Recombinant calcium channels and exocytosis in bovine adrenal chromaffin cells.

Abstract

A novel method for investigating the role of specific proteins in regulated exocytosis in bovine chromaffin cells has been developed. Primary chromaffin cell cultures were transiently transfected with the gene for human growth hormone (GH). Subcellular fractionation and secretion studies showed that transiently overexpressed GH colocalized with endogenous catecholamine and was a valid reporter for regulated secretion from a small population of transfected chromaffin cells. This method was used to study secretion from cells cotransfected with plasmids encoding the genes for GH and a mouse brain L-type Ca$\sp{2+}$ channel $\alpha\sb1$ subunit. The Ca$\sp{2+}$ channels formed with this exogenous subunit caused cells to become supersensitive to both elevated K$\sp+$ depolarization and the nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP), which suggests that secretion is limited by the number of Ca$\sp{2+}$ channels. The recombinant channels had proportionally greater effects on GH release with suboptimal stimuli which suggests that the Ca$\sp{2+}$-sensitive mechanisms of exocytosis are saturable. The exogenous channels increased GH secretion during later interviews but had no effect on secretion before 30 seconds. The mouse brain $\alpha\sb1$ subunit-based channels were responsive to the dihydropyridine L-type Ca$\sp{2+}$ channel agonist, Bay K 8644, but pretreatment did not reverse the latency of enhancement, indicating that the extra Ca$\sp{2+}$ channels functioned differently than the endogenous L-type Ca$\sp{2+}$ channels. Fluorescence immunocytochemistry was used to visualize dopamine-$\beta$-hydroxylase (D$\beta$H) on the stimulated chromaffin cell surface. The appearance of D$\beta$H was uniformly distributed and punctate in DMPP-stimulated intact cells and in digitonin-permeabilized cells stimulated with 1-30 $\mu$M Ca$\sp{2+}$. These data indicate that localized Ca$\sp{2+}$ currents are not responsible for the punctate appearance of D$\beta$H. Quantitative analysis showed the average integrated intensity of surface D$\beta$H spots was one-fourth the mean chromaffin granule intensity and indicated the punctate appearance of D$\beta$H reflects the exocytotic fusion of individual granules. The average surface site area was 0.218 $\mu\rm m\sp2$, similar to the predicted surface area (0.246 $\mu\rm m\sp2$) of a single chromaffin granule and is also consistent with each D$\beta$H site representing the fusion of single chromaffin granules.