Glucose stimulates glucagon release in single rat alpha-cells by mechanisms that mirror the stimulus-secretion coupling in beta-cells

In isolated rat pancreatic alpha-cells, glucose, arginine, and the sulfonylurea tolbutamide stimulated glucagon release. The effect of glucose was abolished by the KATP-channel opener diazoxide as well as by mannoheptulose and azide, inhibitors of glycolysis and mitochondrial metabolism. Glucose inhibited KATP-channel activity by 30% (P<0.05; n=5) and doubled the free cytoplasmic Ca2+ concentration. In cell-attached recordings, azide opened KATP channels. The N-type Ca2+-channel blocker omega-conotoxin and the Na+-channel blocker tetrodotoxin inhibited glucose-induced glucagon release whereas tetraethylammonium, a blocker of delayed rectifying K+ channels, increased secretion. Glucagon release increased monotonically with increasing K+ concentrations. omega-Conotoxin suppressed glucagon release to 15 mM K+, whereas a combination of omega-conotoxin and an L-type Ca2+-channel inhibitor was required to abrogate secretion in 50 mM K+. Recordings of cell capacitance revealed that glucose increased the exocytotic response evoked by membrane depolarization 3-fold. This correlated with a doubling of glucagon secretion by glucose in intact rat islets exposed to diazoxide and high K+. In whole-cell experiments, exocytosis was stimulated by reducing the cytoplasmic ADP concentration, whereas changes of the ATP concentration in the physiological range had little effect. We conclude that glucose stimulates glucagon release from isolated rat alpha-cells by KATP-channel closure and stimulation of Ca2+ influx through N-type Ca2+ channels. Glucose also stimulated exocytosis by an amplifying mechanism, probably involving changes in adenine nucleotides. The stimulatory action of glucose in isolated alpha-cells contrasts with the suppressive effect of the sugar in intact islets and highlights the primary importance of islet paracrine signaling in the regulation of glucagon release.     

Muscarinic control of pancreatic B cell function involves sodium-dependent depolarization and calcium influx

Mouse pancreatic islets were used to investigate the mechanisms and functional significance of the B cell membrane depolarization by acetylcholine (ACh). At low glucose (3mM), ACh (20 microM) increased 22Na+ influx, and slightly depolarized the B cell membrane but did not induce electrical activity or stimulate 45Ca2+ influx. ACh also accelerated 86Rb+ and 45Ca2+ efflux and barely affected basal insulin release. At a stimulatory concentration of glucose (10 mM), ACh stimulated 22Na+ influx, depolarized the B cell membrane, increased glucose-induced electrical activity, and stimulated 45Ca2+ influx. ACh also accelerated 86Rb+ and 45Ca2+ efflux and strongly potentiated insulin release. Omission of extracellular Ca2+ did not impair ACh stimulation of 22Na+ influx or 86Rb+ efflux, slightly modified the acceleration of 45Ca2+ efflux, and almost completely suppressed the increase in insulin release. Na+ omission (with N-methyl-D-glucamine as substitute) prevented the B cell membrane depolarization and the stimulation of 45Ca2+ influx, largely inhibited the acceleration of 86Rb+ efflux and insulin release, and suppressed the late phase of 45Ca2+ efflux otherwise produced by ACh. On the other hand, ACh stimulation of 3H efflux from islets prelabeled with myo-[2-3H]inositol was not affected by Na+ omission. All effects of ACh were blocked by atropine and unaffected by nicotinic antagonists. It is concluded that activation of muscarinic receptors depolarized the B cell membrane by increasing its permeability to Na+. When the membrane is already depolarized by glucose, this further depolarization augments Ca2+ influx and, hence, potentiates insulin release.     

