Digitonin-treated Chromaffin Cells Research Articles

Effects of trypsin on secretion stimulated by micromolar Ca2+ and phorbol ester in digitonin-permeabilized adrenal chromaffin cells.

1. Catecholamine secretion from digitonin-treated chromaffin cells is stimulated directly by micromolar Ca2+ in the medium. The permeabilized cells are leaky to proteins. 2. In this study trypsin (30-50 micrograms/ml) added to cells after digitonin treatment completely inhibited subsequent Ca2+-dependent catecholamine secretion. The same concentrations of trypsin did not inhibit secretion from permeabilized cells if trypsin was present only prior to cell permeabilization. 3. The data indicate that trypsin entered digitonin-treated chromaffin cells which were capable of undergoing secretion and that an intracellular, trypsin-sensitive protein is involved in secretion. Chymotrypsin was less potent but had effects similar to those of trypsin. 4. The enhancement of Ca2+-dependent secretion from permeabilized chromaffin cells induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by trypsin added simultaneously with Ca2+ to permeabilized cells at concentrations (3-10 micrograms/ml) which had little or no effect on Ca2+-dependent secretion from cells untreated with TPA. Ca2+-dependent secretion in TPA-treated cells was reduced by trypsin only to the level that would have occurred in cells not treated with TPA. Trypsin reduced the large TPA-induced increment of membrane-bound protein kinase C.

Effects of purified myosin light chain kinase on myosin light chain phosphorylation and catecholamine secretion in digitonin-permeabilized chromaffin cells

Many non-muscle cells including chromaffin cells contain actin and myosin. The 20,000 dalton light chain subunits of myosin can be phosphorylated by a Ca2+/calmodulin-dependent enzyme, myosin light chain kinase. In tissues other than striated muscle, light chain phosphorylation is required for actin-induced myosin ATPase activity. The possibility that actin and myosin are involved in catecholamine secretion was investigated by determining whether increased phosphorylation in the presence of [gamma-32P]ATP of myosin light chain by myosin light chain kinase enhances secretion from digitonin-treated chromaffin cells. In the absence of exogenous myosin light chain kinase, 1 microM Ca2+ caused a 30-40% enhancement of the phosphorylation of a 20 kDa protein. This protein was identified on 2-dimensional gels as myosin light chain by its comigration with purified myosin light chain. Purified myosin light chain kinase (400 micrograms/ml) in the presence of calmodulin (10 microM) caused little or no enhancement of myosin light chain phosphorylation in the absence of Ca2+ in digitonin-treated cells. In the presence of 1 microM Ca2+, myosin light chain kinase (400 micrograms/ml) caused an approximately two-fold increase in myosin light chain phosphorylation in digitonin-treated cells in 5 min. The phosphorylation required permeabilization of the cells by digitonin and occurred within the cells rather than in the medium. Myosin light chain kinase-induced phosphorylation of myosin light chain was maximal at 1 microM Ca2+. Under identical conditions to those of the phosphorylation experiments, secretion was unaltered by myosin light chain kinase. The experiments indicate that the phosphorylation of myosin light chain by myosin light chain kinase is not a limiting factor in secretion in digitonin-treated chromaffin cells and suggest that the activation of myosin is not directly involved in secretion from the cells. The experiments also demonstrate the feasibility of investigation of effects of exogenously added proteins on secretion in digitonin-treated cells.

Protein phosphorylation and secretion in digitonin-permeabilized adrenal chromaffin cells. Effects of micromolar Ca2+, phorbol esters, and diacylglycerol.

