.16-2.6 mM) in Ca2+-free solution containing 100 mM K+. Histamine (3 microM) shifted the [Ca2+]i-force relationship to the left and increased the maximum Ca2+-induced force. Under the same conditions, whether in the presence or absence of 3 microM histamine, cilostazol (3-10 microM) shifted the [Ca2+]i-force relationship to the right without producing a change in the maximum Ca2+-induced force. 8. It is concluded that, in smooth muscle of the peripheral part of the rabbit middle cerebral artery, cilostazol attenuates the histamine-induced contraction both by inhibiting histamine-induced Ca2+ mobilization and by reducing the myofilament Ca2+ sensitivity. It is suggested that the increase in the cellular concentration of cyclic AMP that will follow the inhibition of PDE III may play an important role in the cilostazol-induced inhibition of the histamine-contraction.     

Delayed rectifier K+ channel overexpression in transgenic islets and beta-cells associated with impaired glucose responsiveness

Glucose stimulation of pancreatic beta-cell insulin secretion is closely coupled to alterations in ion channel conductances and intracellular Ca2+ ([Ca2+]i). To further examine this relationship after augmentation of voltage-dependent K+ channel expression, transgenic mice were produced which specifically overexpress a human insulinoma-derived, tetraethylammonium (TEA)-insensitive delayed rectifier K+ channel in their pancreatic beta-cells as shown by immunoblot of isolated islets and immunohistochemical analysis of pancreas sections. Whole-cell current recordings confirmed the presence of high amplitude TEA-resistant K+ currents in transgenic islet cells, whose expression correlated with hyperglycemia and hypoinsulinemia. Stable overexpression of the channel in insulinoma cells attenuated glucose-activated increases in [Ca2+]i and prevented the induction of TEA-dependent [Ca2+]i oscillations. These results, employing the first ion channel transgenic mouse, demonstrate the importance of membrane potential regulation in excitation-secretion coupling in the pancreatic beta-cell.     

Adenosine modulates cell proliferation in human colonic adenocarcinoma. I. Possible involvement of adenosine A1 receptor subtypes in HT29 cells

The modality of the insulinotropic action of 1,1-dimethyl-2-[2-morpholinophenyl]guanidine fumarate (BTS 67 582), a new antidiabetic agent, was investigated in rat pancreatic islets. At a 0.1 mM concentration, which was sufficient to cause a close-to-maximal secretory response, BTS 67 582 failed to affect the utilization and oxidation of exogenous D-glucose, but slightly augmented 14CO2 production from islets prelabelled with either L-[U-14C]glutamine or [U-14C]palmitate. BTS 67 582 (0.1 mM) also failed to affect biosynthetic activity in islets incubated with L-[4-3H]phenylalanine. It augmented insulin release from islets incubated for 90 min in the absence or presence of D-glucose (2.8 to 16.7 mM), this coinciding with stimulation of 45Ca net uptake. In perifused islets deprived of extracellular D-glucose for 45 min, BTS 67 582 (0.1 mM) decreased 86Rb outflow from prelabelled islets, but failed to increase 45Ca efflux and insulin release. In the presence of D-glucose (7.0 mM), BTS 67 582, whilst failing to decrease 86Rb+ outflow, provoked rapid, sustained and rapidly reversible increases of both 45Ca2+ efflux and insulin output. The latter increases were attenuated, but not totally suppressed, in the absence of extracellular Ca2+. BTS 67 582 (0.1 mM) suppressed the inhibitory action of diazoxide (0.25 mM) upon glucose-stimulated insulin release, but nevertheless augmented insulin output from islets incubated in the presence of 90 mM K+. These findings support the view that the insulinotropic action of BTS 67 582 is mainly attributable to the inactivation of ATP-sensitive K+ channels. An intracellular redistribution of Ca2+ ions may also participate, however, to the islet functional response to BTS 67 582.     

Defective pancreatic beta-cell glycolytic signaling in hepatocyte nuclear factor-1alpha-deficient mice

Mutations in the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene cause maturity onset diabetes of the young type 3, a form of type 2 diabetes mellitus. In mice lacking the HNF-1alpha gene, insulin secretion and intracellular calcium ([Ca2+]i) responses were impaired following stimulation with nutrient secretagogues such as glucose and glyceraldehyde but normal with non-nutrient stimuli such as potassium chloride. Patch clamp recordings revealed ATP-sensitive K+ currents (KATP) in beta-cells that were insensitive to suppression by glucose but normally sensitive to ATP. Exposure to mitochondrial substrates suppressed KATP, elevated [Ca2+]i, and corrected the insulin secretion defect. NAD(P)H responses to glucose were substantially reduced, and inhibitors of glycolytic NADH generation reproduced the mutant phenotype in normal islets. Flux of glucose through glycolysis in islets from mutant mice was reduced, as a result of which ATP generation in response to glucose was impaired. We conclude that hepatocyte nuclear factor-1alpha diabetes results from defective beta-cell glycolytic signaling, which is potentially correctable using substrates that bypass the defect.     