Regulation of calcium fluxes in pancreatic islets: the role of membrane depolarization

The effect of K+-induced depolarization on calcium fluxes and insulin release from isolated islets were investigated in order to elucidate the mechanism by which glucose initially reduces and later increases 45Ca efflux from prelabeled and perifused rat pancreatic islets. Raising the extracellular K+ concentration from 5.0 to 20.0 mM produced a 2- to 3-fold increase in 45Ca net uptake and efflux from isolated islets. The latter effect was dependent on the presence of extracellular Ca2+, suggesting that it resulted from the entry of calcium into the islet cells. In the presence of 20 mM K+, 16.7 mM glucose failed to stimulate 45Ca efflux, while 20 mM K+ further enhanced 45Ca efflux from islets perifused in the presence of the high concentration of glucose. These findings suggest that the effect of glucose to stimulate 45Ca efflux from perifused islets depends mainly on the glucose-induced depolarization of the cell membrane. In the absence of extracellular calcium, 20 mM K+ failed to mimick the effect of glucose to reduce 45Ca efflux. Glucose (16.7 mM) decreased 45Ca efflux from islets perifused in the presence of 20 mM K+ and antagonized the effect of 20 mM K+ to stimulate 45Ca efflux from perifused islets. It is concluded that K+-induced plasma membrane depolarization reproduces the effect of glucose to stimulate but not to inhibit 45Ca efflux from perifused islets.     

Glucose regulation of insulin secretion independent of the opening or closure of adenosine triphosphate-sensitive K+ channels in beta cells

Two major pathways are implicated in the stimulation of insulin secretion by glucose. The K+-ATP channel-dependent pathway involves closure of these channels, depolarization of the beta-cell membrane, acceleration of Ca2+ influx, and a rise in cytosolic free Ca2+ ([Ca2+]i). The K+-ATP channel-independent pathway potentiates the stimulation of exocytosis by high [Ca2+]i. To determine whether this second pathway is influenced by the configuration of the channel, we compared the effects of glucose on [Ca2+]i and insulin secretion in mouse islets under three conditions. First, in the presence of 20, 25, and 30 mM K+, i.e. without pharmacological action on K+-ATP channels, [Ca2+]i and insulin secretion were already elevated at 3 mM glucose. High glucose (20 mM) caused a transient decrease in [Ca2+]i followed by an ascent to slightly above control levels, and rapidly stimulated insulin secretion. Second, opening of K+-ATP channels with diazoxide did not influence [Ca2+]i and insulin secretion at 3 mM glucose and high K+. However, high glucose now caused a sustained lowering of [Ca2+]i accompanied by a slow increase in secretion that augmented with the K+ concentration. Third, when K+-ATP channels were blocked and beta-cells depolarized by high concentrations of tolbutamide or glibenclamide, [Ca2+]i and insulin secretion were elevated even in low glucose. High glucose transiently lowered [Ca2+]i, which then increased to or slightly above control levels, while insulin secretion was rapidly stimulated. Under all conditions the correlation between [Ca2+]i and insulin secretion was excellent at low and high glucose levels, and high glucose increased release at all [Ca2+]i. The potentiation of Ca2+-induced exocytosis by glucose is thus independent of the closed or open state of K+-ATP channels. It is only when the channels are opened by diazoxide that the increase in release is a strict amplification of the action of Ca2+. When the channels are closed (sulfonylureas) or still closable (high K+ alone), the effect of glucose on secretion also comprises a slight increase in [Ca2+]i and, in the latter case, is not strictly K+-ATP channel independent.     

Modulation of the effect of acetylcholine on insulin release by the membrane potential of B cells

Mouse islets were used to test the hypothesis that the B cell membrane must be depolarized for acetylcholine to increase insulin release. The resting membrane potential of B cells (at 3 mM glucose) was slightly decreased (5 mV) by acetylcholine, but no electrical activity appeared. This depolarization was accompanied by a Ca-independent acceleration of 86Rb and 45Ca efflux but no insulin release. When the B cell membrane was depolarized by a stimulatory concentration of glucose (10 mM), acetylcholine potentiated electrical activity, accelerated 86Rb and 45Ca efflux, and increased insulin release. This latter effect, but not the acceleration of 45Ca efflux, was totally dependent on extracellular Ca. If glucose-induced depolarization of the B cell membrane was prevented by diazoxide, acetylcholine lost all effects but those produced at low glucose. In contrast, when the B cell membrane was depolarized by leucine or tolbutamide (at 3 mM glucose), acetylcholine triggered a further depolarization with appearance of electrical activity, accelerated 86Rb and 45Ca efflux, and stimulated insulin release. Acetylcholine produced similar effects (except for electrical activity) in the presence of high K or arginine which, unlike the above test agents, depolarize the B cell membrane by a mechanism other than a decrease in K+ permeability. Omission of extracellular Ca abolished the releasing effect of acetylcholine under all conditions but only partially decreased the stimulation of 45Ca efflux. The results show thus that acetylcholine stimulation of insulin release does not result from mobilization of cellular Ca but requires that the B cell membrane be sufficiently depolarized to reach the threshold potential where Ca channels are activated. This may explain why acetylcholine alone does not initiate release but becomes active in the presence of a variety of agents.     

