Galanin can inhibit insulin release by a mechanism other than membrane hyperpolarization or inhibition of adenylate cyclase

Studies on the mode of action of galanin to inhibit insulin release in RINm5F cells have shown that basal and glyceraldehyde-stimulated release were both inhibited. Galanin was inhibitory at concentrations in the low nanomolar range. Binding studies with 125I-labeled galanin indicated that the RINm5F cells exhibit a single set of sites estimated to be of the order of 30,000 sites/cell. Displacement of 125I-galanin by galanin from the receptor sites occurred over a similar concentration range to that which inhibited insulin release. Half-displacement was achieved with 2 nM galanin. Measurements of bis-(1,3-diethylthiobarbiturate) trimethineoxonol (bis-oxonol) fluorescence showed that galanin hyperpolarized the RINm5F cell plasma membrane. Measurements of intracellular free calcium, [Ca2+]i by means of the fluorescent indicator fura-2 showed that galanin decreased [Ca2+]i. As galanin did not inhibit either basal or glyceraldehyde-stimulated insulin release in the presence of the Ca2+ channel blocker nitrendipine, the hyperpolarization and reduction of Ca2+ entry appear to be a possible explanation for the galanin effects. However, quantitatively, the effects on membrane potential and [Ca2+]i appear to be insufficient to account for the potent inhibition of insulin release. Furthermore, evidence for an additional mechanism of action was obtained from experiments with 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester which stimulates insulin secretion by at least two mechanisms, one Ca2+ dependent and one Ca2+ independent. TPA-stimulated insulin release was inhibited by galanin over the same concentration range as for the inhibition of glyceraldehyde-stimulated release. Galanin inhibited TPA-stimulated release in the presence of maximally effective concentrations of nitrendipine and in the absence of extracellular Ca2+. These effects cannot be explained by hyperpolarization of the plasma membrane and consequent reduction of Ca2+ entry via the voltage-dependent Ca2+ channels. One suggested mechanism for the action of galanin is inhibition of adenylate cyclase. However, it was found that galanin inhibits insulin release even in the presence of 8-Br-cAMP, an agent which effectively bypasses adenylate cyclase. Therefore, an additional mechanism for the inhibitory effect of galanin must be present. All of the effects of galanin were sensitive to pertussis toxin. These data suggest two G-protein-dependent actions of galanin, one to hyperpolarize the plasma membrane and one at a distal point in stimulus-secretion coupling, close to the exocytotic event.     

ATP-sensitive K+ channels reveal the effects of intracellular chloride variations on cytoplasmic ATP concentrations and mitochondrial function

Replacement of intracellular Cl- by impermeant anions, as well as treatment of insulinoma cells by the Cl- channel blocker, NPPB, leads to activation of ATP-dependent K+ (KATP) channels. Activation of KATP channels by C1- substitution is eliminated (i) when intracellular ATP is replaced by non-hydrolyzable ATP analogs, (ii) when the perfusion medium contains an ATP regenerating system, (iii) when the mitochondrial ATPase is blocked by oligomycin. Dinitrophenol and GDP have the same activating effects on KATP channels as NPPB or intracellular Cl- substitution. Our interpretation of the results is that NPPB and intracellular Cl- replacement produce an uncoupling of oxidative phosphorylation by acting on mitochondrial anion channels, which leads to rapid degradation of ATP and to activation of KATP channels. KATP channels are useful sensors of cytoplasmic ATP variations.     

