Effects of Na+, K+ and Mg2+ on45Ca2+ uptake by pancreatic islets

Microdissected pancreatic islets of noninbredob/ob-mice were used to study ionic effects on the lanthanum-nondisplaceable⁴⁵Ca²⁺ uptake by islet cells. Omission of Mg²⁺ from the incubation medium had no effect, but the⁴⁵Ca²⁺ uptake was increased by omission of Na⁺ and decreased by omission of K⁺. Excess Mg²⁺ (1.2–15 mM) inhibited and excess K⁺ (4.7–25 mM) stimulated the⁴⁵Ca²⁺ uptake in a concentration-dependent manner. Stimulation of⁴⁵Ca²⁺ uptake in Na⁺-deficient islets was associated with an enhancement of the basal insulin release. Total abolishment of glucose-stimulated⁴⁵Ca²⁺ uptake in K⁺-deficient islets did not preclude a significant secretory response to glucose. It is concluded that the lanthanum-nondisplaceable⁴⁵Ca²⁺ uptake shows a partial correlation to insulin release.     

Insulin inhibits phagocytosis in normal human neutrophils via PKCα/β-dependent priming of F-actin assembly

Objective: This study investigated the effects of insulin on the phagocytosis of C3bi – and IgG-opsonized yeast particles in normal human neutrophils. Methods: Neutrophils were incubated in different insulin concentrations for 30 minutes and stimulated by C3bi – or IgG-opsonized yeast particles. Phagocytosis was quantified by both light microscopy and FACscan flow cytometry. Laser confocal microscopy was used for quantification of F-actin levels. Results: Elevated insulin concentrations decreased neutrophil phagocytosis of both types of targets. This defect was shown to be in part due to a delayed phagocytosis in the presence of insulin. Following a 30 minute incubation, insulin was found to increase the accumulation of cortical F-actin, without affecting the total cellular F-actin content. The specific PKCα/β inhibitor, Go6976, abolished the insulin-mediated increase in cortical F-actin content and both Go6976 and the PKCα/β/δ/ε-specific inhibitor GF109203X reversed the inhibitory effects of insulin on phagocytosis. Conclusion: Hyperinsulinemia in vitro can inhibit phagocytosis of opsonized targets in normal human neutrophils. This effect of insulin is dependent on activation of PKCα and/or PKCβ, and these insulin signals may interfere with the dynamic assembly/disassembly and/or distribution of F-actin, which is required for the phagocytosis process. Received 8 July 2005; accepted 13 October 2005 without revision I. Ahnfelt-R?nne     

Evidence that an L-fucose-containing component in the beta-cell plasma membrane is involved in the regulation of glucose-induced insulin release

The effect of the L-fucose-selective lectin Ulex Europeus I (UEA I), a blocker of the Na+, K+, Cl- co-transport system in the kidney, was tested on insulin secretion from isolated beta-cell-rich pancreatic islets. UEA I at doses from 50 to 100 micrograms ml-1 significantly reduced the glucose-induced (20 mmol l-1) insulin release whereas the basal (3 mmol l-1) release was unaffected. The inhibitory effect of 100 micrograms ml l-1 UEA I was completely abolished by 10 mmol l-1 L-fucose. The data suggest that an L-fucose-containing structure in the beta-cell plasma membrane participates in the regulation of glucose-induced insulin release. This structure may be similar to the L-fucose-containing glycoprotein in the kidney tubules that is believed to be the Na+, K+, Cl- cotransporter.     

Perchlorate stimulates insulin secretion by shifting the gating of L-type Ca2+ currents in mouse pancreatic B-cells towards negative potentials

The effects of the chaotrophic anion perchlorate (ClO4-) on glucose-induced electrical activity, exocytosis and ion channel activity in mouse pancreatic B-cells were investigated by patch-clamp recordings and capacitance measurements. ClO4- stimulated glucose-induced electrical activity and increased the action potential frequency by 70% whilst not affecting the membrane potential when applied in the presence of a subthreshold concentration of the sugar. ClO4- did not influence ATP-dependent K (KATP) channel activity and voltage-gated delayed K+ current. Similarly, ClO4- had no effect on Ca2+-dependent exocytosis. The stimulation of electrical activity and insulin secretion was instead attributable to an enhancement of the whole-cell Ca2+ current. This effect was particularly pronounced at voltages around the threshold for action potential initiation and a doubling of the current amplitude was observed at -30 mV. This was due to a 7-mV shift in the gating of the Ca2+ current towards negative voltages. The action of ClO4- was more pronounced when added in the presence of 0.1 mM BAY K8644, whereas no stimulation was observed when applied at a maximal concentration of the agonist (1 mM). Single-channel recordings revealed that the effect of ClO4- on whole-cell currents was principally due to a 60% increase in the mean duration of the long openings and the number of active channels. We propose that ClO4- stimulates insulin secretion and electrical activity by exerting a BAY K8644-like action on Ca2+ channel gating.     