Interactions of maitotoxin with voltage-sensitive calcium channels in cultured neuronal cells.

The dinoflagellate toxin maitotoxin (MTX) stimulated 45Ca2+ uptake in cultured NG108-15 neuroblastoma X glioma cells. Depolarizing stimuli (e.g., 50 mM K+) produced an immediate stimulation in Ca2+ uptake, whereas that produced by MTX occurred only after a lag period of about 2 min. MTX did not stimulate Ca2+ uptake into fibroblasts. Both 50 mM K+- and MTX-stimulated Ca2+ uptake was blocked by organic calcium channel antagonists (nitrendipine, D-600, diltiazem) at very low concentrations. Cd2+ was also a potent blocker. The novel dihydropyridine BAY K8644 enhanced Ca2+ uptake in the presence of 50 mM K+ but had no effect in 5 mM Ca2+. However, in the presence of MTX, BAY K8644 stimulated Ca2+ uptake in 5 mM K+. The effects of MTX were not blocked by tetrodotoxin but were decreased in Na+-free medium. MTX did not stimulate Na+ uptake into NG108-15 cells and did not alter [3H]nitrendipine binding to rat brain cortical synaptosomes. It is concluded that MTX may alter the voltage dependence of calcium-channel activation.     

Structure, function and transforming potential of the epidermal growth factor receptor

We have examined the pharmacology of the voltage-sensitive Ca2+ channels (VSCCs) that mediate gonadotropin secretion from primary cultures of rat pituitary cells, stimulated by either cell depolarization or by binding of gonadotropin-releasing hormone (GnRH). We also measured single-cell [Ca2+]i transients using fura-2 in gonadotropes identified by a reverse hemolytic plaque assay employing an antiserum to luteinizing hormone (LH). Cell depolarization evoked by either 50 mM K+ or 30 microM veratridine induced 2- to 6-fold increases in gonadotropin secretion over basal levels. GnRH caused 6- to 20-fold increases in follicle-stimulating hormone (FSH) and LH secretion, respectively, with maximal stimulation at 100 nM GnRH. K(+)- or GnRH-induced FSH release was largely prevented by co-incubation with 1 mM CdCl. Tetrodotoxin (TTX, 5 microM) prevented the veratridine-, but not the K(+)- or GnRH-induced, stimulation of FSH secretion. Nitrendipine (Ntd, 1 microM) produced 35-50% inhibition (NS) of both FSH and LH release stimulated by either 50 mM K+ or 100 nM GnRH. Ntd also inhibited the K(+)-induced [Ca2+]i rise (greater than 90%), as well as the secondary, plateau phase of the GnRH-induced elevation of [Ca2+]i (100% inhibition). Omega-conotoxin (omega-CgTx, 100 nM) partially suppressed FSH and LH release (NS) due to both K+ (33% each) and GnRH (44% and 18%, respectively). omega-CgTx showed variable effects on [Ca2+]i transients evoked by K+ or GnRH ranging from clear inhibition to no effect. We conclude that influx of extracellular Ca2+ is one of several fundamental events underlying the depolarization- or receptor-activated release of LH and FSH, and that this influx can be inhibited by dihydropyridine-sensitive ('L') Ca2+ channels. Two classes of L-channels may exist in gonadotropes, that differ in their sensitivity to omega-CgTx.     

