Ca2+ clearance mechanisms in neurohypophysial terminals of the rat

The importance of intracellular calcium ([Ca2+](i)) in the release of vasopressin (AVP) and oxytocin from the central nervous system neurohypopyhysial nerve terminals has been well-documented. To date, there is no clear understanding of Ca2+ clearance mechanisms and their interplay with transmembrane Ca2+ entry, intracellular [Ca2+](i) transients, cytoplasmic Ca2+ stores and hence the release of AVP at the clearance in freshly isolated nerve terminals of the rat neurohypophysis level of a sin-le nerve terminal. Here. we studied the mechanism of Ca2+ using Fura-2 Ca2+ imaging and measured the release of AVP by radioimmuno assay. An increase in the K+ concentration in the perfusion solution from 5 to 50 mM caused a rapid increase in [Ca2+](i) and AVP release. Returning K+ concentration to 5 mM led to rapid restoration of both responses to basal level. The K+-evoked [Ca2+](i) and AVP increase was concentration-dependent, reliable, and remained of constant amplitude and time course upon successive applications. Extracellular Ca2+ removal completely abolished the K+-evoked responses. The recovery phase was not affected upon replacement of NaCl with sucrose or drup known to act on intracellular Ca2+ stores suchas thapsigargin, cyclopiazonic acid. caffeine or a combination of caffeine and ryanodine did not affect either resting or K+-evoked [Ca2+], or AVP release. By contrast, the plasma membrane Ca2+ pump inhibitor, [Ca2+](i) markedly slowed down the recovery phase. The mitochondrial respiration uncoupler. carbonyl cyanide 3-chlorophenylhydrazone (CCCP), slightly but significantly increased the basal [Ca2+] and also slowed down the recoverv phase of both [Ca2+](i) and release responses. In conclusion, we show in nerve terminals that (i) Ca2+ extrusion through the Ca2+ pump in the plasma membrane plays a major role in the Ca2+ clearance mechanisms of (ii) Ca2+ uptake by mitochondria also contributes to the Ca2+ clearance and (iii) neither Na+/Ca2+ exchangers nor Ca2+ stores are involved in the Ca'+ clearance or in the maintenance of basal [Ca2+](i) or release of AVP. (C) 2004 Elsevier Ltd. All rights reserved.