The Relationship Between Arachidonic Acid Release and Catecholamine Secretion from Cultured Bovine Adrenal Chromaffin Cells

Increased arachidonic acid release occurred during activation of catecholamine secretion from cul- tured bovine adrenal medullary chromaffin cells. The nicotinic agonist l,l-dimethyl-4-phenylpiperazinium (DMPP) caused an increased release of prcincubated [ 3 H]arachidonic acid over a time course which corre- sponded to the stimulation of catecholamine secretion. Like catecholamine secretion, the DMPP-induced [ 3 H]arachidonic acid release was calcium-dependent and was blocked by the nicotinic antagonist mecamylamine. Depolarization by elevated K + , which induced catechol amine secretion, also stimulated arachidonic acid release. Because arachidonic acid release from cells probably re- sults from phospholipase A 2 activity, our findings indicate that phospholipase A 2 may be activated in chromaffin cells during secretion.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66068/1/j.1471-4159.1984.tb06690.x.pd

Effects of Osmolality and Ionic Strength on Secretion from Adrenal Chromaffin Cells Permeabilized with Digitonin

Hyperosmotic solutions inhibit exocytosis of catecholamine from adrenal chromaffin cells at a step after Ca 2+ entry into the cells. The possibility that the inhibition resulted from an inability of shrunken secretory granules to undergo exocytosis was investigated in cells with plasma membranes permeabilized by digitonin. The osmoticants and salts used in this study rapidly equilibrated across the plasma membrane and bathed the intracellular organelles. When sucrose was the osmoticant, secretion was not significantly inhibited unless the osmolality was raised above 1,000 mOs. When the osmolality was raised with the tetrasaccharide stachyose or a low-molecular-weight maltodextrin fraction (average size a tetrasaccharide), one-half maximal inhibition occurred at 900–1,000 mOs. Prior treatment of permeabilized cells with Ca 2+ in hyperosmotic solution did not result in enhanced secretion when cells were restored to normal osmolality. Increased concentrations of potassium glutamate or sodium isethionate were more potent than carbohydrate in inhibiting secretion. Half-maximal inhibition occurred at 600–700 mOs or when the ionic strength was approximately doubled. The inhibition by elevated potasium glutamate also occurred when the osmolality was kept constant with sucrose. Increasing the ionic strength did not alter the Ca 2+ sensitivity of the secretory response. Reducing the ionic strength by substituting sucrose for salt reduced the Ca 2+ concentration required for half-maximal stimulated secretion from approximately 1.2 Μ M . Chromaffin granules, the secretory granules, are known to shrink in hyperosmotic solution. The experiments indicate that shrunken chromaffin granules can undergo exocytosis and suggest that in intact cells elevated ionic strength rather than chromaffin granule shrinkage contributes to the inhibition of secretion by hyperosmotic solutions. The experiments place limits on the possible osmotic mechanisms that could be involved in exocytosis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66041/1/j.1471-4159.1986.tb08502.x.pd

Phorbol Esters Enhance Exocytosis from Chromaffin Cells by Two Mechanisms

Treatment with phorbol esters such as 12- O -tet-radecanoylphorbol acetate (TPA) rapidly enhances [ 3 H]norepinephrine secretion from digitonin-permeabilized adrenal chromaffin cells. When TPA treatment was prolonged for several hours, a second distinct enhancing effect was observed. This later enhancement was most prominent at intracellular Ca 2+ concentrations of 3–30 ΜM , and did not require the continued presence of membrane-bound protein kinase C for its expression. The effect could be elicited by as little as 30-min exposure to TPA, followed by several hours in TPA- free medium. This effect of TPA was blocked by actinomycin D and cycloheximide, indicating a requirement for RNA and protein synthesis. Similar effects were seen when intact cells that had been pretreated with TPA were stimulated to secrete by depolarizing concentrations of K + . Thus, protein kinase C enhances secretion by two mechanisms. One is rapid and probably reflects the effects of immediate protein phosphorylation. The other occurs over several hours and requires gene transcription and protein synthesis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66341/1/j.1471-4159.1990.tb13302.x.pd