The loss of 45Ca2+ associated with prolactin release from the tilapia (Oreochromis mossambicus) rostral pars distalis

The relationship between tritium 3H-labeled prolactin (PRL) release and the loss of tissue-associated 45Ca2+ was examined in the tilapia rostral pars distalis (RPD) using perifusion incubation under conditions which inhibit or stimulate PRL release. Depolarizing [K+] (56 mM) and hyposmotic medium (280 mOsmolal) increased both the release of [3H]PRL and the loss of 45Ca2+. The responses to high [K+] were faster and shorter in duration than those produced by reduced osmotic pressure. The depletion of Ca2+ from the incubation medium with 2 mM EGTA suppressed the [3H]PRL response evoked by high [K+] or reduced osmotic pressure. Exposing the tissues to Ca(2+)-depleted medium in the absence of high [K+] or reduced osmotic pressure produced a sharp, but brief, increase in 45Ca2+ loss. Cobalt (10(-3) M), a competitive inhibitor of calcium-mediated processes, inhibited the [3H]PRL response to hyposmotic medium and to high [K+]. Cobalt also diminished the increased loss of 45Ca2+ evoked by exposure to reduced osmotic pressure, but was ineffective in altering responses to high [K+]. Methoxyverapamil (D600; 10(-5) M), a blocker of certain voltage-sensitive Ca2+ channels, did not alter either the [3H]PRL or the 45Ca2+ responses to high [K+] and reduced osmotic pressure. Taken together with our earlier studies, the present findings suggest that exposure to high [K+] or hyposmotic medium produces rapid changes in the Ca2+ metabolism of the tilapia RPD that are linked to the stimulation of PRL secretion. Nevertheless, the increased 45Ca2+ loss, but not [3H]PRL release, upon exposure to Ca(2+)-depleted media suggests that Ca2+ loss may not always reflect intracellular events that lead to PRL release.     

Mobilization of extracellularly bound Ca2+ during high K+ and norepinephrine stimulation of the rabbit aorta

The effects of alpha-adrenergic stimulation and high K+-induced membrane depolarization on 45Ca2+ uptake and tension generation in the rabbit aorta were investigated. Tension and unidirectional 45Ca2+ uptake were increased by both stimulants. Moreover, this 'steady-state' uptake remained elevated for as long as the stimulants were present. When tissues were preincubated in 45Ca2+-containing PSS prior to the Ne or high K+ challenge the resulting Ca2+ uptake showed an 'initial burst' of uptake which was not observed in the 'steady-state' experiments. The magnitude of the 'initial burst' increased with time displaying a t1/2 for exchange of 1.25 min for both high K+ and Ne, suggesting that this Ca2+ source is shared by both stimulants. The 'initial burst' became Ca2+ saturated when [Ca2+]o was between 0.5 and 1.5 mM, was enhanced by 45Ca2+ preincubation in a solution of lowered ionic strength and was inhibited (approximately 70%) by D600 (5 X 10(-6) M). In contrast, the 'steady-state' uptake was linearly dependent on [Ca2+]o up to 6.4 mM, was 90% inhibited by 5 X 10(-6) M D600 and was unaffected by lowered ionic strength. It is concluded that the properties displayed by the Ca2+ source responsible for the 'initial burst' and 'steady-state' uptake suggest that they are of distinct origin; the 'initial burst' being derived from a bound extracellular Ca2+ pool and the 'steady-state' uptake resulting from the uptake of free Ca2+ dissolved in the extracellular space.     

Effects of verapamil and nifedipine on gliclazide-induced increase in cytosolic free Ca2+ in pancreatic islet cells

The changes in cytosolic free calcium [Ca2+]i induced by the sulfonylurea gliclazide and potassium in normal rat pancreatic islet cells were measured using the fluorescent Ca2+ indicator fura-2. Both in the absence or presence of 5.6 mM glucose, gliclazide caused a rapid and sustained increase in [Ca2+]i. The phenylalkylamine verapamil reduced these increases, but the Ca2+ channel blocker was more potent in the presence than in the absence of glucose. In contrast, nifedipine, a Ca2+ channel blocker of another chemical type, reduced to a similar extent the increase in [Ca2+]i evoked by gliclazide in the absence and presence of glucose. In the absence of glucose, a rise in extracellular K+ concentration from 5 to 20 or 30 mM also induced a rapid and sustained rise in [Ca2+]i. Verapamil more markedly reduced the rise in [Ca2+]i induced by 30 mM than by 20 mM K+. It is concluded that gliclazide increases Ca2+ inflow into normal islet cells primarily, if not exclusively, by opening voltage-sensitive Ca2+ channels. The differential sensitivity toward verapamil of gliclazide-induced rise in [Ca2+]i can be explained by the use-dependent block exerted by Ca2+ channel blockers of the phenylalkylamine type.     

