The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.

The perforated patch whole‐cell configuration of the patch‐clamp technique was applied to superficial glucagon‐secreting α‐cells in intact mouse pancreatic islets. α‐cells were distinguished from the β‐ and δ‐cells by the presence of a large TTX‐blockable Na+ current, a TEA‐resistant transient K+ current sensitive to 4‐AP (A‐current) and the presence of two kinetically separable Ca2+ current components corresponding to low‐ (T‐type) and high‐threshold (L‐type) Ca2+ channels. The T‐type Ca2+, Na+ and A‐currents were subject to steady‐state voltage‐dependent inactivation, which was half‐maximal at −45, −47 and −68 mV, respectively. Pancreatic α‐cells were equipped with tolbutamide‐sensitive, ATP‐regulated K+ (KATP) channels. Addition of tolbutamide (0·1 mm) evoked a brief period of electrical activity followed by a depolarisation to a plateau of −30 mV with no regenerative electrical activity. Glucagon secretion in the absence of glucose was strongly inhibited by TTX, nifedipine and tolbutamide. When diazoxide was added in the presence of 10 mm glucose, concentrations up to 2 μm stimulated glucagon secretion to the same extent as removal of glucose. We conclude that electrical activity and secretion in the α‐cells is dependent on the generation of Na+‐dependent action potentials. Glucagon secretion depends on low activity of KATP channels to keep the membrane potential sufficiently negative to prevent voltage‐dependent inactivation of voltage‐gated membrane currents. Glucose may inhibit glucagon release by depolarising the α‐cell with resultant inactivation of the ion channels participating in action potential generation.

␣-Cells were identified in preparations of dispersed mouse islets by immunofluorescence microscopy. A high fraction of ␣-cells correlated with a small cell size measured as the average cell diameter (10 µm) and whole-cell capacitance (<4 pF). The ␣-cells generated action potentials at a low frequency (1 Hz) in the absence of glucose. These action potentials were reversibly inhibited by elevation of the glucose concentration to 20 mmol/l. The action potentials originated from a membrane potential more negative than -50 mV, had a maximal upstroke velocity of 5 V/s, and peaked at +1 mV. Voltage-clamp experiments revealed the ionic conductances underlying the generation of action potentials. ␣-Cells are equipped with a delayed tetraethyl-ammonium-blockable outward current (activating at voltages above -20 mV), a large tetrodotoxin-sensitive Na + current (above -30 mV; peak current 200 pA at +10 mV), and a small Ca 2+ current (above -50 mV; peak current 30 pA at +10 mV). The latter flowed through -conotoxin GVIA (25%)-and nifedipine-sensitive (50%) Ca G lucagon is a major catabolic and hyperglycemic hormone of 29 amino acids and is secreted from the ␣-cells of the islets of Langerhans (1). Its main biological effect is the regulation of glucose metabolism by enhancing synthesis and mobilization of glucose in the liver. Normally, secretion of the hormone is stimulated by low blood glucose (2), amino acids (3), and a variety of hormones and neurotransmitters, such as adrenaline, glucose-dependent insulinotropic polypeptide, and glucagon-like peptide-1 (4,5). Hyperglycemia and fatty acids are the main inhibitors (6), but the islet hormones insulin and somatostatin (4) also appear to reduce glucagon secretion, possibly by a paracrine mechanism (7). Whereas insulin levels are inadequately low in hyperglycemic diabetic subjects, glucagon levels are actually elevated, and this increase exacerbates the disease (8). The reason for this abnormality is unknown, and studies on ␣-cells are complicated by the scarcity of islet tissue and the low occurrence of ␣-cells compared with ␤-cells. Therefore, how glucose physiologically regulates secretion in the ␣-cell remains unknown. Electrical activity in glucagon-secreting cells has been observed using several experimental approaches (5,9,10), and it is, at least in part, attributable to voltage-gated Ca 2+ channels. Available evidence also suggests that glucagon release is a Ca 2+ -dependent process. Indeed, capacitance measurements on single rat ␣-cells revealed a close relationship between N-type Ca 2+ channels and the secretory granules under basal conditions, whereas L-type Ca 2+ channels appeared more important when secretion was stimulated with adrenaline (5). The finding that glucagon secretion is Ca 2+ -dependent, coupled with the fact that glucagon release is suppressed by glucose, suggests that the ␣-cells must be electrically silent at elevated glucose concentrations, contrary to the situation in the ␤-cell. Thus, it is surprising that ATP-sensitive potassium channels (i.e...

