TRANSIENT TRANSFECTION STUDIES OF SECRETION IN BOVINE CHROMAFFIN CELLS AND PC12 CELLS - GENERATION OF KAINATE-SENSITIVE CHROMAFFIN CELLS

We have developed a transient transfection method to measure protein s ecretion from non-dividing, primary bovine chromaffin cells and from t he continuous cell line, PC12. A plasmid coding human growth hormone ( GH) was expressed in sufficient amounts in bovine chromaffin and PC12 cells to allow precise measurements of secretion from the small fracti on (less than 1%) of transfected cells in a dish. GH was secreted in a similar proportion to endogenous catecholamine upon nicotinic stimula tion, depolarization with elevated K+, and upon permeabilization with digitonin and subsequent stimulation with micromolar Ca2+. GH in homog enates from GH-transfected chromaffin cells cosedimented with catechol amine on discontinuous sucrose gradients. The data indicate that trans iently expressed human GH in chromaffin and PC12 cells is localized pr edominantly in secretory vesicles in the regulated secretory pathway. With transient transfection there is a high probability of coexpressio n in the same cell of two plasmids which are cotransfected. Coexpressi on of a plasmid for GH and a plasmid for the non-N-methyl-D-aspartate glutamate receptor, GluR1, created chromaffin cells in which Ca2+-depe ndent GH secretion could be stimulated by the glutamatergic agonist ka inate. The ability to coexpress a plasmid of interest with a plasmid f or GH will allow the investigation of the role of other cloned protein s in the regulated secretory pathway in differentiated, non-dividing c ells.