Effect of secretagogues on cytosolic free Ca2+ and insulin release at different extracellular Ca2+ concentrations in the hamster clonal beta-cell line HIT-T15

We have examined the relationship between extracellular Ca2+, cytosolic free Ca2+ and insulin release in the clonal beta-cell line HIT-T15. Glucose-stimulated insulin release was dependent on the extracellular Ca2+ concentration in a dose-related manner; the threshold medium Ca2+ concentration for glucose-stimulated insulin release was 0.5 mM. Both forskolin and 12-O-tetradecanoylphorbol 13-acetate (TPA) increased insulin release in the presence of glucose at all extracellular Ca2+ concentration tested (0.1-2.5 mM) but not in the absence of Ca2+. Thus, the threshold medium Ca2+ concentration for glucose-stimulated insulin release was reduced to 0.1 mM by forskolin or TPA. Step-wise increases in the medium Ca2+ concentration in the presence of an initiator of insulin release resulted in a dose-related increase in cytosolic free Ca2+. In the presence of 10 mM glucose, cytosolic free Ca2+ in HIT cells was increased from 60 +/- 5 nM in Ca2+-free medium to 290 +/- 46 nM in medium containing 2.5 mM Ca2+. The effects of increasing extracellular Ca2+ in the presence of 40 mM K+ were similar but considerably more pronounced. Inclusion of either TPA or forskolin in the incubation medium had no significant effect on the steady-state cytosolic free Ca2+ levels in the absence of glucose but in the presence of 10 mM glucose forskolin caused modest (11-18%) increases in steady-state cytosolic free Ca2+ levels at extracellular Ca2+ concentrations of 0.25 mM or above. In contrast, in the presence of glucose TPA significantly reduced the steady-state levels of cytosolic free Ca2+ by 17-21% at extracellular Ca2+ concentrations of 0.25 mM or above. These data provide further evidence that insulin release mediated by activation of beta-cell protein kinases involves primarily an increase in sensitivity of the secretory system to intracellular Ca2+.     

Effect of W-7 on ionic fluxes and electrical activity of mouse pancreatic islets

W-7 (N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide) (0.1 mM), a calmodulin inhibiting compound, suppressed the reincrease of 86Rb+ efflux from pancreatic islets normally seen in response to lowering the glucose concentration from stimulated to basal value. Ionophore (A23187)-induced increase was completely abolished. W-7 inhibited 45Ca2+ uptake and stimulation of 45Ca2+ efflux in response to glucose (11.1 mM) but did not affect K+ (20 mM)-induced 45Ca2+ uptake. Electrical activity of B-cells at 11.1 mM glucose showed a prolongation in burst length in the presence of 0.1 mM W-7. The data suggest that W-7 affects the opening properties of K+ channels resulting in a delayed repolarisation of the cells possibly through its inhibitory action on Ca2(+)-activated calmodulin.     

Phorbol esters potentiate rapid dopamine release from median eminence and striatal synaptosomes

In the present study, we investigated the ability of phorbol esters to potentiate Ca2+-dependent depolarization-induced release of tritium-labeled dopamine ([3H]DA) from median eminence and striatal synaptosomes. Phorbol esters potentiated [3H]DA release in a concentration-dependent manner in both kinds of dopaminergic nerve terminals and with a potency series similar to that reported for stimulation of protein kinase-C (PKC) activity in other cell systems. Evoked [3H]DA release was increased by 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-7) M) after 1, 3, 5, and 10 sec of depolarization. The effect of TPA was suppressed by sphingosine, a PKC inhibitor. TPA enhanced [3H]DA release evoked by high K+, veratridine or the Ca2+ ionophore A23187. Phorbol ester potentiation was found to be depolarization dependent, as it was present from 30-75 mM, but not at 5-20 mM external K+. Potentiation was seen at all external Ca2+ concentrations studied between 0.01-3 mM. However, in the absence of external free Ca2+ (i.e. with 0.1 mM EGTA), the phorbol effect was not present. These data indicate that an increase in intrasynaptosomal Ca2+ concentration is necessary for the enhancement of [3H]DA release by phorbol esters to occur. The combination of TPA and the Ca2+ ionophore A23187 does not show the marked synergism observed in some other systems, that is maximal release was not reinstated. This suggests that in dopaminergic nerve terminals, activation of PKC has a modulatory, rather than a mediating, effect on release. Recently, we have shown that hyperprolactinemia stimulated [3H]DA release from median eminence synaptosomes by an external Ca2+-independent mechanism which might involve the PKC pathway. However, in the present work we found that the TPA and PRL effects on evoked [3H]DA release were additive, suggesting that two independent mechanisms are involved. A marked difference in the sensitivity of median eminence and striatal synaptosomes to calcium ionophore was discovered. The concentration of A23187 required to support significant [3H]DA release from median eminence synaptosomes was 3-fold greater than that in striatal synaptosomes. This suggests that some difference in calcium homeostatic processes exists, such as a higher resting striatal Ca2+ concentration, in these two kinds of dopaminergic nerve terminals. These data support the hypothesis that PKC activation potentiates the intrasynaptosomal stimulus-secretion coupling mechanism(s) and that nigrostriatal and tuberoinfundibular dopaminergic nerve terminals are affected by phorbol esters in a similar manner.     