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

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

Reconstitution of the ATP-sensitive potassium channel of skeletal muscle. Activation by a G protein-dependent process

Potassium channels inhibited by adenosine-5'-trisphosphate, K(ATP), found in the transverse tubular membrane of rabbit skeletal muscle were studied using the planar bilayer recording technique. In addition to the single-channel properties of K(ATP) we report its regulation of Mg2+ and by the guanosine-5'-trisphosphate analogue, GTP-y(gamma)-S. The K(ATP) channel (a) has a conductance of 67 pS in 250 mM internal, 50 mM external KCl, and rectifies weakly at holding potentials more positive than 50 mV, (b) is not activated by internal Ca2+ or membrane depolarization, (c) has a permeability ratio PK/PNa greater than 50, and (d) is inhibited by millimolar internal ATP. Activity of K(ATP), measured as open channel probability as a function of time, was unstable at all holding potentials and decreases continuously within a few minutes after a recording is initiated. After a decrease in activity, GTP-y-S (100 microM) added to the internal side reactivated K(ATP) channels but only transiently. In the presence of internal 1 mM Mg2+, GTP-y-S produced a sustained reactivation lasting 20-45 min. Incubation of purified t-tubule vesicles with AlF4 increased the activity of K(ATP) channels, mimicking the effect of GTP-y-S. The effect of AlF4 and the requirement of GTP-y-S plus Mg2+ for sustained channel activation suggests that a nucleotide-binding G protein regulates ATP-sensitive K channels in the t-tuble membrane of rabbit skeletal muscle.     

Galanin: structural requirements for binding and signal transduction in RINm5F insulinoma cells.

Receptors for galanin, a neuropeptide inhibiting insulin release, have been described on RINm5F insulinoma cells. To characterize structural requirements for binding and biological activity of galanin, we studied binding and inhibition of hormone stimulated intracellular cAMP-production of N-terminal galanin fragments and -analogues in RINm5F cells. Half-maximal binding and potency were the same for all peptides used. Active peptides had the following rank of potency: galanin = galanin(1-22(23)Cys) greater than galanin(1-29(4)NLe) greater than galanin(1-18) greater than galanin(1-29(7)DAla) greater than galanin(1-29(2)DTrp4NLe7DAla) greater than galanin(1-29(2)DTrp). Galanin(3-29) was inactive. Therefore the first two amino acids of the galanin molecule with the indole side chain of the tryptophane residue in the right steric position are crucial for receptor binding.     

Regulation of immunoreactive-insulin release from a rat cell line (RINm5F).

1. An insulin-producing cell line, RINm5F, derived from a rat insulinoma was studied. 2. The cellular content of immunoreactive insulin was 0.19 pg/cell, which represents approx. 1% of the insulin content of native rat beta-cells, whereas that of immunoreactive glucagon and somatostatin was five to six orders of magnitude less than that of native alpha- or delta-cells respectively. 3. RINm5F cells released 7-12% of their cellular immunoreactive-insulin content at 2.8 mM-glucose during 60 min in Krebs-Ringer bicarbonate buffer. 4. Glucose utilization was increased by raising glucose from 2.8 to 16.7 mM. There was, however, no stimulation of immunoreactive-insulin release even when glucose was increased from 2.8 to 33.4 mM. A small stimulation of release was, however, found when glucose was raised from 0 to 2.8 mM. 5. Glyceraldehyde stimulated the release of immunoreactive insulin in a dose-dependent manner. 6. At 20 mM, leucine or arginine stimulated release at 2.8 mM-glucose. 7. Raising intracellular cyclic AMP by glucagon or 3-isobutyl-1-methylxanthine stimulated release at 2.8 mM-glucose with no additional stimulation at 16.7 mM-glucose. 8. Stimulation of immunoreactive-insulin release by K+ was dose-related between 2 and 30 mM. Another depolarizing agent, ouabain, also stimulated release. 9. Adrenaline (epinephrine) inhibited both basal (2.8 mM-glucose) release and that stimulated by 30 mM-K+. 10. Raising Ca2+ from 1 to 3 mM stimulated immunoreactive-insulin release, whereas a decrease from 1 to 0.3 or to 0.1 mM-Ca2+ lowered the release. 11. These findings could reflect a relatively specific impairment in glucose handling by RINm5F cells, contrasting with the preserved response to other modulators of insulin release.     