Effects of alpha-adrenoceptor agonists and antagonists on insulin secretion, calcium uptake, and rubidium efflux in mouse pancreatic islets

Epinephrine, norepinephrine or the more selective alpha-2 adrenoceptor agonist, clonidine, inhibited insulin release from isolated pancreatic islets of lean mice or obese mice homozygous for the gene ob. Clonidine was highly effective at 0.1 mumol/l. In contrast, the preferential alpha-1 adrenoceptor agonist, phenylephrine, had no or only a modest effect at 10 mumol/l. The effects of norepinephrine or clonidine were counteracted by yohimbine, a preferential blocker of alpha-2 receptors, but not by prazosine, an alpha-1 receptor blocker. The glucose-stimulated uptake of 45Ca2+ in the islets was only consistently inhibited by epinephrine. This effect was counteracted by yohimbine. Clonidine had no effect on the release of 86Rb+ from preloaded islets. It is concluded that insulin secretion is suppressed by alpha-2 receptor agonism in the pancreatic beta-cells and that this effect is mediated by mechanisms other than the transmembrane fluxes of calcium or potassium ions.     

Anion-selective amplification of glucose-induced insulin secretion

The functional roles of anions on glucose-induced insulin secretion are poorly understood. We investigated the effects of the monovalent anions thiocyanate, iodide, bromide, nitrate and chloride on the dynamics of insulin secretion in isolated pancreatic islets from non-inbred Ume? ob/ob mice. All anion species (12 mM), except Cl-, significantly amplified glucose-induced (20 mM) first- and second-phase insulin secretion (selectivity sequence: SCN->NO3->I->Br->Cl-). Simultaneously, the anions reduced the lag-time prior to the initiation of the secretion (SCN-=I-=NO3->Br->Cl-). The results indicate that pancreatic beta-cell activation can be initiated and amplified by an anion-selective mechanism showing increasing degrees of activation in the order of the anion series of Hofmeister. On the basis of the strikingly similar anion selectivity of amplified secretion and shortened lag-phase, we suggest that both types of anion effects are caused by action at a single site on the beta-cell.     

Effects of 5-hydroxytryptamine on rubidium ion fluxes and insulin release in cultured pancreatic islets

We have incubated pancreatic islets isolated from noninbred ob/ob mice and NMRI mice for 3 days with or without 5-hydroxytryptamine (5-HT) in the medium and tested the effect of such long term treatment on subsequent insulin release and 86Rb+ accumulation and efflux. Two tenths millimolars of 5-HT abolished insulin release in response to 20 mM glucose. Two tenths millimolars of 5-HT also diminished the ability of islets to accumulate 86Rb+ and the effect of 10 mM glucose on 86Rb+ efflux. One one-hundredth millimolars of 5-HT had no effect on insulin release or 86Rb+ fluxes. Clearly, islets subjected to 5-HT for 3 days at concentrations that do not elicit demonstrable effects in short term incubations show a reduced secretory response. However, the physiological role of the high affinity uptake system for 5-HT in islet cells [Michaelis-Menten constant (Km) = 1.6 microM] remains unknown.     

D-glucose stimulates the Na+/K+ pump in mouse pancreatic islet cells

To determine the effect of D-glucose on the beta-cell Na+/K+ pump, 86Rb+ influx was studied in isolated, -cell-rich islets of Ume?-ob/ob mice in the absence or presence of 1mM ouabain. D-glucose (20mM) stimulated the ouabain-sensitive portion of 86Rb+ influx by 65%, whereas the ouabain-resistant portion was inhibited by 48%. The Na+/K+ ATPase activity in homogenates of islets of Ume?-ob/ob mice or normal mice was determined to search for direct effects of D-glucose. Thus, ouabain-sensitive ATP hydrolysis in islet homogenates was measured in the presence of different D-glucose concentrations. No effect of D-glucose (3-20mM) was observed in either ob/ob or normal islets at the optimal Na+/K+ ratio for the enzyme (135mM Na+ and 20mM K+). Neither D-glucose (3-20mM) nor L-glucose or 3-O-methyl-D-glucose (20mM) affected the enzyme activity at a high Na+/K+ ratio (175 mM Na+ and 0.7 mM K+). Diphenylhydantoin (150 microM) decreased the enzyme activity at optimal Na+/K+ ratio, whereas 50 microM of the drug had no effect. The results suggest that D-glucose induces a net stimulation the Na+/K+ pump of beta-cells in intact islets and that D-glucose does not exert any direct effect on the Na+/K+ ATPase activity.