Is there a role for osmotic events in the exocytotic release of insulin?

The possible role of an osmotic lysis of insulin granules during exocytosis has been studied in perifused mouse pancreatic islets. Raising the osmolarity of the extracellular medium by addition of 400 mM sucrose reversibly inhibited glucose-stimulated insulin release. This inhibition was accompanied by a decrease in the rates of 86Rb+ or 45Ca2+ efflux from the islets. Increasing the osmolarity and restoring a normotonic medium in the presence of a nonstimulatory concentration of glucose accelerated 86Rb+ and 45Ca2+ efflux and augmented basal insulin release in both the presence and absence of Ca2+. Hyperosmolarity did not prevent a rise in glucose concentration from decreasing 86Rb+ efflux from islet cells or from inhibiting 45Ca2+ efflux in Ca2+-free medium. However, the stimulation of 45Ca2+ efflux otherwise produced by glucose in the presence of Ca2+ was abolished, and the stimulation of insulin release was almost suppressed. Hyperosmolarity also strongly impaired the release of insulin during stimulation by eight experimental conditions known to act through at least partially different mechanisms. The changes in 45Ca2+ efflux brought about by these different agents were also altered by hyperosmolarity, whether they resulted from direct mobilization of intracellular Ca2+ or were secondary to increased Ca2+ influx. The blockade of insulin release by hyperosmolarity, whatever the mode of action of the stimulus, is compatible with the participation of osmotic events in exocytosis. However, the marked alterations in Ca2+ handling that occur concomitantly and might account for the inhibition of release make it impossible to demonstrate their exact role in intact islet cells.     

Effect of low temperatures on glucose-induced insulin secretion and ionic fluxes in rat pancreatic islets

The direct effect of cold on the inhibition of B cell secretion is well known in hibernating and experimentally hypothermic mammals. This temperature dependency may result from the inhibition of ion transport across the membranes. In order to verify this hypothesis, ionic effluxes and insulin secretion from rat islets loaded with 86Rb+ and 45Ca+ were measured during perifusion. At 37 degrees C, the rise in glucose concentration from zero to 16.7 mmol/l provoked a rapid decrease in 86Rb+ efflux, an early fall and subsequent rise in 45Ca2+ efflux and a typical biphasic pattern of insulin secretion. At 27 degrees C, glucose induced only a very slight increase in insulin secretion, while the fluxes of radioactive ions were not significantly modified in amplitude but were clearly delayed. At 17 degrees C, no insulin response to glucose was observed and the decrease in K+ conductance indicated by 86Rb+ flux decrease was less temperature-dependent than the movement of Ca2+. After supplementary stimulation with a high extracellular concentration of Ca2+, insulin secretion was enhanced at 27 degrees C and reached levels induced by glucose alone at 37 degrees C. An increase in hormone secretion occurred even at 17 degrees C, but only during a first phase of secretion. Regular increases in temperature potentiated insulin secretion and provoked changes in ionic fluxes which suggest that B cell depolarization (86Rb+ flux decrease) induced by glucose can occur at 15 degrees C but cannot induce the opening of voltage-dependent Ca2+ channels (increase in 45Ca2+ efflux) until temperatures higher than 27 degrees C are reached.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of Ca2+ channels in the ability of membrane depolarization to prevent neuronal death induced by trophic-factor deprivation: evidence that levels of internal Ca2+ determine nerve growth factor dependence of sympathetic ganglion cells.