The effects of phorbol esters, dioctanoylglycerol (DiC8), and micromolar Ca2+ on protein phosphorylation and catecholamine secretion in digitonin-treated chromaffin cells were investigated. [gamma-32P]ATP was used as a substrate for phosphorylation in the permeabilized cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) enhanced Ca2+-dependent catecholamine secretion from digitonin-permeabilized cells. The enhancement required MgATP. Only those phorbol esters which activate protein kinase C in vitro enhanced both catecholamine secretion and protein phosphorylation. DiC8, which activates protein kinase C in vitro and mimics phorbol ester effects in situ, also enhanced both catecholamine secretion and protein phosphorylation. Preincubation of intact cells with TPA or DiC8 was necessary for maximal effects on both catecholamine secretion and protein phosphorylation in subsequently digitonin-treated chromaffin cells. The TPA-induced enhancement of protein phosphorylation was almost entirely Ca2+-independent, whereas DiC8-induced enhancement of protein phosphorylation was mainly Ca2+-dependent. Micromolar Ca2+ alone also enhanced the phosphorylation of a large number of proteins. Most of the proteins phosphorylated in response to TPA or potentiated by DiC8 in combination with Ca2+ were also phosphorylated by micromolar Ca2+ in the absence of exogenous protein kinase C activators. In intact cells, 1,1-dimethyl-4-phenylpiperazinium (DMPP) induced Ca2+-dependent phosphorylation of at least 17 proteins which were detected by two-dimensional gel electrophoresis. All of the proteins phosphorylated upon incubation with 1,1-dimethyl-4-phenylpiperazinium were phosphorylated upon incubation with micromolar Ca2+ in digitonin-treated cells. These results demonstrate that TPA- or DiC8-enhanced Ca2+-dependent catecholamine secretion is associated with enhanced protein phosphorylation which is probably mediated by protein kinase C and that activation of protein kinase C modulates catecholamine secretion from digitonin-treated chromaffin cells.

Ascorbic acid and catecholamine release from digitonin-treated chromaffin cells.

The subcellular localization of catecholamines and ascorbic acid in cultured bovine adrenal chromaffin cells was studied by permeabilizing the cells with digitonin, a steroid glycoside. Catecholamine release from permeabilized chromaffin cells was dependent on the free calcium concentration and the temperature of the incubation mixture. By contrast, [14C]ascorbic acid, preloaded into the cells, was released by digitonin treatment in a manner independent of the concentration of free calcium and with only moderate regard to the incubation temperature. The sensitivity of ascorbic acid release to digitonin treatment was identical to that of calcium-dependent catecholamine release. These results thus suggest that ascorbic acid preloaded into the cells may directly efflux from the cell cytoplasm as a result of the permeabilization of the plasma membrane. Dimethylepinephrine, a permanently positively charged catecholamine analog which is known to be excluded from vesicular fractions, was also released by digitonin treatment in a manner independent of calcium. The time course of dimethylepinephrine release was very similar to that of ascorbic acid release. Thus, newly accumulated ascorbic acid in chromaffin cells may be localized to a free pool in the cell cytoplasm rather than in a vesicular compartment.

Plasma membrane and chromaffin granule characteristics in digitonin-treated chromaffin cells.

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 microM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 microM reserpine, 30 microM NH4+, or 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules with digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 microM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-beta-hydroxylase. The studies demonstrate that 20 microM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.

Effects of phorbol ester on catecholamine secretion and protein phosphorylation in adrenal medullary cell cultures.

The effects of phorbol 12-myristate 13-acetate (PMA) on catecholamine secretion and protein phosphorylation from intact and digitonin-treated chromaffin cells were investigated. PMA (10-300 nM), an activator of protein kinase C, caused a slow Ca2+-dependent release of catecholamine from intact chromaffin cells that was potentiated by the Ca2+ ionophore ionomycin. PMA also enhanced secretion induced by Ba2+. In cells with plasma membranes rendered permeable by digitonin to Ca2+, ATP, and protein, PMA (100 nM) enhanced Ca2+-dependent secretion approximately 70% at 0.5 microM Ca2+ and 30% at 10 microM Ca2+. PMA enhanced the maximal response to Ca2+ approximately 25% and decreased the Ca2+ concentration required for half-maximal secretion approximately 30%. The effects of PMA on chromaffin cells were associated with a 2- to 3-fold increase in the phosphorylation of a 56-kDa protein that may be tyrosine hydroxylase. Other proteins were phosphorylated to a lesser extent. The experiments suggest that PMA increases protein kinase activity and secretion in chromaffin cells and raise the possibility that protein kinase C modulates catecholamine secretion in chromaffin cells.

Arachidonic acid release and catecholamine secretion from digitonin-treated chromaffin cells: effects of micromolar calcium, phorbol ester, and protein alkylating agents.

