Abstract
PC12 cells, a cloned rat pheochromocytoma cell line, were treated with digitonin to render the plasma membrane permeable to ions and proteins. At a cell density of 2-6 X 10(5) cells/cm2, incubation with 7.5 microM digitonin permitted a Ca2+-dependent release of 25-40% of the catecholamine within 18 min in the presence of 10 microM Ca2+. Half-maximal secretion occurred at 0.5-1 microM Ca2+. PC12 cultures at lower cell densities were more sensitive to digitonin and gave more variable results. Secretion in the presence of digitonin and Ca2+ began after a 2-min lag and continued for up to 30 min. When cells were treated for 3 min in digitonin and then stimulated with Ca2+ in the absence of digitonin, secretion occurred in the same manner but without the initial lag. Optimal secretion from PC12 cells was also dependent upon the presence of Mg2+ and ATP. Permeabilized PC12 cells exhibited a slow time-dependent loss of secretory responsiveness which was correlated with the release of a cytosolic marker, lactate dehydrogenase (134 kDa). This suggests that digitonin permeabilization allows soluble constituents necessary for secretion to leave the cell in addition to allowing Ca2+ and ATP access into the cell interior. Ca2+-dependent secretion was completely inhibited by exposure of digitonin-permeabilized cells to 100 micrograms/ml trypsin (27 kDa), whereas secretion was only slightly inhibited by trypsin exposure prior to digitonin treatment. Thus, an intracellular, trypsin-sensitive protein is probably involved in secretion. The data also indicate that the same population of digitonin-treated cells which responded to Ca2+ was permeable to a 27-kDa protein. 1,2-Dioctanoylglycerol and phorbol esters which activate protein kinase C enhanced the Ca2+-dependent and Ca2+-independent secretion in digitonin-permeabilized PC12 cells. Thus, protein kinase C appears to be involved in the regulation of catecholamine secretion from permeabilized PC12 cells.
Highlights
PC12 cells, a cloned rat pheochromocytomacell line, receptors, and a high affinity, saturable norepinephrine upwere treatedwith digitonin to render tphleasma mem- take mechanism
At a cell density causes differentiation into cells similar to sympathetic neuof 2-6 X lo6cells/cm2,incubation with7.5 @M digitonin rons with extensive neurite development, loss of cell replicapermitted a Ca2+-dependentrelease of 25-40% of the tion, enhancedresponsiveness to acetylcholine, and induction catecholamine within 18 min in the presence of 10 PM of several adrenergic and cholinergic synthetic enzymes
The phorbol ester TPA and a relatively watersecretion wascompletely inhibited by exposure of dig- soluble diacylglycerol, l-oleoyl-2-acetylglycerolw,hich are acitonin-permeabilized cells to 100 pg/ml trypsin(27 tivators of protein kinase C, induce a slow and sustained kDa), whereas secretion woansly slightly inhibitedby catecholamine release from PC12 cells andpotentiate the trypsin exposure prior to digitonin treatment
Summary
PC12 cells, a cloned rat pheochromocytomacell line, receptors, and a high affinity, saturable norepinephrine upwere treatedwith digitonin to render tphleasma mem- take mechanism. This suggesttshat digitonin per- Most important, TPA activates protein kinase C and protein meabilization allows soluble constituents necessary for phosphorylation in intact cells and enhances secretion [8,9]. The phorbol ester TPA and a relatively watersecretion wascompletely inhibited by exposure of dig- soluble diacylglycerol, l-oleoyl-2-acetylglycerolw,hich are acitonin-permeabilized cells to 100 pg/ml trypsin(27 tivators of protein kinase C (lo), induce a slow and sustained kDa), whereas secretion woansly slightly inhibitedby catecholamine release from PC12 cells andpotentiate the trypsin exposure prior to digitonin treatment.
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