In B cells from dispersed rat islet of Langerhans we have identified an inward rectifying voltageindependent K+ channel whose behavior parallels the metabolically regulated potassium permeability (PK) found in tracer flux and microelectrode recording studies. In cell-attached patches of membrane, the channel is closed when any one of several substrates (glucose, mannose, leucine, or glyceraldehyde) is added to the cell's bathing solution but is reopened on addition of an appropriate metabolic inhibitor, which prevents utilization of that substrate. In inside-out excised patches, a K+ channel with nearly identical kinetic features is closed by addition of micromolar concentrations of ATP to the "cytoplasmic" solution. The ATP sensitivity of channel activity is modified by addition of ADP, suggesting competition at a nucleotide binding site. These results suggest the presence of a metabolically regulated K+ channel gated by intracellular concentrations of ATP or the ratio of ATP/ADP concentrations.A popular hypothesis for stimulus-secretion coupling in pancreatic islet B cells is: nutrient metabolism by B cells decreased K+ permeability (PK) -* membrane depolarization --voltage-dependent Ca2 entry --insulin granule exocytosis (1, 2). Using single-channel recording techniques, several groups have identified, in cell-attached patches of pancreatic islet cells, a potassium-selective channel whose activity is reduced by raising the glucose concentration of the medium bathing the rest of the cell (3-6). Others have reported the existence in inside-out excised patches of a potassium channel of similar conductance whose opening frequency is reduced by micromolar concentrations of ATP (4-8). Here we demonstrate that both channels represent the same "metabolite-regulated" K+ channel in two different recording configurations and describe some regulatory effects of cytoplasmic nucleotides on channel gating. METHODS AND MATERIALSRat pancreatic islets, isolated by collagenase digestion of chopped pancreases of adult male Sprague-Dawley rats were dispersed into small clumps of cells by incubation with the enzyme "dispase" (9). Cells prepared in this manner increase their insulin secretion nearly 3-fold above baseline in the presence of 10 mM glucose media (10). Clumps of cells were added to 35-mm tissue culture dishes "seeded" with appropriately sized glass coverslips and were cultured for 1-10 days at 37°C in modified CMRL medium (GIBCO) containing 10% heat-inactivated fetal bovine serum, 0.5% penicillin, and 0.5% streptomycin in 5% C02/95% air. Individual coverslip chips were then transferred to the recording chamber containing modified physiological saline solutions at 20-23°C. The basic zero-glucose extracellular saline solution (zeroglucose ES solution) consisted of 138 mM NaCl, 5.5 mM KCl, 0.5 mM CaCl2, 2 mM MgCl2, and 20 mM Hepes titrated with NaOH to pH 7.35. The solution used to fill patch pipettes as well as bathe inside-out excised patches (IS solution) consisted of 138 mM KCl, 2 mM Mg2+, 5-20 AM CaCl2, and 2...

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Single-vesicle release of catecholamines from chromaffin cells can be detected in real time as current spikes by the electrochemical method of amperometry. About 70% of spikes are preceded by a small "foot," the trickle of transmitter out of the early fusion pore. In addition, 20-50% of foot signals exhibit rapid fluctuations that we interpret as flickering of the fusion pore. There are also "stand-alone" foot signals, which may reflect transient fusions, in which the vesicles do not collapse completely into the plasma membrane. The number and frequency of the foot flickering are affected by intracellular Ca2+ concentration.

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Recent studies suggest a role for autocrine insulin signaling in beta cells, but the mechanism and function of insulin-stimulated Ca 2؉ signals is uncharacterized. We examined Ca 2؉ -dependent insulin signaling in human beta cells. calcium signals ͉ ryanodine ͉ autocrine ͉ CD38 ͉ diabetes mellitus D iscovery of insulin receptors on pancreatic beta cells and the characterization of beta cell-specific insulin receptor null mice with a diabetes-like phenotype suggest a physiological role for autocrine insulin signaling (1). Although results from in vivo and whole islet experiments suggested a negligible or inhibitory effect of insulin on insulin release, recent experiments with dispersed rodent beta cells suggested a stimulatory effect on calcium signaling, insulin expression, and exocytosis (2-7). Unlike glucose signaling, insulin-evoked Ca 2ϩ signals in mouse beta cells required intracellular Ca 2ϩ stores sensitive to the sarcoplasmic͞endoplasmic Ca 2ϩ -ATPase (SERCA) inhibitor thapsigargin (4). Insulin action was not blocked by a phospholipase C inhibitor, suggesting indirectly that inositol 1,4,5-trisphosphate (IP 3 )-sensitive Ca 2ϩ stores were not involved (5). The mechanisms of autocrine insulin feedback are unknown in human beta cells.Ca 2ϩ signals control multiple functions in secretory cells, and at least three distinct biochemical classes of Ca 2ϩ stores coexist (8, 9). Aside from the phospholipase C͞IP 3 pathway that is commonly activated by G-protein-coupled receptors, Ca 2ϩ can be mobilized through ryanodine receptors, activated by Ca 2ϩ or cyclic ADP-ribose (cADPr). A third class of Ca 2ϩ store, mobilized by nicotinic acid adenine dinucleotide phosphate (NAADP), functions in oocytes, Jurkat T lymphocytes, and mouse pancreatic acini (8, 10, 11). The production of NAADP and cADPr are catalyzed by CD38 and related ADP-ribosyl cyclases (12, 13). CD38 is found in many cell types, including human beta cells. Glucose-stimulated Ca 2ϩ mobilization and insulin release (in vivo and in vitro) were enhanced by CD38 overexpression and reduced by CD38 knockout (14,15). Beta cells with poor glucose-stimulated insulin production͞release (ob͞ob, GK, RINm5F) have less CD38 (16,17). CD38 autoantibodies, found in Ϸ14% of diabetic patients, evoked Ca 2ϩ signals and insulin release from human islet cells (18).In this study, we tested the hypothesis that NAADP-sensitive Ca 2ϩ stores regulate beta cell function, in the context of autocrine insulin signaling. We report that insulin generates complex Ca 2ϩ signals by mobilizing novel intracellular Ca 2ϩ stores in primary cultures of dispersed human islet cells. NAADPsensitive Ca 2ϩ stores initiate insulin signaling, whereas subsequent oscillatory behavior is sensitive to the removal of extracellular Ca 2ϩ and to putative IP 3 receptor inhibitors. These prolonged insulin-stimulated Ca 2ϩ signals regulate cellular insulin levels, but do not measurably stimulate overall secretion. Materials and MethodsDrugs and Solutions. Reagents were from Sigma unless otherwise stated. Stocks ...