Nitric oxide opens ATP-sensitive K+ channels through suppression of phosphofructokinase activity and inhibits glucose-induced insulin release in pancreatic beta cells

Nitric oxide (NO) is known to be a potent messenger in the intracellular signal transduction system in many tissues. In pancreatic beta cells, NO has been reported to be formed from L-arginine through NO synthase. To elucidate the effect of NO on insulin secretion and to investigate the intracellular mechanism of its effect, we have used sodium nitroprusside (SNP) as a NO donor. SNP inhibited glucose-induced insulin secretion in a dose-dependent manner, and its effect was reversed by hemoglobin, a known NO scavenger. However, glyceraldehyde- induced insulin secretion was not affected by SNP. Since the closure of ATP-sensitive K+ channels (KATP channel) has been established as a key step in glucose-induced insulin secretion, we have directly assessed the effect of SNP on KATP channel activity using the patch clamp technique. The KATP channel activity reduced by glucose was found to be reversibly activated by the addition of SNP, and this activation was able to be similarly reproduced by applying S-Nitroso-N-acetyl-DL- penicillamine (SNAP), another NO generator. Furthermore, these activating effects were completely eliminated by hemoglobin, in accordance with the reversibility in inhibition of glucose-induced insulin release. However, SNP could not affect the KATP channel suppression by ATP applied to the inside of the plasma membrane. The activation of the KATP channel by NO, therefore, seems to be due to the decreased ATP production attributable to impairment of glucose metabolism in beta cells. Since SNP exhibited no effect on glyceraldehyde-induced KATP channel inhibition, NO may disturb a glycolytic step before glyceraldehyde-3-phosphate. The KATP channel activation by 2-deoxyglucose through presumable ATP consumption due to its phosphorylation by glucokinase was, however, not affected even in the presence of SNP. But in the permeabilized beta cells made by exposure to a low concentration (0.02 U/ml) of streptolysin O (open cell-attached configuration), SNP reopens KATP channels which have been eliminated by fructose-6-phosphate, while this effect was not observed in the KATP channels inhibited by fructose-1,6-bisphosphate. On the other hand, in rat ventricular myocyte KATP channels were not activated by SNP even under a low concentration of glucose. From these observations, the inhibition of phosphofructokinase activity is probably the site responsible for the impairment of glucose metabolism induced by NO in pancreatic beta cells. NO, therefore, seems to be a factor in the deterioration of glucose-induced insulin secretion from pancreatic beta cells through a unique intracellular mechanism.     

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 muM) stimulated glucagon secretion, whereas high concentrations (>10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.     

Effects of galnon, a non-peptide galanin-receptor agonist, on insulin release from rat pancreatic islets

Galanin is a neurotransmitter peptide that suppresses insulin secretion. The present study aimed at investigating how a non-peptide galanin receptor agonist, galnon, affects insulin secretion from isolated pancreatic islets of healthy Wistar and diabetic Goto-Kakizaki (GK) rats. Galnon stimulated insulin release potently in isolated Wistar rat islets; 100 microM of the compound increased the release 8.5 times (p<0.001) at 3.3 mM and 3.7 times (p<0.001) at 16.7 mM glucose. Also in islet perifusions, galnon augmented several-fold both acute and late phases of insulin response to glucose. Furthermore, galnon stimulated insulin release in GK rat islets. These effects were not inhibited by the presence of galanin or the galanin receptor antagonist M35. The stimulatory effects of galnon were partly inhibited by the PKA and PKC inhibitors, H-89 and calphostin C, respectively, at 16.7 but not 3.3 mM glucose. In both Wistar and GK rat islets, insulin release was stimulated by depolarization of 30 mM KCl, and 100 microM galnon further enhanced insulin release 1.5-2 times (p<0.05). Cytosolic calcium levels, determined by fura-2, were increased in parallel with insulin release, and the L-type Ca2+-channel blocker nimodipine suppressed insulin response to glucose and galnon. In conclusion, galnon stimulates insulin release in islets of healthy rats and diabetic GK rats. The mechanism of this stimulatory effect does not involve galanin receptors. Galnon-induced insulin release is not glucose-dependent and appears to involve opening of L-type Ca2+-channels, but the main effect of galnon seems to be exerted at a step distal to these channels, i.e., at B-cell exocytosis.     

