Voltage-dependent entry and generation of slow Ca2+ oscillations in glucose-stimulated pancreatic beta-cells.

The role of voltage-dependent Ca2+ entry for glucose generation of slow oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i) was evaluated in individual mouse pancreatic beta-cells. Like depolarization with K+, a rise of the glucose concentration resulted in an enhanced influx of Mn2+, which was inhibited by nifedipine. This antagonist of L-type Ca2+ channels also blocked the slow oscillations of [Ca2+]i induced by glucose. The slow oscillations occurred in synchrony with variations in Mn2+ influx and bursts of action currents, with the elevation of [Ca2+]i being proportional to the frequency of the action currents. A similar relationship was obtained when Ca2+ was replaced with Sr2+. Occasionally, the slow [Ca2+]i oscillations were superimposed with pronounced spikes temporarily arresting the action currents. It is concluded that the glucose-induced slow oscillations of [Ca2+]i are caused by periodic depolarization with Ca2+ influx through L-type channels. Ca2+ spiking, due to intracellular mobilization, may be important for chopping the slow oscillations of [Ca2+]i into shorter ones characterizing beta-cells situated in pancreatic islets.

Generation of glucose-dependent slow oscillations of cytoplasmic Ca2+ in individual pancreatic beta cells.

Individual pancreatic beta cells respond to glucose stimulation with large amplitude (300-500 nM) oscillations in the cytoplasmic Ca2+ concentration ([Ca2+]i). These oscillations (frequency 0.05-0.5/min) depend on rhythmical depolarization of the plasma membrane, with influx of Ca2+ through voltage-operated channels, but do not require intracellular mobilization of Ca2+. Patch clamp analyses of the activity of ATP-sensitive K+ channels indicate that oscillations in beta-cell metabolism underlie the rhythmical depolarizations, causing the large amplitude oscillations of [Ca2+]. The oscillatory responses of adjacent beta cells are synchronized by gap-junctional coupling in cellular microdomains. With increasing glucose concentration, previously unresponsive domains are activated, and their oscillations entrained with those of other active domains. In pancreatic islets, glucose-induced large amplitude oscillations occur in parallel with insulin release pulses, the amplitudes of which are determined by the number of beta cells recruited into the secretory state.

Induction of a glucose-dependent insulin secretory response by the nonmetabolizable amino acid alpha-aminoisobutyric acid.

The effects of the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB) on insulin release were evaluated using beta cell-rich pancreatic islets from ob/ob mice. Both AIB and L-alanine promptly induced transient insulin release during column perifusion of islet cells. The secretory response was dependent on an elevated level of glucose and effectively suppressed by removal of Na+. The insulin release elicited by AIB fulfilled the criteria of a physiological event in being suppressed by clonidine or lowering of the temperature to 22 degrees C. AIB effectively promoted the increase in sodium (total as well as ionized cytoplasmic) obtained with ouabain blockage of the Na/K pump. When added to a medium containing 11 mM glucose, AIB altered cytoplasmic Ca2+ in terms of both an initial transitory rise and transformation of existing oscillations into a sustained elevation. It is concluded that amino acids can stimulate insulin release from mature beta cells by virtue of being cotransported with Na+.

Local changes of medium in studies of individual cells.

A procedure is described for changing the medium surrounding individual cells attached to the bottom of a cell chamber. A small hole at the "apex" of a plastic U-tube allowed application and withdrawal of medium. The medium to be applied was perfused through the U-tube by pressure at one end and suction at the other. To prevent premature delivery of new medium from the U-tube, suction of the outlet dominated resulting in a net withdrawal of medium from the cell chamber. The flow of medium through the hole could be reversed rapidly by arresting the suction with an electromechanical valve. In this way it was possible to obtain 95% replacement of medium within 60 ms. A pressure transient arising from the closure of the valve was damped by the presence of a small air bubble in the system. To secure a precise deposition of medium and minimize the risk of mechanical disturbances to the cell it was essential to be able to inspect the medium changes visually. For this purpose the fluorescent indicator rhodamine B bound to dextran proved satisfactory. Free rhodamine B could not be used because it had biological effects, as was evident from studying ATP-regulated K+ channels in pancreatic beta-cells. When using a purpose-designed syringe pump for perfusing the U-tube, the technique allows well controlled exposure of individual cells to test substances added together with dextran-linked rhodamine B.

Variations in ATP-sensitive K+ channel activity provide evidence for inherent metabolic oscillations in pancreatic beta-cells.

The cell-attached configuration of the patch clamp technique was used for studying slow variations in the activity of the ATP-sensitive K+ channels in pancreatic beta-cells isolated from mouse and man. In 0 or 3 mM glucose, the fraction of time the channels were open exhibited oscillations with frequencies in the 0.25-0.40/min range. This phenomenon is a strong argument for inherent fluctuations in the ATP production of the beta-cells. Variations in metabolism may thus be a major determinant for the characteristic large amplitude oscillations of cytoplasmic Ca2+ with equivalent frequency.

The role of Ca2+ in the release of pancreatic islet hormones.

The role of Ca2+ in initiating exocytosis of granule-bound secretory products was evaluated with respect to pancreatic islet hormones. Apart from stimulating the transfer of the granules to the plasma membrane and their subsequent extrusion, a rise of the cytoplasmic Ca2+ concentration ([Ca2+]i) may under certain conditions also have depressive effects on insulin release. Glucose has a bidirectional action on [Ca2+]i by stimulating both the entry of the ion and its removal by organelle sequestration and outward transport. The recognition of glucose as a secretory stimulus is based on sudden transitions between oscillatory and steady-state [Ca2+]i at threshold concentrations of the sugar characteristic for the individual beta-cell. The intrinsic ability of each beta-cell to generate oscillations of [Ca2+]i and the subsequent synchronization of these signals result in a pulsatile release of insulin from isolated islets. Glucose regulation of this process is manifested as alterations of the amplitudes of the insulin pulses without effects on the frequency. It is suggested that electrical signalling from the beta-cells in combination with direct effects of glucose are important for regulating the release of glucagon and somatostatin.

Glucose has regulatory effects on insulin release also in the virtual absence of extracellular Ca2+.

The glucose effect on insulin release in a Ca(2+)-deficient medium was analyzed in perifusion experiments with aggregates of cells prepared by dispersal of the beta-cell-rich pancreatic islets of ob/ob-mice. Hyperosmolar additions of 20 mM D-glucose or its poorly metabolized transport analogue 3-0-methyl-D-glucose resulted in 50% suppression of the secretory rate. However, after isosmolar additions of the sugars, replacing non-penetrating sucrose, there was a stimulation of insulin release. Whereas D-glucose was less effective than 3-O-methyl-D-glucose in stimulating insulin release after isosmolar addition, the opposite was found for the enhanced secretory response obtained when the sugars were excluded from the perifusion medium. The studies indicate that D-glucose has regulatory actions on insulin release also in the virtual absence of extracellular Ca2+. This effect is not only due to osmolar influences but involves also direct suppression of the secretory activity probably mediated by the metabolism of the sugar.