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 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.
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0626.
P F
PF
Wick
R A
RA
Senter
L A
LA
Parsels
M D
MD
Uhler
R W
RW
Holz
D000313:Adrenal Medulla / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002395:Catecholamines / Q000378:metabolism*; D002417:Cattle; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002837:Chromaffin Granules / Q000378:metabolism; D013006:Growth Hormone / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007608:Kainic Acid / Q000494:pharmacology*; D007700:Kinetics; D009638:Norepinephrine / Q000378:metabolism*; D016716:PC12 Cells; D011188:Potassium / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection / Q000379:methods*
D002395:Catecholamines; D013006:Growth Hormone; D011188:Potassium; D007608:Kainic Acid; D002118:Calcium; D009638:Norepinephrine