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 Ca 2+ , ATP, and proteins. Digitonin-treated cells undergo exocytosis of catecholamine in response to micromolar Ca 2+ in the medium. The addition of micromolar Ca 2+ to digitonin-treated chromaffin cells that had been prelabeled with [ 3 H]arachidonic acid caused a marked increase in the release of [ 3 H]arachidonic acid. The time course of [ 3 H]arachidonic acid release paralleled catecholamine secretion. Although [ 3 H]arachidonic acid release and exocytosis were both activated by free Ca 2+ in the micromolar range, the activation of [ 3 H]arachidonic acid release occurred at Ca 2+ 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 Μ M Ca 2+ -stimulated [ 3 H]arachidonic acid release and exocytosis. The IC 50 of NEM for both [ 3 H]arachidonic acid release and exocytosis was 40 Μ M. The IC 50 of BPB for both events was 25 Μ M. High concentrations (5–20 m M ) of Mg 2+ caused inhibition of catecholamine secretion without altering [ 3 H]arachidonic acid release. A phorbol ester that activates protein kinase C, 12- O -tetradecanoylphorbol-13-acetate (TPA), caused enhancement of both [ 3 H]arachidonic acid release and exocytosis. The findings demonstrate that [ 3 H]arachidonic acid release is stimulated during catecholamine secretion from digitonin-treated chromaffin cells and they are consistent with a role for phospholipase A 2 in exocytosis from chromaffin cells. Furthermore the data suggest that protein kinase C can modulate both arachidonic acid release and exocytosis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65534/1/j.1471-4159.1985.tb07140.x.pd

Cholinergic Stimulation of Inositol Phosphate Formation in Bovine Adrenal Chromaffin Cells: Distinct Nicotinic and Muscarinic Mechanisms

The ability of cholinergic agonists to activate phospholipase C in bovine adrenal chromaffin cells was examined by assaying the production of inositol phosphates in cells prelabeled with [ 3 H]inositol. We found that both nicotinic and muscarinic agonists increased the accumulation of [ 3 H]inositol phosphates (mainly inositol monophos-phate) and that the effects mediated by the two types of receptors were independent of each other. The production of inositol phosphates by nicotinic stimulation required extracellular Ca 2+ and was maximal at 0.2 m M Ca 2+ . Increasing extracellular Ca 2+ from 0.22 to 2.2 m M increased the sensitivity of inositol phosphates formation to stimulation by submaximal concentrations of 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) but did not enhance the response to muscarine. Elevated K + also stimulated Ca 2+ -dependent [ 3 H]inositol phosphate production, presumably by a non-receptor-mediated mechanism. The Ca 2+ channel antagonists D600 and nifedipine inhibited the effects of DMPP and elevated K + to a greater extent than that of muscarine. Ca 2+ (0.3–10 Μ M ) directly stimulated the release of inositol phosphates from digitonin-permeabilized cells that had been prelabeled with [ 3 H]inositol. Thus, cholinergic stimulation of bovine adrenal chromaffin cells results in the activation of phospholipase C by distinct muscarinic and nicotinic mechanisms. Nicotinic receptor stimulation and elevated K + probably increased the accumulation of inositol phosphates through Ca 2+ influx and a rise in cytosolic Ca 2+ . Because Ba 2+ caused catechol-amine secretion but did not enhance the formation of inositol phosphates, phospholipase C activation is not required for exocytosis. However, diglyceride and wyo -inositol 1,4,5-trisphosphate produced during cholinergic stimulation of chromaffin cells may modulate secretion and other cellular processes by activating protein kinase C and/or releasing Ca 2+ from intracellular stores.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65738/1/j.1471-4159.1987.tb01037.x.pd

Effects of Tetanus Toxin on Catecholamine Release from Intact and Digitonin-Permeabilized Chromaffin Cells

Tetanus exotoxin inhibited Ca 2+ -dependent cate-cholamine secretion in a dose-dependent manner in digito-nin-permeabilized chromaffin cells. The inhibition was specific for tetanus exotoxin and the B fragment of tetanus toxin; the C fragment had no effect. Inhibition required the introduction of toxin into the cell, and was not seen when intact cells were preincubated with the toxin or toxin fragments. The degree of inhibition was related to the length of preincubation with toxin, as well as the concentration of toxin used. A short preincubation with toxin was sufficient to inhibit secretion, and the continued presence of toxin in the incubation medium was not required during the incubation with Ca 2+ . The inhibition of secretion by tetanus toxin or the B fragment was not overcome with increasing Ca 2+ concentrations. Tetanus toxin also inhibited catechol-amine secretion enhanced by phorbol ester-induced activation of protein kinase C. Thus, the toxin or a proteolytic fragment of the toxin can enter digitonin-permeabilized cells to interact with a component of the Ca 2+ -dependent exocytotic pathway to inhibit secretion.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66452/1/j.1471-4159.1988.tb01059.x.pd

Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca 2+ , ATP, and proteins and allows micromolar Ca 2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 Μ M ) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [ 3 H]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 [ 3 H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [ 3 H]norepinephrine uptake was inhibited by 1 Μ M reserpine, 30 m M NH 4 + , or 1 Μ M carbonyl cyanide p -trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [ 3 H]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 within 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 Μ M or greater. Catecholamine released into the medium by micromolar Ca 2+ 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-Β-hydroxylase. The studies demonstrate that 20 Μ M 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 Ca 2+ , the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65144/1/j.1471-4159.1985.tb07226.x.pd