Sympathetic neurons depend on nerve growth factor (NGF) for their survival both in vivo and in vitro; these cells die upon acute deprivation of NGF. We studied the effects of agents that cause membrane depolarization on neuronal survival after NGF deprivation. High-K+ medium (greater than or equal to 33 mM) prevented cell death; the effect of K+ was dose-dependent (EC50 = 21 mM). The protection by high K+ was abolished either by withdrawal of extracellular Ca2+ or by preloading the cells with a Ca2+ chelator. The involvement of Ca2+ flux across membranes in high-K+ saving of NGF-deprived neurons was also supported by experiments using Ca2+-channel antagonists and agonists. The Ca2+ antagonists nimodipine and nifedipine effectively blocked the survival-promoting effect of high K+. The Ca2+ agonists Bay K 8644 and (S)-202-791 did not by themselves save neurons from NGF deprivation but did strongly augment the effect of high K+; EC50 was shifted from 21 mM to 13 mM. These data suggest that dihydropyridine-sensitive L-type Ca2+ channels play a major role in the high-K+ saving. The depolarizing agents choline (EC50 = 1 mM) and carbamoylcholine (EC50 = 1 microM), acting through nicotinic cholinergic receptors, also rescued NGF-deprived neurons. The saving effect of nicotinic agonists was not blocked by withdrawal of extracellular Ca2+ but was counteracted by a chelator of intracellular Ca2+, suggesting the possible involvement of Ca2+ release from internal stores. Based on these findings we propose a "Ca2+ set-point hypothesis" for the degree of trophic-factor dependence of sympathetic neurons in vitro.     

Effects of glucose, leucine and adenosine on insulin release, 45Ca2+ net uptake, NADH/NAD ratios and oxygen consumption of islets isolated from fed and starved mice

In order to elucidate further the effects of starvation on islet metabolism and insulin release, pancreatic islets of mice were isolated and incubated in the presence of various nutrient secretagogues. Starvation for 60 h completely blocked the insulin release in response to either 16.7 mM glucose or 10 mM leucine. The further addition of 20 mM adenosine partly restored the insulin response. Glucose, adenosine, glucose + adenosine, glucose + leucine or leucine + adenosine all increased the NADH/NAD ratios over basal values in islets from both fed and starved mice. No effects of starvation were observed on islet NADH/NAD ratios in any of the above media, but when islets of starved animals were incubated in the absence of any metabolic substrates the NADH/NAD ratios were decreased. In the absence of exogenous substrates the respiratory rate was also lower in islets from starved animals. Respiratory stimulation evoked by either 16.7 mM glucose or 10 mM leucine + 10 mM glutamine was lower after starvation, whereas glucose + adenosine, glucose + leucine and adenosine all induced normal respiratory responses. No differences between the 45Ca2+ uptake of islets from either starved or fed mice were observed under any conditions. It is concluded that, in starvation, a dissociation between islet insulin release and metabolism (measured as NADH/NAD ratios, oxygen consumption and 45Ca2+ uptake) may exist in the presence of certain nutrient secretagogues.     

