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ims/hypothesis: Evidence is accumulating that Ca-regulated K (K) channels are important for beta cell function. We used BK channel knockout (BK-KO) mice to examine the role of these K channels for glucose homeostasis, beta cell function and viability. Methods: Glucose and insulin tolerance were tested with male wild-type and BK-KO mice. BK channels were detected by single-cell RT-PCR, cytosolic Ca concentration ([Ca]) by fura-2 fluorescence, and insulin secretion by radioimmunoassay. Electrophysiology was performed with the patch-clamp technique. Apoptosis was detected via caspase 3 or TUNEL assay. Results: BK channels were expressed in murine pancreatic beta cells. BK-KO mice were normoglycaemic but displayed markedly impaired glucose tolerance. Genetic or pharmacological deletion of the BK channel reduced glucose-induced insulin secretion from isolated islets. BK-KO and BK channel inhibition (with iberiotoxin, 100 nmol/l) broadened action potentials and abolished the after-hyperpolarisation in glucose-stimulated beta cells. However, BK-KO did not affect action potential frequency, the plateau potential at which action potentials start or glucose-induced elevation of [Ca]. BK-KO had no direct influence on exocytosis. Importantly, in BK-KO islet cells the fraction of apoptotic cells and the rate of cell death induced by oxidative stress (HO, 10-100 μmol/l) were significantly increased compared with wild-type controls. Similar effects were obtained with iberiotoxin. Determination of HO-induced K currents revealed that BK channels contribute to the hyperpolarising K current activated under conditions of oxidative stress. Conclusions/interpretation: Ablation or inhibition of BK channels impairs glucose homeostasis and insulin secretion by interfering with beta cell stimulus-secretion coupling. In addition, BK channels are part of a defence mechanism against apoptosis and oxidative stress. [ABSTRACT FROM AUTHOR]

Pancreatic beta-cells of sulfonylurea receptor type 1 knock-out (SUR1−/−) mice exhibit an oscillating membrane potential ( Vm) demonstrating that hyper-polarisation occurs despite the lack of KATP channels. We hypothesize that glucose activates the Na+/K+-ATPase thus increasing a hyper-polarising current. Elevating glucose in SUR1−/− beta-cells resulted in a transient fall in Vm and [Ca2+]c independent of sarcoplasmic and endoplasmic reticulum Ca2+-activated ATPase (SERCA) activation. This was not affected by K+ channel blockade but inhibited by ATP depletion and by ouabain. Increasing glucose also reduced [Na+]c, an effect reversed by ouabain. Exogenously applied insulin decreased [Na+]c and hyper-polarised Vm. Inhibiting insulin signalling in SUR1−/− beta-cells blunted the glucose-induced decrease of [Ca2+]c. Tolbutamide (1 mmol/l) disclosed the SERCA-independent effect of glucose on [Ca2+]c in wild-type beta-cells. The data show that in SUR1−/− beta-cells, glucose activates the Na+/K+-ATPase presumably by increasing [ATP]c. Insulin can also stimulate the pump and potentiate the effect of glucose. Pathways involving the pump may thus serve as potential drug targets in certain metabolic disorders. [ABSTRACT FROM AUTHOR]

In a previous study, we demonstrated that a creatine kinase (CK) modulates KATP channel activity in pancreatic beta cells. To explore phosphotransfer signalling pathways in more detail, we examined whether KATP channel regulation in beta cells is determined by a metabolic interaction between adenylate kinase (AK) and CK. Single channel activity was measured with the patch–clamp technique in the inside-out (i/o) and open-cell attached (oca) configuration. The ATP sensitivity of KATP channels was higher in i/o patches than in permeabilised beta cells (oca). One reason for this observation could be that the local ATP:ADP ratio in the proximity of the channels is determined by factors not active in i/o patches. AMP (0.1 mmol/l) clearly increased open channel probability in the presence of ATP (0.125 mmol/l) in permeabilised cells but not in excised patches. This suggests that AK-catalysed ADP production in the vicinity of the channels is involved in KATP channel regulation. The observation that the stimulatory effect of AMP on KATP channels was prevented by the AK inhibitor P 1, P 5-di(adenosine-5′)pentaphosphate (Ap5A; 20 μmol/l) and abolished in the presence of the non-metabolisable ATP analogue adenosine 5′-(β,γ-imido)triphosphate tetralithium salt (AMP-PNP; 0.12 mmol/l) strengthens this idea. In beta cells from AK1 knockout mice, the effect of AMP was less pronounced, though not completely suppressed. The increase in KATP channel activity induced by AMP in the presence of ATP was outweighed by phosphocreatine (1 mmol/l). We suggest that this is due to an elevation of the ATP concentration by CK. We propose that phosphotransfer events mediated by AK and CK play an important role in determining the effective concentrations of ATP and ADP in the microenvironment of pancreatic beta cell KATP channels. Thus, these enzymes determine the open probability of KATP channels and eventually the actual rate of insulin secretion. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Islets or beta cells from Sur1-/- mice were used to determine whether changes in plasma membrane potential (Vm) remain coupled to changes in cytosolic Ca2+ ([Ca2+]i) in the absence of KATP channels and thus provide a triggering signal for insulin secretion. The study also sought to elucidate whether [Ca2+]i influences oscillations in Vm in sur1-/- beta cells. Methods: Plasma membrane potential and ion currents were measured with micro electrodes and the patch-clamp technique. [Ca2+]i was monitored with the fluorescent dye fura-2. Insulin secretion from isolated islets was determined by static incubations. Results: Membrane depolarisation of Sur1-/ islets by arginine or increased extracellular K+, elevated [Ca2+]i and augmented insulin secretion. Oligomycin completely abolished glucose-stimulated insulin release from Sur1-/ islets. Oscillations in Vm were influenced by [Ca2+]i as follows: (1) elevation of extracellular Ca2+lengthened phases of membrane hyperpolarisation; (2) simulating a burst of action potentials induced a Ca2+-dependent outward current that was augmented by increased Ca2+ influx through L-type Ca2+ channels; (3) Ca2+ depletion of intracellular stores by cyclopiazonic acid increased the burst frequency in Sur1-/- islets, elevating [Ca2+]i and insulin secretion; (4) store depletion activated a C2+ influx that was not in- inhibitable by the L-type C2+ channel blocker D600. Conclusions/interpretation: Although Vm is largely uncoupled from glucose metabolism in the absence of KATP channels, increased electrical activity leads to elevations of [Ca2+ that are sufficient to stimulate insulin secretion. In Sur1-/- - beta cells, [Ca2+]i exerts feedback mechanisms on Vm by activating a hyperpolarising outward current and by depolarising Vm via store-operated ion channels. [ABSTRACT FROM AUTHOR]