Aluminum enhances the stimulatory effect of NaF on prostaglandin E2 synthesis in a clonal osteoblast-like cell line, MOB 3-4, in vitro

The effect of NaF on prostaglandin E2 (PGE2) synthesis in a clonal osteoblast-like cell line, MOB 3-4, was examined in the presence of Al3+. The MOB 3-4 cell line, which was derived from neonatal mouse calvaria, displays many osteoblastic characteristics, including the biosynthesis of PGE2. In the absence of Al3+, 1 mM NaF increased PGE2 synthesis (per well) to about 340% of the control level, whereas NaF at lower concentrations (below 0.1 mM) did not show such a significant effect. In the presence of 10 microM Al3+, NaF concentrations ranging from 0.01 to 1 mM increased PGE2 synthesis in a dose-dependent manner, though 10 microM Al3+ had no effect by itself. Similar effects were observed on alkaline phosphatase (ALP) activity per well, but a stimulatory effect of NaF on protein synthesis was observed only in the presence of 10 microM Al3+. These data demonstrated that PGE2 synthesis per protein was increased by NaF alone, and this effect was markedly enhanced by the addition of AlCl3. ALP activity per protein was, however, significantly increased by NaF in the absence of AlCl3. Taken together with our previous finding that Al3+ enhances the NaF-induced Ca2+ mobilization in MOB 3-4 cells, these results suggest that F- combined with Al3+ (i.e., AlF4-) is a more potent stimulator of PGE2 synthesis in cells than F- alone, and that the AlF4- -enhanced PGE2 synthesis may be caused by an increase in cytosolic free Ca2+ concentration during activation of the G protein by AlF4-.     

AlF4- reversibly inhibits ''P''-type cation-transport ATPases, possibly by interacting with the phosphate-binding site of the ATPase.

The only known cellular action of AlF4- is to stimulate the G-proteins. The aim of the present work is to demonstrate that AlF4- also inhibits 'P'-type cation-transport ATPases. NaF plus AlCl3 completely and reversibly inhibits the activity of the purified (Na+ + K+)-ATPase (Na+- and K+-activated ATPase) and of the purified plasmalemmal (Ca2+ + Mg2+)-ATPase (Ca2+-stimulated and Mg2+-dependent ATPase). It partially inhibits the activity of the sarcoplasmic-reticulum (Ca2+ + Mg2+)-ATPase, whereas it does not affect the mitochondrial H+-transporting ATPase. The inhibitory substances are neither F- nor Al3+ but rather fluoroaluminate complexes. Because AlF4- still inhibits the ATPase in the presence of guanosine 5'-[beta-thio]diphosphate, and because guanosine 5'-[beta gamma-imido]triphosphate does not inhibit the ATPase, it is unlikely that the inhibition could be due to the activation of an unknown G-protein. The time course of inhibition and the concentrations of NaF and AlCl3 required for this inhibition differ for the different ATPases. AlF4- inhibits the (Na+ + K+)-ATPase and the plasmalemmal (Ca2+ + Mg2+)-ATPase noncompetitively with respect to ATP and to their respective cationic substrates, Na+ and Ca2+. AlF4- probably binds to the phosphate-binding site of the ATPase, as the Ki for inhibition of the (Na+ + K+)-ATPase and of the plasmalemmal (Ca2+ + Mg2+)-ATPase is shifted in the presence of respectively 5 and 50 mM-Pi to higher concentrations of NaF. Moreover, AlF4- inhibits the K+-activated p-nitrophenylphosphatase of the (Na+ + K+)-ATPase competitively with respect to p-nitrophenyl phosphate. This AlF4- -induced inhibition of 'P'-type cation-transport ATPases warns us against explaining all the effects of AlF4- on intact cells by an activation of G-proteins.     

