Molecular mechanisms of glutamate release by bovine chromaffin cells in primary culture

Abstract

Previous work indicated that glutamate could be involved in the regulation of catecholamine secretion in bovine chromaffin cells. Thus, the question arises on the source of this putative regulatory glutamate. In this work we have examined the possibility that glutamate could be released from chromaffin cells. Data from this study indicate that chromaffin cells are able to release glutamate when they are stimulated by different depolarising agents such as 60 mM KCl, 1 mM 4-aminopyridine or 50 μM veratridine. The amount of glutamate released by these compounds was 0.32 nmol/10 6 cells (9.24% of cellular glutamate content), 0.275 (7.86%) and 0.158 (4.52%) for KCl, 4-AP and veratridine stimulation, respectively. All these catecholamine-secretagogues induced glutamate secretion by two mechanisms: 1) a Ca 2+-dependent, probably exocytotic, mechanism and 2) a Ca 2+-independent mechanism mediated by reversion of the electrogenic glutamate transporter. Analysis of Ca 2+-dependent and independent releases for different compounds carried out by several experimental approaches, indicate that Ca 2+-dependent release was the predominant mechanism for release induced by 4-aminopyridine (84% of total release) and high KCl (63%) whilst Ca 2+-independent release was predominant for veratridine (67%). The Ca 2+-dependent glutamate release evoked by depolarisation of chromaffin cells with high KCl and 4-AP could be split into both a fast and a slow kinetic component, which might correspond to the release of docked and mobilised chromaffin granules, respectively. On the other hand, depolarisation of cells with veratridine result in glutamate release with only the fast kinetic component. In the case of 60 mM KCl-evoked glutamate release, the fast component exhibited a decay time of <1 s and accounted for 0.63 nmol glu/6×10 6 cells (70% of total exocytotic release), whereas the slow component, which exhibited a decay time of 231 s, accounted for the release of 0.27 nmol glu/6×10 6 cells (30% of total exocytotic release). By contrast in the case of 4-aminopyridine the fast component of exocytosis only represents a 19% of total secretion and the slow a 81% with a decay time of 94 s. These data are very similar to those found in neurones and support the possible intracellular origin of glutamate having a role in the regulation of catecholamine secretion from chromaffin cells. In support of this, we have found that glutamate secretion could be evoked by stimulation of the nicotinic cholinergic receptors.