Direct effects of diazoxide on mitochondria in pancreatic B-cells and on isolated liver mitochondria

1. The direct effects of diazoxide on mitochondrial membrane potential, Ca2+ transport, oxygen consumption and ATP generation were investigated in mouse pancreatic B-cells and rat liver mitochondria. 2. Diazoxide, at concentrations commonly used to open adenosine 5'-triphosphate (ATP)-dependent K+-channels (K(ATP) channels) in pancreatic B-cells (100 to 1000 microM), decreased mitochondrial membrane potential in mouse intact perifused B-cells, as evidenced by an increase of rhodamine 123 fluorescence. This reversible decrease of membrane potential occurred at non-stimulating (5 mM) and stimulating (20 mM) glucose concentrations. 3. A decrease of mitochondrial membrane potential in perifused B-cells was also caused by pinacidil, but no effect could be seen with levcromakalim (500 microM each). 4. Measurements by a tetraphenylphosphonium-sensitive electrode of the membrane potential of rat isolated liver mitochondria confirmed that diazoxide decreased mitochondrial membrane potential by a direct action. Pretreatment with glibenclamide (2 microM) did not antagonize the effects of diazoxide. 5. In Fura 2-loaded B-cells perifused with the Ca2+ channel blocker, D 600, a moderate, reversible increase of intracellular Ca2+ concentration could be seen in response to 500 microM diazoxide. This intracellular Ca2+ mobilization may be due to mitochondrial Ca2+ release, since the reduction of membrane potential of isolated liver mitochondria by diazoxide was accompanied by an accelerated release of Ca2+ stored in the mitochondria. 6. In the presence of 500 microM diazoxide, ATP content of pancreatic islets incubated in 20 mM glucose for 30 min was significantly decreased by 29%. However, insulin secretion from mouse perifused islets induced by 40 mM K+ in the presence of 10 mM glucose was not inhibited by 500 microM diazoxide, suggesting that the energy-dependent processes of insulin secretion distal to Ca2+ influx were not affected by diazoxide at this concentration. 7. The effects of diazoxide on oxygen consumption and ATP production of liver mitochondria varied depending on the respiratory substrates (5 mM succinate, 10 mM alpha-ketoisocaproic acid, 2 mM tetramethyl phenylenediamine plus 5 mM ascorbic acid), indicating an inhibition of respiratory chain complex II. Pinacidil, but not levcromakalim, inhibited alpha-ketoisocaproic acid-fuelled ATP production. 8. In conclusion, diazoxide directly affects mitochondrial energy metabolism, which may be of relevance for stimulus-secretion coupling in pancreatic B-cells.     

Glucose and tolbutamide induce apoptosis in pancreatic beta-cells. A process dependent on intracellular Ca2+ concentration

High concentrations of glucose are considered to be toxic for the pancreatic beta-cell. However, the mechanisms underlying beta-cell dysfunction and resulting cell death are not fully characterized. In the present study we have demonstrated that incubation of pancreatic islets and beta-cells from ob/ob mice and Wistar rats with glucose induced a process of apoptotic beta-cell death, as shown by DNA laddering, TdT-mediated dUTP-biotin nick end-labeling (TUNEL) technique, and by using DNA-staining dye HOECHST 33342. The obtained results show that the percentage of apoptotic cells was dependent on glucose concentration, being minimal at 11 mM glucose. At a concentration of 100 microM, aurintricarboxylic acid, an inhibitor of endonuclease activity, almost completely inhibited apoptosis triggered by 17 mM glucose. We have also shown that long term incubation with 100 microM sulfonylurea, tolbutamide, triggered apoptosis in pancreatic beta-cells. The process of beta-cell death induced by high glucose concentration and tolbutamide were Ca2+-dependent, because introduction to the culture medium of 50 microM D-600 or 200 microM diazoxide, which blocked glucose- and tolbutamide-induced [Ca2+]i increase, inhibited apoptosis. Thus, this study shows for the first time that high glucose concentrations and tolbutamide induce apoptosis in pancreatic beta-cells, and that this process is Ca2+-dependent.     

Studies on the microsomal cytochrome P450s in the livers of carcinogen-resistant and sensitive rats during long-term administration of 3''-methyl-4-dimethylaminoazobenzene