Electrical properties of isolated rat adrenal glomerulosa and fasciculata cells

Passive and active electrical properties of isolated rat adrenal glomerulosa and fasciculata cells were studied by intracellular voltage-recording and constant current stimulation. The average resting membrane potential was -78.9 +/- 4.2 mV for glomerulosa cells and -77.8 +/- 5.0 mV for fasciculata cells. The response of the membrane potential to changes in external K+ concentration was stable and reversible for changes up to 28 mM and was independent of external Cl-. The relationship between membrane potential and the log of external K+ concentration was linear between 4 and 28 mM, and the membrane potential could be predicted by a simplified form of the constant field equation with a [K]i of 138.5 mM and a PNa/PK of 0.015 for glomerulosa cells and a [K]i of 112.4 mM and a PNa/PK of 0.011 for fasciculata cells. Under current clamp conditions, both cells demonstrated a nonlinear relationship between membrane voltage and applied current for depolarizing current steps. Depolarizing current pulses elicited a regenerative response and were followed by a rectifying steady state potential. The maximum rate of rise and the peak amplitude of the regenerative response were increased by prior hyperpolarization below the resting membrane potential and decreased by depolarization. The regenerative response was unaffected by the removal of Na+. Elevated Ca2+ concentrations increased the rate of rise, peak amplitude, and rate of fall, but decreased the duration of the regenerative response. The regenerative response was maintained upon replacement of Ca2+ with Sr2+ or Ba2+, but was inhibited by Mn2+ or Co2+. Regenerative responses elicited in both glomerulosa and fasciculata cells exhibited similar characteristics. The results suggest the ionic mechanism underlying the regenerative response to be a voltage-dependent Ca2+ conductance. Both adrenal glomerulosa and fasciculata cells demonstrate electrical properties in common with other excitable cells. They are good K+ sensors with regard to their membrane potential, approaching the maximum sensitivity expected for a membrane exclusively permeable to K+. In addition, the Ca2+ regenerative response, which has been identified in both adrenal glomerulosa and fasciculata cells, may be involved in secretagogue stimulation of steroidogenesis.     

Gonadotropin-releasing hormone-induced Ca2+ transients in single identified gonadotropes require both intracellular Ca2+ mobilization and Ca2+ influx.

We examined the effects of gonadotropin-releasing hormone (GnRH) on the intracellular free Ca2+ concentration ([Ca2+]i) in single rat anterior pituitary gonadotropes identified by a reverse hemolytic plaque assay. Concentrations of GnRH greater than 10 pM elicited increases in [Ca2+]i in identified cells but not in others. In contrast, depolarization induced by 50 mM K+ increased [Ca2+]i in all cells. Ca2+ transients induced by GnRH exhibited a complex time course. After an initial rapid rise, the [Ca2+]i fell to near basal levels only to be followed by a secondary extended rise and fall. Analysis of the Ca2+ transients on a rapid time base revealed that responses frequently consisted of several rapid oscillations in [Ca2+]i. Removal of extracellular Ca2+ or addition of the dihydropyridine Ca2+-channel blocker nitrendipine completely blocked the secondary rise in [Ca2+]i but had no effect whatsoever on the initial spike. Nitrendipine also blocked 50 mM K+-induced increases in [Ca2+]i in identified gonadotropes. The secondary rise induced by GnRH could be enhanced by a phorbol ester in a nitrendipine-sensitive fashion. Multiple spike responses to GnRH stimulation of the same cell could only be obtained if subsequent Ca2+ influx was permitted either by allowing a secondary rise to occur or by producing a Ca2+ transient by depolarizing the cells with 50 mM K+. It therefore appears that the response to GnRH consists of an initial phase of Ca2+ mobilization, probably mediated by inositol trisphosphate, and a subsequent phase of Ca2+ influx through nitrendipine-sensitive Ca2+ channels that may be activated by protein kinase C. The relative roles of these phases in the control of gonadotropin secretion are discussed.     

Time-dependent potentiation of the beta-cell is a Ca2+-independent phenomenon

When isolated rat pancreatic islets are treated with 16.7 mM glucose, a time-dependent potentiation (TDP) of insulin release occurs that can be detected by subsequent treatment with 50 mM KCl. It has been thought that TDP by glucose is a Ca2+-dependent phenomenon and only occurs when exposure to glucose is carried out in the presence of Ca2+. In contrast to this, we now demonstrate TDP under stringent Ca2+-free conditions (Ca2+-free buffer containing 1 mM EGTA). In fact, under these Ca2+-free conditions glucose caused an even stronger TDP than in the presence of Ca2+. TDP induced by glucose in the absence of extracellular Ca2+ was unaffected by inhibitors of protein kinase C (PKC). However, cerulenin or tunicamycin, two inhibitors of protein acylation, eradicated TDP without affecting glucose metabolism. The TDP by glucose was not associated with an increase in the cytosolic free Ca2+ concentration ([Ca2+]i) during subsequent treatment with high K+. Exposure of islets to forskolin under Ca(2+)-free conditions did not cause TDP despite a large increase in the cellular cAMP levels. In conclusion, glucose alone induces TDP under stringent Ca2+-free conditions when [Ca2+]i was significantly lowered. Protein acylation is implicated in the underlying mechanism of TDP.