Sulfur dioxide relaxes rat aorta by endothelium-dependent and -independent mechanisms

This study aimed to investigate the vasoactivity of sulfur dioxide (SO2), a novel gas identified from vascular tissue, in rat thoracic aorta. The thoracic aorta was isolated, cut into rings, and mounted in organ-bath chambers. After equilibrium, the rings were gradually stretched to a resting tension. Isometric tension was recorded under the treatments with vasoconstrictors, SO2 derivatives, and various drugs as pharmacological interventions. In endothelium-intact aortic rings constricted by 1 microM phenylephrine (PE), SO2 derivatives (0.5-8 mM) caused a dose-dependent relaxation. Endothelium removal and a NOS inhibitor L-NAME reduced the relaxation to low doses of SO2 derivatives, but not that to relatively high doses (>or=2 mM). In endothelium-denuded rings, SO2 derivatives attenuated vasoconstriction induced by high K+ (60 mM) or CaCl2 (0.01-10 mM). The relaxation to SO2 derivatives in PE-constricted rings without endothelium was significantly inhibited by blockers of ATP-sensitive K+(KATP) and Ca2+-activated K+ (KCa) channels, but not by those of voltage-dependent K+ channels, Na+- K+-ATPase or Na+-Ca2+ exchanger. SO2 relaxed vessel tone via endothelium-dependent mechanisms associated with NOS activation, and via endothelium-independent mechanisms dependent on the inhibition of voltage-gated Ca2+ channels, and the opening of KATP and KCa channels.     

ATP-sensitive K+ channels in insulinoma cells are activated by nonesterified fatty acids.

Both 86Rb+ efflux experiments and electrophysiological studies have shown that arachidonic acid and other nonesterified fatty acids activate ATP-sensitive K+ channels in insulinoma cells (HIT-T15). Activation was observed with arachidonic, oleic, linoleic, and docosahexaenoic acid but not with myristic, stearic, and elaidic acids. Fatty acid activation of ATP-sensitive K+ channels was blocked by antidiabetic sulfonylureas such as glibenclamide. The activating effect of arachidonic acid was unaltered by indomethacin and by nordihydroguaiaretic acid, indicating that it is not due to metabolites of arachidonic acid via cyclooxygenase or lipoxygenase pathways. Moreover, the nonmetabolizable analogue of arachidonic acid, eicosatetraynoic acid, was an equally potent activator. Activation of ATP-sensitive K+ channels by fatty acids was potentiated by diacylglycerol and was inhibited by calphostin C, an inhibitor of protein kinase C. These findings indicate that fatty acid activation of ATP-sensitive K+ channels is most likely due to the participation of arachidonic acid (and other fatty acid)-activated protein kinase C isoenzymes. Activation of ATP-sensitive K+ channels by nonesterified fatty acids is not involved in the control of insulin secretion since arachidonic acid stimulates insulin secretion from insulinoma cells instead of inhibiting it.     

Galanin inhibits glucose-stimulated insulin release by a mechanism involving hyperpolarization and lowering of cytoplasmic free Ca2+ concentration

The intrapancreatic neuropeptide galanin has been demonstrated to lower plasma insulin levels in vivo. The effects of this peptide on insulin secretion, cytoplasmic free Ca2+ concentration and membrane potential have now been studied in vitro. Glucose-stimulated insulin secretion was inhibited by galanin under these conditions, indicating a direct effect of the peptide on the beta-cells. The neuropeptide reversed both the increase in membrane potential and cytoplasmic free Ca2+ in response to glucose stimulation. At a non-stimulatory concentration of the sugar, galanin induced a slight hyperpolarization without any effect on cytoplasmic free Ca2+. Galanin did not affect K+-induced increase in cytoplasmic free Ca2+, excluding a direct inhibitory effect on the voltage-activated Ca2+ channels. The results indicate that galanin inhibition of glucose-stimulated insulin release involves hyperpolarization with a subsequent decrease in cytoplasmic free Ca2+.     