The fluorescent indicator Fura-2 was used to characterize the store-operated Ca2+ entry in insulin-releasing pancreatic beta-cells. To avoid interference with voltage-dependent Ca2+ entry, the cells were hyperpolarized with 400 microM diazoxide and the channel blocker methoxyverapamil was also present in some experiments. The cytoplasmic Ca2+ concentration ([Ca2+]j) of hyperpolarized mouse beta-cells was strikingly resistant to changes in external Ca2+. In cells exposed to 20 mM glucose, stimulation with 100 microM carbachol induced an initial [Ca2+]j peak followed by a sustained increase due to store-operated influx of the cation. Store-operated influx was also induced by the intracellular Ca(2+)-ATPase inhibitor thapsigargin. In the presence of store-operated influx, [Ca2+]j became markedly sensitive to variations in external Ca2+, but this sensitivity was blocked by La3+. In beta-cells exposed to both Ca2+ and Mn2+ there was slow Mn2+ quenching of the Fura-2 fluorescence, which was accelerated upon stimulation of store-operated influx. This acceleration was reversed by glucose-stimulated filling of the internal Ca2+ stores. The store-operated Ca2+ entry increased markedly during culture of the beta-cells. Activation of protein kinase C by the phorbol ester 12-O-tetradecanoylphorbol-13 acetate, inhibition of serine/threonine phosphatase by okadaic acid and inhibition of tyrosine kinase by genistein had little effect on the store-operated influx of Ca2+. In beta-cells equilibrated in 5 mM Sr2+, carbachol exposure resulted in a pronounced cytoplasmic Sr2+ ([Sr2+]j) peak due to intracellular mobilization, but little or no sustained elevation. Moreover, after activating the store-operated pathway by exposure to thapsigargin, variations in extracellular Sr2+ between 0-2 mM had only marginal effects on [Sr2+]j. Although the store-operated influx apparently accounts for a minor fraction of the Ca2+ entry, its depolarizing influence may under certain conditions be up-regulated with resulting distortion of the beta-cell function.     

Dendritic cell development: multiple pathways to nature''s adjuvants

The endothelin (ET) isoforms ET-1, ET-2 and ET-3 applied at 100 nM triggered a transient increase in [Ca2+]i in Bergmann glial cells in cerebellar slices acutely isolated from 20-25 day-old mice. The intracellular calcium concentration ([Ca2+]i) was monitored using Fura-2-based [Ca2+]i microfluorimetry. The ET-triggered [Ca2+]i transients were mimicked by ETB receptor agonist BQ-3020 and were inhibited by ETB receptor antagonist BQ-788. ET elevated [Ca2+]i in Ca(2+)-free extracellular solution and the ET-triggered [Ca2+]i elevation was blocked by 500 nM thapsigargin indicating that the [Ca2+]i was released from InsP3-sensitive intracellular pools. The ET-triggered [Ca2+]i increase in Ca(2+)-free solution was shorter in duration. Restoration of normal extracellular [Ca2+] briefly after the ET application induced a second [Ca2+]i increase indicating the presence of a secondary Ca2+ influx which prolongs the Ca2+ signal. Pre-application of 100 microM ATP or 10 microM noradrenaline blocked the ET response suggesting the involvement of a common Ca2+ depot. The expression of ETB receptor mRNAs in Bergmann glial cells was revealed by single-cell RT-PCR. The mRNA was also found in Purkinje neurones, but no Ca2+ signalling was triggered by ET. We conclude that Bergmann glial cells are endowed with functional ETB receptors which induce the generation of intracellular [Ca2+]i signals by activation of Ca2+ release from InsP3-sensitive intracellular stores followed by a secondary Ca2+ influx.     

Mastoparan stimulates exocytosis at a Ca(2+)-independent late site in stimulus-secretion coupling. Studies with the RINm5F beta-cell line

Mastoparan, a tetradecapeptide from wasp venom, stimulated exocytosis in a concentration-dependent manner, which was enhanced by pertussis toxin pre-treatment, in the insulin secreting beta-cell line RINm5F. Mastoparan (3-20 microM) also elevated cytosolic free calcium concentration ([Ca2+]i), a rise that was not attenuated by nitrendipine. Divalent cation-free Krebs-Ringer bicarbonate (KRB) medium with 0.1 mM EGTA nullified the mastoparan-induced increase in [Ca2+]i, suggesting that the peptide increased Ca2+ influx but not through the L-type voltage-dependent Ca2+ channel. Depletion of the intracellular Ca2+ pool did not affect the mastoparan-induced elevation of [Ca2+]i. Remarkably, in divalent cation-free KRB medium with 0.1 mM EGTA and 2 microM thapsigargin in which mastoparan reduced [Ca2+]i, the mastoparan-stimulated insulin release was similar to that in normal Ca(2+)-containing KRB medium. Inhibitors of protein kinase C, such as bisindolylmaleimide, staurosporine, and 1-O-hexadecyl-2-O-methyl-rac-glycerol did not suppress the mastoparan-stimulated insulin release. Mastoparan at 10-20 microM did not increase cellular cAMP levels, nor did mastoparan at 5-10 microM affect [3H]arachidonic acid release. In conclusion, although mastoparan increased [Ca2+]i, this increase was not involved in the stimulation of insulin release. Rather, the data suggest that mastoparan directly stimulates exocytosis in a Ca(2+)-independent manner. As GTP-binding proteins (G proteins) are thought to be involved in the process of exocytosis and as mastoparan is known to exert at least some of its effects by activation of G proteins, an action of mastoparan to activate the putative stimulatory Ge (exocytosis) protein is likely.