Internal Ca 2+ mobilization and secretion in bovine adrenal chromaffin cells

Abstract

Since secretion from intact bovine adrenal chromaffin cells in response to depolarization by nicotine is triggered by a rise in the concentration of intracellular Ca 2+ ([Ca 2+] i) to about 200–300 nM above basal, it has been assumed that the failure of the inositol 1,4,5-trisphosphate (InsP 3)-mobilizing muscarinic agonists to induce secretion reflects the fact that the 50 nM rise in [Ca 2+] i they elicit is insufficient to trigger the exocytotic machinery. A recent report [11], however, has demonstrated that some of the nicotine-induced rise in [Ca 2+] i could originate from the InsP 3-releasable Ca 2+ store. The role of this Ca 2+ store in secretion from bovine adrenal chromaffin cells is therefore unclear. In order to investigate in more detail the role of the InsP 3-sensitive Ca 2+ store in secretion from these cells, we have used a combination of an InsP 3-mobilizing muscarinic agonist and the sesquiterpene lactone thapsigargin (TG), which releases internal Ca 2+ without concomitant breakdown of inositol lipids or protein kinase C activation, to examine the events which follow depletion of the releasable Ca 2+ store in these cells. Monitoring [Ca 2+] i using Fura-2 demonstrated that TG released Ca 2+ from the InsP 3-sensitive store and, additionally, that the Ca 2+ response to TG was composed of two distinct, temporally separated, components: a) a slow (1 min) increase in [Ca 2+] i to ∼50 nM above basal that was independent of extracellular Ca 2+ and b) the maintenance of this level at a new steady-state that was dependent on the continual entry of extracellular Ca 2+. Since the entry of external Ca 2+ always followed on from the release of stored Ca 2+, it is conceivable that Ca 2+ flows into the cell via the store after it has first been depleted by TG. A link similar to that proposed by Putney [28] may therefore exist between the Ca 2+ store and the external milieu in these cells. The results also showed that, despite raising [Ca 2+] i by only 50 nM, TG was able to elicit a secretory response. This result demonstrates that it is possible to induce secretion from these cells by a modest elevation in [Ca 2+] i resulting from internal Ca 2+ release, provided that mobilization is followed by Ca 2+ influx. These results, therefore, argue against the lack of efficacy of muscarinic agonists being a result of their inability to sufficiently elevate [Ca 2+] i, and point towards Ca 2+ influx distal to internal release as important for secretion due to some stimuli.