Glucose generates sub-plasma membrane ATP microdomains in single islet beta-cells. Potential role for strategically located mitochondria

Increases in the concentration of free ATP within the islet beta-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K+ (KATP) channels and activating Ca2+ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free [ATP] in subdomains of single living MIN6 and primary beta-cells. Resting [ATP] in the cytosol ([ATP]c), in the mitochondrial matrix ([ATP]m), and beneath the plasma membrane ([ATP]pm) were similar ( approximately 1 mM). Elevations in extracellular glucose concentration (3-30 mM) increased free [ATP] in each domain with distinct kinetics. Thus, sustained increases in [ATP]m and [ATP]pm were observed, but only a transient increase in [ATP]c. However, detectable increases in [ATP]c and [ATP]pm, but not [ATP]m, required extracellular Ca2+. Enhancement of glucose-induced Ca2+ influx with high [K+] had little effect on the apparent [ATP]c and [ATP]m increases but augmented the [ATP]pm increase. Underlying these changes, glucose increased the mitochondrial proton motive force, an effect mimicked by high [K+]. These data support a model in which glucose increases [ATP]m both through enhanced substrate supply and by progressive Ca2+-dependent activation of mitochondrial enzymes. This may then lead to a privileged elevation of [ATP]pm, which may be essential for the sustained closure of KATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration.     

Evidence that Glucose-Induced Electrical Activity in Rat Pancreatic b-Cells Does Not Require KATP Channel Inhibition

KATP-channel activity, recorded in cell-attached patches from isolated rat pancreatic beta-cells, was found to be maximally inhibited in the presence of a substimulatory concentration (5 mM) of glucose, with no further effect of higher, stimulatory glucose concentrations. KATP channel-independent effects of glucose on electrical activity were therefore investigated by incubating cells in the presence of a supramaximal concentration of tolbutamide. Addition of tolbutamide (500 mM) to cells equilibrated in the absence of glucose resulted in a rapid depolarization and electrical activity followed by a gradual repolarization and disappearence of electrical activity. Repolarization was not due to desensitization of KATP channels to the sulfonylurea, but was probably the result of activation of another K+ conductance. The subsequent application of 16 mM glucose in the continued presence of tolbutamide depolarized the cells again, leading to renewed electrical activity. Input conductance of the cells was markedly reduced by tolbutamide, reflecting KATP-channel inhibition, but was not significantly affected by the addition of glucose in the presence of the drug. In cells voltage-clamped at -70 mV, addition of glucose in the presence of tolbutamide generated a noisy inward current, probably representing activation of the volume-sensitive anion channel. KATP channel-independent activation of electrical activity by glucose was inhibited by the anion channel inhibitor 4,4'-dithiocyanatostilbene-2,2'-disulphonic acid. It is concluded that the induction of electrical activity in rat pancreatic b-cells does not require inhibition of KATP channels. The KATP channel-independent mechanism could involve, at least in part, activation by glucose of the volume-sensitive anion channel.     

Somatostatin activates glibenclamide-sensitive and ATP-regulated K+ channels in insulinoma cells via a G-protein

Somatostatin, an hyperglycemia-inducing hormone, was studied in rat insulinoma (RINm5F) cells using 86Rb+ efflux techniques. 86Rb+ efflux is stimulated by somatostatin in a dose-dependent manner. The half-maximum value of activation is 0.7 nM. Somatostatin-induced 86Rb+ efflux is abolished by the hypoglycemia-inducing sulfonylurea, glibenclamide, a known blocker of ATP-regulated K+ channels. Somatostatin activation is prevented by pretreatment of insulinoma cells with pertussis toxin. 86Rb+ efflux studies show that somatostatin activates an ATP-dependent K+ channel.     