The relationship between catecholamine secretion and arachidonic acid release from digitonin-treated chromaffin cells was investigated. Digitonin renders permeable the plasma membranes of bovine adrenal chromaffin cells to Ca2+, ATP, and proteins. Digitonin-treated cells undergo exocytosis of catecholamine in response to micromolar Ca2+ in the medium. The addition of micromolar Ca2+ to digitonin-treated chromaffin cells that had been prelabeled with [3H]arachidonic acid caused a marked increase in the release of [3H]arachidonic acid. The time course of [3H]arachidonic acid release paralleled catecholamine secretion. Although [3H]arachidonic acid release and exocytosis were both activated by free Ca2+ in the micromolar range, the activation of [3H]arachidonic acid release occurred at Ca2+ concentrations slightly lower than those required to activate exocytosis. Pretreatment of the chromaffin cells with N-ethylmaleimide (NEM) or p-bromophenacyl bromide (BPB) resulted in dose-dependent inhibition of 10 microM Ca2+-stimulated [3H]arachidonic acid release and exocytosis. The IC50 of NEM for both [3H]arachidonic acid release and exocytosis was 40 microM. The IC50 of BPB for both events was 25 microM. High concentrations (5-20 mM) of Mg2+ caused inhibition of catecholamine secretion without altering [3H]arachidonic acid release. A phorbol ester that activates protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA), caused enhancement of both [3H]arachidonic acid release and exocytosis. The findings demonstrate that [3H]arachidonic acid release is stimulated during catecholamine secretion from digitonin-treated chromaffin cells and they are consistent with a role for phospholipase A2 in exocytosis from chromaffin cells. Furthermore the data suggest that protein kinase C can modulate both arachidonic acid release and exocytosis.

Calcium-evoked secretion from digitonin-permeabilized adrenal medullary chromaffin cells.

The effects of the steroid glycoside digitonin on cultured bovine adrenal chromaffin cells were studied as a way to gain access to the intracellular sites of calcium action in stimulus-secretion coupling. Chromaffin cells treated with digitonin secreted catecholamines by an exocytotic mechanism when exposed to free calcium concentrations of greater than 1 microM. At free calcium concentrations of less than 100 microM, an apparently saturable release of catecholamines was observed with a half-maximal effect (EC50) of 3-4 microM. Catecholamine release at these low calcium concentrations was limited to 10-15% of the cellular content, was partially dependent on the presence of MgATP, and was inhibited by magnesium (EC50 = 6 mM). At calcium concentrations between 0.1 and 10 mM, a second, apparently saturable component of catecholamine release from digitonin-treated chromaffin cells was observed (EC50 = 0.3 mM). This release at the higher calcium concentrations was unaffected by the addition of MgATP, magnesium, antipsychotic drugs known to bind and inactivate calmodulin, or calcium channel blockers, but was temperature-dependent and was inhibited by N-ethylmaleimide (EC50 = 140 microM). Strontium and manganese can also evoke catecholamine secretion from digitonin-treated chromaffin cells. The results suggest that digitonin will be a useful preparation in elucidating the molecular mechanisms of the secretory process.

Catecholamine secretion from digitonin-treated adrenal medullary chromaffin cells.

The plasma membrane of cultured chromaffin cells from bovine adrenal medulla was rendered leaky by incubation in low concentrations of digitonin. Digitonin (20 microM) induced Ca2+-dependent release of 10-20% of the catecholamine in the presence of 10 microM Ca2+ without addition of secretagogue. Half-maximal catecholamine release occurred at approximately 1 microM Ca2+ x Mg2+ could not substitute for Ca2+. Cells incubated with digitonin rapidly lost their ability to exclude trypan blue. Digitonin caused release of the cytosolic markers lactic dehydrogenase and phenylethanolamine-N-methyltransferase, but, in contrast to release of catecholamine, the release of the cytosolic proteins was inhibited by Ca2+. Soluble dopamine-beta-hydroxylase, a protein marker of granule contents, was released proportionally with catecholamine in a Ca2+ dependent manner. Catecholamine release was optimal in solutions containing MgATP. Hence, digitonin-treated cells, although they lose soluble cytosolic proteins and presumably low molecular weight cytosolic constituents, maintain the Ca2+-dependent reactions of exocytosis in the presence of MgATP. Digitonin-treated chromaffin cells may be a powerful system in which to study the biochemical mechanisms underlying exocytosis.