A readily releasable pool (RRP) of granules has been proposed to underlie the first phase of insulin secretion. In the present study we combined electron microscopy, insulin secretion measurements and recordings of cell capacitance in an attempt to define this pool ultrastructurally. Mouse pancreatic B-cells contain approximately 9,000 granules, of which 7% are docked below the plasma membrane. The number of docked granules was reduced by 30% (200 granules) during 10 min stimulation with high K+. This stimulus depolarized the cell to -10 mV, elevated cytosolic [Ca2+] ([Ca2+](i)) from a basal concentration of 130 nM to a peak of 1.3 microM and released 0.5 ng insulin/islet, corresponding to 200-300 granules/cell. The Ca2+ transient decayed towards the prestimulatory concentration within approximately 200 s, presumably reflecting Ca2+ channel inactivation. Renewed stimulation with high K+ failed to stimulate insulin secretion when applied in the absence of glucose. The size of the RRP, derived from the insulin measurements, is similar to that estimated from the increase in cell capacitance elicited by photolytic release of caged Ca2+. We propose that the RRP represents a subset of the docked pool of granules and that replenishment of RRP can be accounted for largely by chemical modification of granules already in place or situated close to the plasma membrane.

The perforated patch whole‐cell configuration of the patch‐clamp technique was applied to superficial cells in intact pancreatic islets. Immunostaining in combination with confocal microscopy revealed that the superficial cells consisted of 35 % insulin‐secreting B‐cells and 65 % non‐B‐cells (A‐ and D‐cells). Two types of cell, with distinct electrophysiological properties, could be functionally identified. One of these generated oscillatory electrical activity when the islet was exposed to 10 mm glucose and had the electrophysiological characteristics of isolated B‐cells maintained in tissue culture. The Ca2+ current recorded from B‐cells in situ was 80 % larger than that of isolated B‐cells. It exhibited significant (70 %) inactivation during 100 ms depolarisations. The inactivation was voltage dependent and particularly prominent during depolarisations evoking the largest Ca2+ currents. Voltage‐dependent K+ currents were observed during depolarisations to membrane potentials above −20 mV. These currents inactivated little during a 200 ms depolarisation and were unaffected by varying the holding potential between −90 and −30 mV. The maximum resting conductance in the absence of glucose, which reflects the conductance of ATP‐regulated K+ (KATP) channels, amounted to ≈4 nS. Glucose produced a concentration‐dependent reduction of KATP channel conductance with half‐maximal inhibition observed with 5 mm glucose. Combining voltage‐ and current‐clamp recording allowed the estimation of the gap junction conductance between different B‐cells. These experiments indicated that the input conductance of the B‐cell at stimulatory glucose concentrations (≈1 nS) is almost entirely accounted for by coupling to neighbouring B‐cells.

Glucose-stimulated insulin secretion consists of a transient first phase followed by a sustained second phase. Diabetes (type II) is associated with abnormalities in this release pattern. Here we review the evidence that biphasic insulin secretion reflects exocytosis of two functional subsets of secretory granules and the implications for diabetes.

Capacitance measurements of exocytosis were applied to functionally identified α-, β-and δ-cells in intact mouse pancreatic islets. The maximum rate of capacitance increase in β-cells during a depolarization to 0 mV was equivalent to 14 granules s −1 , <5% of that observed in isolated β-cells. β-Cell secretion exhibited bell-shaped voltage dependence and peaked at +20 mV. At physiological membrane potentials (up to ∼−20 mV) the maximum rate of release was ∼4 granules s −1 . Both exocytosis (measured by capacitance measurements) and insulin release (detected by radioimmunoassay) were strongly inhibited by the L-type Ca 2+ channel blocker nifedipine (25 µM) but only marginally (<20%) affected by the Rtype Ca 2+ channel blocker SNX482 (100 nM). Exocytosis in the glucagon-producing α-cells peaked at +20 mV. The capacitance increases elicited by pulses to 0 mV exhibited biphasic kinetics and consisted of an initial transient (150 granules s −1 ) and a sustained late component (30 granules s −1 ). Whereas addition of the N-type Ca 2+ channel blocker ω-conotoxin GVIA (0.1 µM) inhibited glucagon secretion measured in the presence of 1 mM glucose to the same extent as an elevation of glucose to 20 mM, the L-type Ca 2+ channel blocker nifedipine (25 µM) had no effect. Thus, glucagon release during hyperglycaemic conditions depends principally on Ca 2+ -influx through N-type rather than L-type Ca 2+ channels. Exocytosis in the somatostatinsecreting δ-cells likewise exhibited two kinetically separable phases of capacitance increase and consisted of an early rapid (600 granules s −1 ) component followed by a sustained slower (60 granules s −1 ) component. We conclude that (1) capacitance measurements in intact pancreatic islets are feasible; (2) exocytosis measured in β-cells in situ is significantly slower than that of isolated cells; and (3) the different types of islet cells exhibit distinct exocytotic features.

We have applied the perforated patch whole-cell technique to β cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K+ conductance. The current was dependent on Ca2+ influx but unaffected by apamin and charybdotoxin, two blockers of Ca2+-activated K+ channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K+ channels) but partially (>60%) blocked by high (10–20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca2+-activated K+ current plays an important role in the generation of oscillatory electrical activity in the β cell.