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

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The Relationship Between Arachidonic Acid Release and Catecholamine Secretion from Cultured Bovine Adrenal Chromaffin Cells Journal of Neurochemistry Raven Press, New York 0 1984 International Society for Neurochemistry The Relationship Between Arachidonic Acid Release and Catecholamine Secretion from Cultured Bovine Adrenal Chromaffin Cells Roy A. Frye and Ronald W. Holz Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan, U.S.A. ~ ~ Abstract: Increased arachidonic acid release occurred amine secretion, also stimulated arachidonic acid release. during activation of catecholamine secretion from cul- Because arachidonic acid release from cells probably re- tured bovine adrenal medullary chromaffin cells. The sults from phospholipase A, activity, our findings indicate nicotinic agonist l,l-dimethyl-4-phenylpiperazinium that phospholipase A, may be activated in chromaffin (DMPP) caused an increased release of preincubated cells during secretion. Key Words: Arachidonic acid- [3H]arachidonic acid over a time course which corre- Catecholamine secretion-Chromaffin cells-Phospho- sponded to the stimulation of catecholamine secretion. lipase A,. Frye R. A. and Holz R. W. The relationship Like catecholamine secretion, the DMPP-induced between arachidonic acid release and catecholamine se- [3H]arachidonic acid release was calcium-dependent and cretion from cultured bovine adrenal chromaffin cells. J. was blocked by the nicotinic antagonist mecamylamine. Neirrochem. 43, 146-150 (1984). Depolarization by elevated K+, which induced catechol- Prepackaged hormones and neurotransmitters In the present study we have investigated whether are usually released from cells via exocytosis. phospholipase A, activation (measured by release During exocytosis a rise in cytosolic [Ca2+]triggers of preincorporated [3H]arachidonic acid) occurs fusion of the secretory vesicle membrane and the during the stimulation of catecholamine secretion plasma membrane. Phospholipase A,, which may from cultured bovine adrenal medullary chromaffin be activated by a rise in cytosolic [Ca2+](Van den cells. Bosch, 1980), causes the release of cis-unsaturated fatty acids (including arachidonic acid) which are MATERIALS AND METHODS potent membrane fusogens (Creutz, 1981). Activa- tion of phospholipase A, may be a necessary step Cell preparation in the chain of events linking elevated cytosolic Cells disaggregated from bovine adrenal medullae [Ca2+]to the vesicle membrane-plasma membrane (Fenwick et al., 1978; Livett et al., 1979) were added to fusion event. Although we have recently found that 16-mm diameter uncoated culture wells (Costar, Cam- putative phospholipase A, inhibitors block secre- bridge, MA) at a density of 500,000 cells per well in 1 ml tion from cultured chromaffin cells, they also block of Eagle’s Minimum Essential Medium (Gibco, Grand Is- land, NY) supplemented with 10% heat-inactivated fetal calcium uptake into the cells which is necessary to calf serum (Gibco), 10 pM cytosine arabinoside (to inhibit stimulate secretion (Frye and Holz, 1983); thus, the fibroblast proliferation), gentamycin (50 Kglml), penicillin effects of these inhibitors are difficult to interpret. (100 Uiml), streptomycin (100 pg/ml), and Fungizone (2.5 Received September 1, 1983; accepted December 21, 1983. 1,1-Dimethyl-4-phenylpiperazinium; HEPES, N-2-Hydroxy- Address correspondence and reprint requests to Dr. Ronald ethylpiperazine-N’-2-ethanesulfonicacid; Mec, Mecamylamine; W. Holz, Department of Pharmacology, M6322, Medical Science Meth, Methacholine; PC, Phosphdtidylchohe; PE, Phosphati- I, University of Michigan Medical School, Ann Arbor, MI 48109, dylethanolamine; PUPS, Phosphatidylinositol/phosphatidyl- U.S.A. serine; PL, Phospholipid; PSS, Physiological salt solution; TG/ Abbreviations used: AA, Arachidonic acid; Ach, Acetylcho- CE, Triacylglyceridelcholesterol ester. line; BSA, Bovine serum albumin; DG, Diacylglyceride; DMPP, 146 ARACHIDONATE RELEASE AND CATECHOLAMINE SECRETION 147 pg/ml) (Squibb, Princeton, NJ). After 4 days at 34" in 5% wells was solubilized in 0.4 ml of 1% Triton X-100 and C0,/95% air, chromaffin cells formed monolayers con- quantitated by liquid scintillation counting. Results of ex- taining approximately 20 nmol catecholamine per well. periments are expressed as the percent of total radioac- The incubation medium was replaced after 4 days and tivity which was released from the cells during the test experiments were performed on days 5- 10. Cultures period and are expressed as the mean * the standard were incubated in cytosine arabinoside-free medium for error of the mean. Statistical significance was determined 1 day before each experiment. by Student's t test. [3H]Arachidonicacid labeling RESULTS Cells were incubated for 4 h with 0.2 ml per well tissue