Transient transfection studies of secretion in bovine chromaffin cells and PC12 cells. Generation of kainate-sensitive chromaffin cells

P.F Wick,R.A Senter,L.A Parsels,M.D Uhler,R.W Holz

doi:10.1016/s0021-9258(18)82082-8

P.F Wick, R.A Senter + Show 3 more

Open Access

https://doi.org/10.1016/s0021-9258(18)82082-8

Copy DOI

Abstract

We have developed a transient transfection method to measure protein secretion from non-dividing, primary bovine chromaffin cells and from the 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 fraction (less than 1%) of transfected cells in a dish. GH was secreted in a similar proportion to endogenous catecholamine upon nicotinic stimulation, depolarization with elevated K+, and upon permeabilization with digitonin and subsequent stimulation with micromolar Ca2+. GH in homogenates from GH-transfected chromaffin cells cosedimented with catecholamine on discontinuous sucrose gradients. The data indicate that transiently expressed human GH in chromaffin and PC12 cells is localized predominantly in secretory vesicles in the regulated secretory pathway. With transient transfection there is a high probability of coexpression in the same cell of two plasmids which are cotransfected. Coexpression of a plasmid for GH and a plasmid for the non-N-methyl-D-aspartate glutamate receptor, GluR1, created chromaffin cells in which Ca(2+)-dependent GH secretion could be stimulated by the glutamatergic agonist kainate. The ability to coexpress a plasmid of interest with a plasmid for GH will allow the investigation of the role of other cloned proteins in the regulated secretory pathway in differentiated, non-dividing cells.

Highlights

We have developed a transient transfection method nels raises cytosolic Ca2+ andis the trigger for exocytosis of to measure protein secretion from non-dividing, pri- the secretory vesicle, the chromaffin granule[1].Chromaffin mary bovine chromaffin cells and from the continuougsranules contain catecholamine,ATP, opiate peptides, and a cell line, PC12
The Transient Expression and Ca2+-dependentSecretion of Human GH in Bovine Chromaffin Cells-Transfection with Ca2+phosphate precipitation using the method of Ross et al [17] or the Calcium PhosphateTransfection System kit (BRL) followed by 10% glycerol shock resulted in significant expression of a plasmid for human GH (pXGH5 containing the mouse metallothionein-I promoter [27])
To determine whether the GH was stored in the regulated secretory pathway, cells were stimulated to secrete 48-96 h after transfection (Fig. 1).Chromaffin cells that were incubated for 15min with either anicotinic agonist (1,l-dimethyl4-phenylpiperazinium) or elevated K+ secreted 25 or 11%, respectively, of the GH expressed in a small fraction of the cells

Summary

Introduction

We have developed a transient transfection method nels raises cytosolic Ca2+ andis the trigger for exocytosis of to measure protein secretion from non-dividing, pri- the secretory vesicle, the chromaffin granule[1].Chromaffin mary bovine chromaffin cells and from the continuougsranules contain catecholamine,ATP, opiate peptides, and a cell line, PC12. The Transient Expression and Ca2+-dependentSecretion of Human GH in Bovine Chromaffin Cells-Transfection with Ca2+phosphate precipitation using the method of Ross et al [17] or the Calcium PhosphateTransfection System kit (BRL) followed by 10% glycerol shock resulted in significant expression of a plasmid for human GH (pXGH5 containing the mouse metallothionein-I promoter [27]).

Results

Conclusion