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Crotamine, a neurotoxin present in the venom of the South American rattlesnake Crotalus durrisus terrificus exists as several polymorphic variants, as demonstrated by recombinant DNA technology (Smith and Schmidt, Toxicon 28 (1990) 575-585). We have isolated native crotamine by chromatography on Sephadex G75, and have purified two crotamine isoforms (F2 and F3) by a single step of RP-HPLC. Native crotamine and RP-HPLC fractions F2 and F3 produced skeletal muscle spasms and spastic paralysis in mice. At low glucose concentrations (2.8-5.6 mmol/l), none of the crotamines altered the insulin secretion by rat isolated islets. In the presence of 16.7 mmol glucose/l, F2 (5 microg/ml), but not F3, increased insulin secretion two-fold, whereas native crotamine (1.5, 5 and 16.5 microg/ml) potentiated the secretion dose-dependently. The increase in insulin secretion induced by F2 fraction (5 microg/ml) was similar to that obtained with 16.5 microg of native crotamine/ml. These results indicate that the mode of action of the F2 and F3 isoforms in beta-cells is different from that in muscle cells. This difference may be related to the binding affinity of each isoform for the Na(+) channels located in the beta-cell membrane. Crotamine isoforms may be valuable tools for studying the involvement of Na(+) channels in the mechanism of insulin secretion.

1. Glucose-induced insulin release from single islets of Langerhans is pulsatile. We have investigated the correlation between changes in cytosolic free calcium concentration ([Ca2+]i) and oscillatory insulin secretion from single mouse islets, in particular examining the basis for differences in secretory responses to intermediate and high glucose concentrations. Insulin release was monitored in real time through the amperometric detection of the surrogate insulin marker 5-hydroxytryptamine (5-HT) via carbon fibre microelectrodes. The [Ca2+]i was simultaneously recorded by whole-islet fura-2 microfluorometry. 2. In 82 % of the experiments, exposure to 11 mM glucose evoked regular high-frequency (average, 3.4 min-1) synchronous oscillations in amperometric current and [Ca2+]i. In the remaining experiments (18 %), 11 mM glucose induced an oscillatory pattern consisting of high-frequency [Ca2+]i oscillations that were superimposed on low-frequency (average, 0.32 min-1) [Ca2+]i waves. Intermittent high-frequency [Ca2+]i oscillations gave rise to a similar pattern of pulsatile 5-HT release. 3. Raising the glucose concentration from 11 to 20 mM increased the duration of the steady-state [Ca2+]i oscillations without increasing their amplitude. In contrast, both the duration and amplitude of the associated 5-HT transients were increased by glucose stimulation. The amount of 5-HT released per secretion cycle was linearly related to the duration of the underlying [Ca2+]i oscillations in both 11 and 20 mM glucose. The slopes of the straight lines were identical, indicating that there is no significant difference between the ability of calcium oscillations to elicit 5-HT/insulin release in 11 and 20 mM glucose. 4. In situ 5-HT microamperometry has the potential to resolve the high-frequency oscillatory component of the second phase of glucose-induced insulin secretion. This component appears to reflect primarily the duration of the underlying [Ca2+]i oscillations, suggesting that glucose metabolism and/or access to glucose metabolites is not rate limiting to fast pulsatile insulin release.

We report a highly sensitive electrochemical approach suitable for the real time measurement of insulin release from single islets of Langerhans, the functional endocrine units in the pancreas. The method is based on the detection of the insulin surrogate 5-hydroxytryptamine (5-HT) by carbon fibre microelectrodes implanted in the islets. Based on the combination of this novel approach with the simultaneous microfluorometric recording of cytosolic free Ca2+ concentration ([Ca2+]i), we demonstrate that glucose-stimulated islets secrete 5-HT/insulin in a pulsatile fashion under physiological conditions, and that this activity is encoded by synchronous [Ca2+]i oscillations. The sensitivity to detect variations in minute amounts of secreted materials is partially conferred by the fact that the tracer is released into a relatively confined space (the intraislet interstitial space).

The study of the influence of intracellular pH (pHi) changes on the mechanism underlying pancreatic beta-cell bursting has been hampered by concomitant effects on the activity of background ATP-dependent K+ (K-ATP) channels. beta-cells were made to burst in the absence of active K-ATP channels by raising external Ca2+ in the presence of 11 mM glucose and tolbutamide. An alkalinizing pHi shift (exposure to 20 mM NH4Cl) increased the burst active phase duration. Conversely, an acidifying shift (NH4Cl withdrawal) suppressed the electrical activity. This is the mirror image of the effects recorded in the absence of tolbutamide. Glibenclamide and quinine suppressed the alkalinization-evoked hyperpolarization. This study emphasizes the differential sensitivity of different beta-cell ion channels to pHi and the prevalent role of K-ATP channels as electrical transducers of cytoplasmic pH changes under regular physiological conditions.

In glucose-stimulated pancreatic beta-cells, the membrane potential alternates between a hyperpolarized silent phase and a depolarized phase with Ca2+ action potentials. The molecular and ionic mechanisms underlying these bursts of electrical activity remain unknown. We have observed that 10.2-12.8 mM Ca2+, 1 microM Bay K 8644 and 2 mM tetraethylammonium (TEA) trigger bursts of electrical activity and oscillations of intracellular free Ca2+ concentration ([Ca2+]i) in the presence of 100 microM tolbutamide. The [Ca2+]i was monitored from single islets of Langerhans using fura-2 microfluorescence techniques. Both the high-Ca(2+)- and Bay-K-8644-evoked [Ca2+]i oscillations overshot the [Ca2+]i recorded in tolbutamide. Nifedipine (10-20 microM) caused an immediate membrane hyperpolarization, which was followed by a slow depolarization to a level close to the burst active phase potential. The latter depolarization was accompanied by suppression of spiking activity. Exposure to high Ca2+ in the presence of nifedipine caused a steady depolarization of approximately 8 mV. Ionomycin (10 microM) caused membrane hyperpolarization in the presence of 7.7 mM Ca2+, which was not abolished by nifedipine. Charybdotoxin (CTX, 40-80 nM), TEA (2 mM) and quinine (200 microM) did not suppress the high-Ca(2+)-evoked bursts. It is concluded that: (1) the channel underlying the burst is sensitive to [Ca2+]i rises mediated by Ca2+ influx through L-type Ca2+ channels, (2) both the ATP-dependent K+ channel and the CTX- and TEA-sensitive Ca(2+)-dependent K+ channel are highly unlikely to provide the pacemaker current underlying the burst.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose depolarizes the pancreatic beta-cell and induces membrane potential oscillations, but the nature of the underlying oscillatory conductance remains unknown. We have now investigated the effects of the Ca2+ ionophore ionomycin and high external Ca2+ concentration ([Ca2+]o) on glucose-induced electrical activity and whole islet intracellular free Ca2+ concentration ([Ca2+]i), under conditions where the K(ATP) channel was blocked (100 microM tolbutamide or 4 microM glibenclamide). Raising [Ca2+]o to 10.2 or 12.8 mM, but not to 5.1 or 7.7 mM, turned continuous electrical activity into bursting activity. High [Ca2+]o (12.8 mM) regenerated a pattern of fast [Ca2+]i oscillations overshooting the levels recorded in tolbutamide. Ionomycin (10 microM) raised the [Ca2+]i and synergized with 5.1 mM Ca2+ to hyperpolarize the beta-cell membrane. The data indicate that a [Ca2+]i-sensitive and sulphonylurea-insensitive oscillatory conductance underlies the beta-cell bursting activity.