Gi2 proteins couple somatostatin receptors to low-conductance K+ channels in rat pancreatic alpha-cells.

Somatostatin hyperpolarized rat pancreatic alpha-cells and inhibited spontaneous electrical activity by activating a low-conductance K+ channel (0.9 pS with physiological ionic gradients). This channel was insensitive to tolbutamide (a blocker of ATP-sensitive K+ channels) and apamin (an inhibitor of small-conductance Ca(2+)-activated K+ channels). Channel activation was prevented by pre-treating the cells with pertussis toxin, indicating the involvement of G-proteins. A direct interaction between an inhibitory G-protein and the somatostatin-activated K+ channel is suggested by the finding that intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma-S) and the G beta gamma subunit of G-proteins resulted in a transient stimulation of the current. Activation of the K+ current by somatostatin was inhibited by intracellular dialysis with specific antibodies to Gi1/2 and was not seen in cells treated with antisense oligonucleotides against G-proteins of the subtype Gi2. We conclude that somatostatin suppresses alpha-cell electrical activity by a Gi2-protein-dependent mechanism, which culminates in the activation of a sulphonylurea- and apamin-insensitive low-conductance K+ channel.

Tolbutamide stimulates exocytosis of glucagon by inhibition of a mitochondrial-like ATP-sensitive K+ (KATP) conductance in rat pancreatic A-cells.

1. Capacitance measurements were used to examine the effects of the sulphonylurea tolbutamide on Ca2+-dependent exocytosis in isolated glucagon-secreting rat pancreatic A-cells. 2. When applied extracellularly, tolbutamide stimulated depolarization-evoked exocytosis 4.2-fold without affecting the whole-cell Ca2+ current. The concentration dependence of the stimulatory action was determined by intracellular application through the recording pipette. Tolbutamide produced a concentration-dependent increase in cell capacitance. Half-maximal stimulation was observed at 33 microM and the maximum stimulation corresponded to a 3.4-fold enhancement of exocytosis. 3. The stimulatory action of tolbutamide was dependent on protein kinase C activity. The action of tolbutamide was mimicked by the general K+ channel blockers TEA (10 mM) and quinine (10 microM). A similar stimulation was elicited by 5-hydroxydecanoate (5-HD; 10 microM), an inhibitor of mitochondrial ATP-sensitive K+ (KATP) channels. 4. Tolbutamide-stimulated, but not TEA-induced, exocytosis was antagonized by the K+ channel openers diazoxide, pinacidil and cromakalim. 5. Dissipating the transgranular K+ gradient with nigericin and valinomycin inhibited tolbutamide- and Ca2+-evoked exocytosis. Furthermore, tolbutamide- and Ca2+-induced exocytosis were abolished by the H+ ionophore FCCP or by arresting the vacuolar (V-type) H+-ATPase with bafilomycin A1 or DCCD. Finally, ammonium chloride stimulated exocytosis to a similar extent to that obtained with tolbutamide. 6. We propose that during granular maturation, a granular V-type H+-ATPase pumps H+ into the secretory granule leading to the generation of a pH gradient across the granular membrane and the development of a positive voltage inside the granules. The pumping of H+ is facilitated by the concomitant exit of K+ through granular K+ channels with pharmacological properties similar to those of mitochondrial KATP channels. Release of granules that have been primed is then facilitated by the addition of K+ channel blockers. The resulting increase in membrane potential promotes exocytosis by unknown mechanisms, possibly involving granular alkalinization.

Acute stimulation with long chain acyl-CoA enhances exocytosis in insulin-secreting cells (HIT T-15 and NMRI beta-cells).