culture medium without cytosine arabinoside which con- Secretory stimuli cause [3H]arachidonic tained 0.5 pCi [5,6,8,9,11 ,12,14,15-3H]arachidonicacid acid release (100 pCi/mmol) (Amersham, Chicago, IL) and 0.5% (wt/ Bovine adrenal chromaffin cell cultures incorpo- vol) fatty acid free bovine serum albumin (BSA) (Sigma, rated [3H]arachidonic acid into cellular lipid pools. St. Louis, MO). Wells were then incubated twice in 0.2 Nearly 80% of the incorporated label was esterified ml medium plus BSA without arachidonate for 10 min into the phospholipid pool with the remainder es- and then in 0.4 ml of the same medium for 1 h. Cells were then incubated in 1.0 ml of physiological salt solution terified into neutral lipids (Table 1); only a small (PSS) containing 145 mM NaCI, 5.6 mM KCl, 2.2 mM fraction (0.15%) of the cell-associated label was CaCI,, 15 mM N-2-hydroxyethylpiperazine-N-2-ethane- present as free [3H]arachidonic acid. In the resting sulfonic acid (HEPES) (pH 7.4), and 0.5% (wt/vol) fatty state the prelabeled cells released [3H]arachidonic acid free BSA for 30 min. acid-labeled products including neutral lipid, free In experiments in which the distribution of labeled cell arachidonic acid, and phospholipid. Upon stimula- lipids was determined, the lipids of the cell monolayer tion with the nicotinic agonist DMPP, the release of were dissolved in 1 ml of ice-cold methanol which was labeled free arachidonic acid increased signifi- added to the well immediately following the removal of cantly, while the release of labeled neutral lipid and the incubation solution. The labeled lipids in the methanol phospholipid was not significantly altered (Fig. 1). extracts were fractionated by one-dimensional, sequen- tial, ascending TLC. The plates were first run for 15 min DMPP did not alter the amount of [3H]arachidonic in chloroform/methanol/water (95:35:6). The plates were acid or other labeled lipids associated with the cells then run for 25 min in hexane/diethylether/ethanol/NH3 (Table 1). The data indicate that the nicotinic ago- (75:20:5:0.5). The lipids were separated with the fol- nist-induced secretion of catecholamine is associ- lowing R,values: phosphatidylinositoUphosphatidylserine ated with a net deacylation of [3H]arachidonic acid (0.05), phosphatidylcholine (0. lo), phosphatidylethanol- from lipid precursors and release of [3H]arachidonic amine (0.25), arachidonic acid (0.45), diacylglyceride acid into the medium. (0.60), and triacylglyceridekholesterol ester (0.80). Depolarization induced by 56 mM K+ stimulated calcium-dependent catecholamine secretion (Holz [3H]Norepinephrine uptake into cells Cells were incubated for 4 h in 0.2 ml incubation me- et al., 1982) and also stimulated [3H]arachidonic dium containing 0.5 pCi/ml l-[7-3H]norepinephrine (New acid release (Fig. 2). In the present experiments the England Nuclear, Boston, MA) and 0.5 mM ascorbate, amount of elevated K+-induced secretion was ap- and were then incubated three times for 10 min with PSS proximately half the DMPP-induced secretion (Fig. without BSA. 2B). Similarly, the elevated K+-induced release of [3H]arachidonic acid was smaller than that induced [3H]Norepinephrine secretion and stimulation of by DMPP (Fig. 2A). Because the DMPP-stimulated [3H]arachidonic acid release release of [3H]arachidonicacid was larger and more Cells prelabeled with [3H]norepinephrine or readily studied, the following experiments focus on [3H]arachidonic acid were rinsed with 0.4 ml PSS plus the relationship between cholinergic agonist-in- 0.5% BSA immediately prior to addition of the test so- lution. Experiments were performed at 25" with 0.4 ml duced [3H]arachidonic acid release and catechol- PSS containing 0.5% BSA with or without 1,l-dimethyl- amine secretion. 4-phenylpiperazinium (DMPP) or elevated K+ (56.0 mM It is unlikely that the DMPP-induced 13H]- KCl and 95 mM NaC1). Following the incubation the test arachidonic acid release is due to a stimulated re- solutions were removed from the [3Hlnorepinephrine-la- lease of phospholipase A, from the chromaffin cells beled cells and added to scintillation vials for determi- and subsequent action of the released enzyme on nation of [3H]norepinephrine secretion. [3H]Arachidonic the cell from the extracellular medium. Solutions acid in lipid extracts (Billah and Lapetina, 1982) of test containing 10 pM DMPP which had been incubated solutions from [3H]arachidonic acid-labeled cells was sep- for 5 min with unlabeled chromaffin cells (i.e., so- arated by TLC with silica gel HL plates (Analtech, lutions which would have contained the putative Newark, DE) with petroleum etheridiethyl etherlacetic acid (70:30: 1). The released radioactivity which comi- released phospholipase A2) did not stimulate grated with authentic arachidonic acid on the TLC plate [3H]arachidonic acid release when added
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