Insulin release from BK virus-induced and in vitro maintained insulinoma cells of Syrian golden hamster

The pancreatic tumor cells (In 111) derived from BK virus-induced insulinoma of Syrian golden hamsters were maintained in culture for several passages and were studied for their insulin secretory ability under various stimulatory conditions. Insulin release was not increased by D-glucose stimulation (27.8 mM), while dibutyryl cyclic AMP (1 mM), theophylline (1 mM), 3-isobutyl-l-methylxanthine (0.1 mM) and elevation of medium calcium from 0.5 to 2.7 mM stimulated insulin release 2.5- to 4-fold. There was a concomitant increase of medium cyclic AMP with addition of theophylline. Streptozotocin (2 mM) treatment for 48 hours significantly reduced insulin release, while alloxan (2 mM), had no inhibitory effect on insulin release. The results indicate that while in vitro-maintained islet tumor cells, In 111, have a cyclic AMP-mediated process involved in insulin secretion analogous to normal beta cells, these cells lack the ability to recognize glucose as an insulin secretagogue probably due to a defect in the cell membrane, though the possibility of alteration in glucose metabolism cannot be fully excluded.     

Subunit composition of G(o) proteins functionally coupling galanin receptors to voltage-gated calcium channels.

The neuropeptide galanin is widely expressed in the central nervous system and other tissues and induces different cellular reactions, e.g. hormone release from pituitary and inhibition of insulin release from pancreatic B cells. By microinjection of antisense oligonucleotides we studied the question as to which G proteins mediate the galanin-induced inhibition of voltage-gated Ca2+ channels in the rat pancreatic B-cell line RINm5F and in the rat pituitary cell line GH3. Injection of antisense oligonucleotides directed against alpha 01, beta 2, beta 3, gamma 2 and gamma 4 G protein subunits reduced the inhibition of Ca2+ channel current which was induced by galanin, whereas no change was seen after injection of cells with antisense oligonucleotides directed against alpha i, alpha q, alpha 11, alpha 14, alpha 15, beta 1, beta 4, gamma 1, gamma 3, gamma 5, or gamma 7 G protein subunits or with sense control oligonucleotides. In view of these data and of previous results, we conclude that the galanin receptors in GH3 and in RINm5F cells couple mainly to the G(0) protein consisting of alpha 01 beta 2 gamma 2 to inhibit Ca2+ channels and use alpha 01beta 3 gamma 4 less efficiently. The latter G protein composition was previously shown to be used by muscarinic M4 receptors to inhibit Ca2+ channels.     

Effect of perchlorate on calcium uptake and insulin secretion in mouse pancreatic islets.

Microdissected beta-cell-rich pancreatic islets of non-inbred ob/ob mice were used in studies of how perchlorate (CIO4-) affects stimulus-secretion coupling in beta-cells. CIO4- at 16 mM potentiated D-glucose-induced insulin release, without inducing secretion at non-stimulatory glucose concentrations. The potentiation mainly applied to the first phase of stimulated insulin release. In the presence of 20 mM-glucose, the half-maximum effect of CIO4- was reached at 5.5 mM and maximum effect at 12 mM of the anion. The potentiation was reversible and inhibitable by D-mannoheptulose (20 mM) or Ca2+ deficiency. CIO4- at 1-8 mM did not affect glucose oxidation. The effects on secretion were paralleled by a potentiation of glucose-induced 45Ca2+ influx during 3 min. K+-induced insulin secretion and 45Ca2+ uptake were potentiated by 8-16 mM-CIO4-. The spontaneous inactivation of K+-induced (20.9 mM-K+) insulin release was delayed by 8 mM-CIO4-. The anion potentiated the 45Ca2+ uptake induced by glibenclamide, which is known to depolarize the beta-cell. Insulin release was not affected by 1-10 mM-trichloroacetate. It is suggested that CIO4- stimulates the beta-cell by affecting the gating of voltage-controlled Ca2+ channels.