The aim of the present study was to evaluate whether HIV protease inhibitors directly interfere with stimulus-secretion coupling in pancreatic β-cells. Insulin secretion was determined by a radioimmunoassay (RIA), cytosolic free Ca2+ concentration ([Ca2+]c) with the fluorescence dye fura-2 and whole-cell membrane currents with the patch-clamp technique. Glucose-induced insulin secretion was inhibited in a concentration-dependent manner by ritonavir and nelfinavir but not by indinavir. Ritonavir and nelfinavir lowered [Ca2+]c in the presence of a stimulatory glucose concentration whereas indinavir again had no effect. Ritonavir and nelfinavir completely inhibited the effect of tolbutamide, which normally increases [Ca2+]c by blocking KATP channels. This observation points to an action of both drugs on KATP channels or a step distal to these channels in stimulus-secretion coupling. Ritonavir was used to further evaluate the direct effects of HIV protease inhibitors on β-cell ion channel currents. Unexpectedly, ritonavir inhibited neither the whole-cell KATP current nor the whole-cell L-type Ca2+ current. Tolbutamide almost completely suppressed the KATP current in the presence of ritonavir excluding that ritonavir alters the tolbutamide sensitivity of the KATP channel. Ritonavir increased the length and decreased the frequency of glucose-induced action potentials. This effect can be attributed to inhibition of voltage-dependent K+ currents. Intracellular stores seem not to be involved in the ritonavir-induced lowering of [Ca2+]c. In conclusion, different HIV protease inhibitors surprisingly reveal distinct effects on insulin secretion. Ritonavir inhibits insulin secretion by lowering [Ca2+]c but this effect is evidently independent of the opening of KATP channels or the closure of voltage-dependent Ca2+ channels, which are commonly considered to play a key role in stimulus-secretion coupling. [ABSTRACT FROM AUTHOR]

Aims/hypothesis. SUR1(ABCC8)-/- mice lacking functional KATP channels are an appropriate model to test the significance of KATP channels in beta-cell function. We examined how this gene deletion interferes with stimulus-secretion coupling. We tested the influence of metabolic inhibition and galanin, whose mode of action is controversial. Methods. Plasma membrane potential (Vm) and currents were measured with microelectrodes or the patch-clamp technique; cytosolic Ca2+ concentrations ([Ca2+]c) and mitochondrial membrane potential (ΔΨ) were measured using fluorescent dyes. Results. In contrast to the controls, SUR1-/- beta cells showed electrical activity even at a low glucose concentration. Continuous spike activity was measured with the patch-clamp technique, but with microelectrodes slow oscillations in Vm consisting of bursts of Ca2+-dependent action potentials were detected. [Ca2+]c showed various patterns of oscillations or a sustained increase. Sodium azide did not hyperpolarize SUR1-/- beta cells. The depolarization of ΔΨ evoked by sodium azide was significantly lower in SUR1-/- than SUR1+/+ cells. Galanin transiently decreased action potential frequency and [Ca2+]c in cells from both SUR1-/- and SUR1+/+ mice. Conclusion/interpretation. The strong dependence of Vm and [Ca2+]c on glucose concentration observed in SUR1+/+ beta cells is disrupted in the knock-out cells. This demonstrates that both parameters oscillate in the absence of functional KATP channels. The lack of effect of metabolic inhibition by sodium azide shows that in SUR1-/- beta cells changes in ATP/ADP no longer link glucose metabolism and Vm. The results with galanin suggest that this peptide affects beta cells independently of KATP currents and thus could contribute to the regulation of beta-cell function in SUR1-/- animals. [ABSTRACT FROM AUTHOR]