Barium and Calcium Stimulate Secretion from Digitonin-Permeabilized Bovine Adrenal Chromaffin Cells by Similar Pathways

We compared the characteristics of secretion stimulated by EGTA-buffered Ba 2+ - and Ca 2+ -containing solutions in digitonin-permeabilized bovine adrenal chromaffin cells. Half-maximal secretion occurred at approximately 100 Μ M Ba 2+ or 1 Μ M Ca 2+ . Ba 2+ -stimulated release was not due to release of sequestered intracellular Ca 2+ because at a constant free Ba 2+ concentration, increasing unbound EGTA did not diminish the extent of release due to Ba 2+ . The maximal extents of Ba 2+ - and Ca 2+ -dependent secretion in the absence of MgATP were identical. MgATP enhanced Ba 2+ -induced secretion to a lesser extent than Ca 2+ -induced secretion. Half-maximal concentrations of Ba 2+ and Ca 2+ , when added together to cells, yielded approximately additive amounts of secretion. Maximal concentrations of Ba 2+ and Ca 2+ when added together to cells for 2 or 15 min were not additive. Tetanus toxin inhibited Ba 2+ - and Ca 2+ -dependent secretion to a similar extent. Ba 2+ , unlike Ca 2+ , did not activate polyphosphoinositide-specific phospholipase C. These data indicate that (1) Ba 2+ directly stimulates exocytosis, (2) Ba 2+ -induced secretion is stimulated to a lesser extent than Ca 2+ -dependent secretion by MgATP, (3) Ba 2+ and Ca 2+ use similar pathways to trigger exocytosis, and (4) exocytosis from permeabilized cells does not require activation of polyphosphoinositide-specific phospholipase C.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66323/1/j.1471-4159.1992.tb09771.x.pd

Cholinergic Receptor-Mediated Phosphorylation and Activation of Tyrosine Hydroxylase in Cultured Bovine Adrenal Chromaffin Cells

We have identified a 56-kilodalton protein in cultured bovine adrenal chromaffin cells that is phos-phorylated when catecholamine secretion is stimulated. Immunodetection on Western blots from both one- and two-dimensional polyacrylamide gels indicated that this protein was tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. Two-dimensional polyacrylamide gel electrophoresis of proteins from unstimulated cells revealed small amounts of phosphorylated protein with a molecular weight of 56K and pI values of 6.37 and 6.27 which were subunits of tyrosine hydroxylase. Nicotinic stimulation of chromaffin cells caused the phosphorylation of three proteins of 56 kilodaltons with pI values of approximately 6.37, 6.27, and 6.15 which were tyrosine hydroxylase. The immunochemical analysis also revealed that there was unphosphorylated tyrosine hydroxylase 56 kilodaltons with a pI of 6.5 which may have decreased on nicotinic stimulation. The phosphorylation of tyrosine hydroxylase was associated with an increase in in situ conversion of [ 3 H]tyrosine to [ 3 H]dihydroxyphenylalanine ([ 3 H]DOPA). Muscarinic stimulation also caused phosphorylation of tyrosine hydroxylase, but to a smaller extent than did nicotinic stimulation. The secretagogues, elevated K + and Ba 2+ , stimulated phosphorylation of tyrosine hydroxylase and [ 3 H]DOPA production. The effects of nicotinic stimulation and elevated K + on tyrosine hydroxylase phosphorylation and [ 3 H]DOPA production were Ca 2+ -dependent. Nicotinic agonists also raised cyclic AMP levels in chromaffin cells after 2 min. Dibutyryl cyclic AMP and forskolin, which have little effect on catecholamine secretion, also caused phosphorylation of tyrosine hydroxylase. These stimulators of cyclic AMP-dependent processes caused the appearance of two phosphorylated subunits of tyrosine hydroxylase with pI values of 6.37 and 6.27. There was also a small amount of phosphorylated subunit with a pI of 6.15. Both agents stimulated [ 3 H]DOPA production. The experiments indicate that tyrosine hydroxylase is phosphorylated and activated when chromaffin cells are stimulated to secrete. The data suggest that the earliest phosphorylation of tyrosine hydroxylase induced by a nicotinic agonist occurs through stimulation of a Ca 2+ -dependent protein kinase. After 2 min phosphorylation by a cyclic AMP-dependent protein kinase may also occur. Phosphorylation of tyrosine hydroxylase is associated with an increase in in situ tyrosine hydroxylase activity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65828/1/j.1471-4159.1986.tb13011.x.pd

Mechanisms Involved in Calcium-Dependent Exocytosis a

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74760/1/j.1749-6632.1991.tb36506.x.pd