Anne Marie F. Salapatek

Supramaximal cholecystokinin displaces Munc18c from the pancreatic acinar basal surface, redirecting apical exocytosis to the basal membrane

Exocytosis at the apical surface of pancreatic acinar cells occurs in the presence of physiological concentrations of cholecystokinin (CCK) but is inhibited at high concentrations. Here we show that Munc18c is localized predominantly to the basal membranes of acinar cells. Supramaximal but not submaximal CCK stimulation caused Munc18c to dissociate from the plasma membrane, and this displacement was blocked by protein kinase C (PKC) inhibitors. Conversely, whereas the CCK analog CCK-OPE alone failed to displace Munc18c from the membrane, this agent caused Munc18c displacement following minimal PKC activation. To determine the physiological significance of this displacement, we used the fluorescent dye FM1-43 to visualize individual exocytosis events in real-time from rat acinar cells in culture. We showed that supramaximal CCK inhibition of secretion resulted from impaired apical secretion and a redirection of exocytic events to restricted basal membrane sites. In contrast, CCK-OPE evoked apical exocytosis and could only induce basolateral exocytosis following activation of PKC. Infusion of supraphysiological concentrations of CCK in rats, a treatment that induced tissue changes reminiscent of mild acute pancreatitis, likewise resulted in rapid displacement of Munc18c from the basal membrane in vivo.

The 25-kDa Synaptosome-associated Protein (SNAP-25) Binds and Inhibits Delayed Rectifier Potassium Channels in Secretory Cells

Delayed-rectifier K+ channels (KDR) are important regulators of membrane excitability in neurons and neuroendocrine cells. Opening of these voltage-dependent K+ channels results in membrane repolarization, leading to the closure of the Ca2+channels and cessation of insulin secretion in neuroendocrine islet β cells. Using patch clamp techniques, we have demonstrated that the activity of the KDR channel subtype, KV1.1, identified by its specific blocker dendrodotoxin-K, is inhibited by SNAP-25 in insulinoma HIT-T15 β cells. A co-precipitation study of rat brain confirmed that SNAP-25 interacts with the KV1.1 protein. Cleavage of SNAP-25 by expression of botulinum neurotoxin A in HIT-T15 cells relieved this SNAP-25-mediated inhibition of KDR. This inhibitory effect of SNAP-25 is mediated by the N terminus of KV1.1, likely by direct interactions with KVα1.1 and/or KVβ subunits, as revealed by co-immunoprecipitation performed in the Xenopus oocyte expression system and in vitro binding. Taken together we have concluded that SNAP-25 mediates secretion not only through its participation in the exocytotic SNARE complex but also by regulating membrane potential and calcium entry through its interaction with KDR channels.

Temperature and redox state dependence of native Kv2.1 currents in rat pancreatic β-cells

In pancreatic β‐cells, voltage‐dependent K+ (Kv) channels repolarise glucose‐stimulated action potentials. Kv channels are therefore negative regulators of Ca2+ entry and insulin secretion. We have recently demonstrated that Kv2.1 mediates the majority of β‐cell voltage‐dependent outward K+ current and now investigate the function of native β‐cell Kv2.1 channels at near‐physiological temperatures (32‐35 °C). While β‐cell voltage‐dependent outward K+ currents inactivated little at room temperature, both fast‐inactivation (111.5 ± 14.3 ms) and slow‐inactivation (1.21 ± 0.12 s) was observed at 32‐35 °C. Kv2.1 mediates the fast‐inactivating current observed at 32‐35 °C, since it could be selectively ablated by expression of a dominant‐negative Kv2.1 construct (Kv2.1N). The surprising ability of Kv2.1N to selectively remove the fast‐inactivating component, together with its sensitivity to tetraethylammonium (TEA), demonstrate that this component is not mediated by the classically fast‐inactivating and TEA‐resistant channels such as Kv1.4 and 4.2. Increasing the intracellular redox state by elevating the cytosolic NADPH/NADP+ ratio from 1/10 to 10/1 increased the rates of both fast‐ and slow‐inactivation. In addition, increasing the intracellular redox state also increased the relative contribution of the fast‐inactivation component from 38.8 ± 2.1 % to 55.9 ± 1.8 %. The present study suggests that, in β‐cells, Kv2.1 channels mediate a fast‐inactivating K+ current at physiological temperatures and may be regulated by the metabolic generation of NADPH.

Assessment of neural inhibition of the lower esophageal sphincter in cats with esophagitis

BACKGROUND The aim of this study was to investigate the inhibitory innervation of the lower esophageal sphincter in the presence of esophagitis. METHODS Esophagitis was produced in five anesthetized cats with intraesophageal perfusion of HCl. Sphincter pressure responses were assessed with a sleeve catheter after administration of bethanechol, cholecystokinin octapeptide, and McNeil-A343 and with intraesophageal balloon distension. RESULTS In the presence of esophagitis (1) resting lower esophageal sphincter pressure decreased; (2) the excitatory response to bethanechol was maintained; (3) there was a reduction in the excitatory response to McNeil-A343 and cholecystokinin at the highest dosages; (4) there was an increase in the potency of cholecystokinin and McNeil-A343 to produce an inhibitory response; (5) the inhibitory response to intraesophageal balloon distension was maintained; and (6) increased inhibitory responses took longer to normalize than the reduced excitatory responses. CONCLUSIONS Esophagitis decreases cholinergic excitation, but neural inhibition to the LES remains intact. These findings suggest that blocking intact inhibition may be a new therapeutic approach for esophagitis caused by gastroesophageal reflux.