Non-insulin-dependent diabetes mellitus is associated with, in addition to impaired insulin release, elevated levels of free fatty acids (FFA) in the blood. Insulin release is stimulated when beta-cells are acutely exposed to FFA, whereas chronic exposure may inhibit glucose-induced insulin secretion. In the present study we investigated the direct effects of long chain acyl-CoA (LC-CoA), the active intracellular form of FFA, on insulin exocytosis. Palmitoyl-CoA stimulated both insulin release from streptolysin-O-permeabilized HIT cells and fusion of secretory granules to the plasma membrane of mouse pancreatic beta-cells, as measured by cell capacitance. The LC-CoA effect was chain length-dependent, requiring chain lengths of at least 14 carbons. LC-CoA needed to be present to stimulate insulin release, and consequently there was no effect following its removal. The stimulatory effect was observed after inhibition of protein kinase activity and in the absence of ATP, even though both kinases and ATP, themselves, modulate exocytosis. The effect of LC-CoA was inhibited by cerulenin, which has been shown to block protein acylation. The data suggest that altered LC-CoA levels, resulting from FFA or glucose metabolism, may act directly on the exocytotic machinery to stimulate insulin release by a mechanism involving LC-CoA protein binding.

Characterisation of sulphonylurea and ATP-regulated K+ channels in rat pancreatic A-cells.

We have monitored whole-cell and single channel ATP-sensitive K+ (KATP) currents in isolated rat glucagon-secreting pancreatic A-cells. Tolbutamide produced a concentration-dependent decrease in the whole-cell KATP conductance (Ki = 6 microM) and initiated action potential firing. The K+ channel opener diazoxide, but not cromakalim or pinacidil, inhibited electrical activity and increased the whole-cell K+ conductance fourfold. ATP applied to the intracellular face of the membrane inhibited KATP channel activity with a Ki of 17 microM, an effect that could be counteracted by Mg-ADP and Mg-GDP. GTP and UTP did not affect KATP channel activity. Phosphatidylinositol 4,5-bisphosphate activated KATP channels inhibited by ATP after a delay of 90 s. In situ hybridisation demonstrated the expression of the mRNA encoding KATP channel subunits Kir6.2 and SUR1 but not Kir6.1 and SUR2. We conclude that rat pancreatic A-cells express KATP channels with the nucleotide-, sulphonylurea- and K+ channel-opener sensitivities expected for a channel formed by Kir6.2 and SUR1 subunits.

Multiple sites of purinergic control of insulin secretion in mouse pancreatic beta-cells.

In mouse pancreatic beta-cells, extracellular ATP (0.1 mmol/l) effectively reduced glucose-induced insulin secretion. This inhibitory action resulted from a direct interference with the secretory machinery, and ATP suppressed depolarization-induced exocytosis by 60% as revealed by high-resolution capacitance measurements. Suppression of Ca2+-dependent exocytosis was mediated via binding to P2Y1 purinoceptors but was not associated with inhibition of the voltage-dependent Ca2+ currents or adenylate cyclase activity. Inhibition of exocytosis by ATP resulted from G-protein-dependent activation of the serine/threonine protein phosphatase calcineurin and was abolished by cyclosporin A and deltamethrin. In contrast to the direct inhibitory action on exocytosis, ATP reduced the whole-cell ATP-sensitive K+ (K(ATP)) current by 30% (via activation of cytosolic phospholipase A2), leading to membrane depolarization and stimulation of electrical activity. The stimulatory effect of ATP also involved mobilization of Ca2+ from thapsigargin-sensitive intracellular stores. We propose that the inhibitory action of ATP, by interacting with the secretory machinery at a level downstream to an elevation in [Ca2+]i, is important for autocrine regulation of insulin secretion in mouse beta-cells.

A device for the intraoperative identification of the recurrent laryngeal nerve in piglets.

The recurrent laryngeal nerve (RLN) is one of the most frequently injured nerves in head and neck surgery. Routine identification of the RLN during thyroid surgery has reduced the injury rate from 10% to less than 4%. Difficulty in identification of the RLN contributes to this surgical morbidity. Devices previously used for intraoperative identification of the RLN have failed to achieve the simplicity and reliability necessary for clinical use. This animal study uses a simple double-ballooned endotracheal tube and pressure transducer system, which assists intraoperative RLN identification through nerve stimulation and graphic documentation of vocal fold (VF) motion. Iatrogenic injury is demonstrated by a dampened stimulation-pressure tracing. The RLNs of three piglets were injured and examined, and the degree of injury was correlated with perioperative nerve stimulation patterns. The piglet proved to be an adequate model for laryngeal research. An FDA-approved multi-institutional prospective human study using this system of identification of the RLN is in progress.