Ca 2+ clearance mechanisms in neurohypophysial terminals of the rat

Abstract

The importance of intracellular calcium ([Ca 2+] 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 Ca 2+ clearance mechanisms and their interplay with transmembrane Ca 2+ entry, intracellular [Ca 2+] i transients, cytoplasmic Ca 2+ stores and hence the release of AVP at the level of a single nerve terminal. Here, we studied the mechanism of Ca 2+ clearance in freshly isolated nerve terminals of the rat neurohypophysis using Fura-2 Ca 2+ 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 [Ca 2+] i and AVP release. Returning K + concentration to 5 mM led to rapid restoration of both responses to basal level. The K +-evoked [Ca 2+] i and AVP increase was concentration-dependent, reliable, and remained of constant amplitude and time course upon successive applications. Extracellular Ca 2+ removal completely abolished the K +-evoked responses. The recovery phase was not affected upon replacement of NaCl with sucrose or drugs known to act on intracellular Ca 2+ stores such as thapsigargin, cyclopiazonic acid, caffeine or a combination of caffeine and ryanodine did not affect either resting or K +-evoked [Ca 2+] i or AVP release. By contrast, the plasma membrane Ca 2+ pump inhibitor, La 3+, markedly slowed down the recovery phase. The mitochondrial respiration uncoupler, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), slightly but significantly increased the basal [Ca 2+] i, and also slowed down the recovery phase of both [Ca 2+] i and release responses. In conclusion, we show in nerve terminals that (i) Ca 2+ extrusion through the Ca 2+ pump in the plasma membrane plays a major role in the Ca 2+ clearance mechanisms of (ii) Ca 2+ uptake by mitochondria also contributes to the Ca 2+ clearance and (iii) neither Na +/Ca 2+ exchangers nor Ca 2+ stores are involved in the Ca 2+ clearance or in the maintenance of basal [Ca 2+] i or release of AVP.