Stimulation of aortic smooth muscle cell mitogenesis by serotonin.

Abstract

Bovine aortic smooth muscle cells in vitro responded to 1 nM to 10 ,M serotonin with increased incorporation of [3H]thymidine into DNA. The mitogenic effect of serotonin was half-maximal at 80 nM and maximal above 1 ,uM. At a concentration of 1 ,uM, serotonin stimulated smooth muscle cell mitogenesis to the same extent as human plateletderived growth factor (PDGF) at 12 ng/ml, Tryptamine was =1/10th as potent as serotonin as a mitogen for smooth muscle cells. Other indoles that are structurally related to serotonin (Dand L-tryptophan, 5-hydroxy-L-tryptophan, N-acetyl-5-hydroxytryptamine, melatonin, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophol) and quipazine were inactive. The stimulatory effect of serotonin on smooth muscle cell DNA synthesis required prolonged (20-24 hr) [3HlThymidine Incorporation. Bovine aortic smooth muscle cells from frozen stock (passages 3-12) were suspended in Dulbecco's modified Eagle's medium (DMEM) (pH 7.4) supplemented with 10% calf serum/6 mM glutamine/ penicillin (100 units/ml)/streptomycin (100 pug/ml) for seeding into 16-mm 96-well microtiter plates at a density of w50,000 cells per well. After 48 hr, the growth medium was replaced with serum-free quiescing medium consisting of DMEM and Ham's F12 (1:1; pH 7.4) supplemented with ovalbumin (1 mg/ml)/ascorbic acid (25 ,ug/ml)/selenium (25 ng/ml)/dialyzed Cohn fraction IV (10 ug/ml)/epidermal growth factor (10 ng/ml)/transferrin 10 ,tg/ml) 50 ,uM hydrocortisone/6 mM glutamine/penicillin (100 units/ml)/ streptomycin (100 ,ug/ml) (8). Smooth muscle cells were incubated in the quiescing medium for 96 hr. The medium was changed once during this period. Compounds to be tested were added to smooth muscle cells in quiescing medium 1 hr before serotonin and/or PDGF. The cultures were exposed to serotonin and/or PDGF for 20 hr before [3H]thymidine (2.0 ,uCi/ml; 1 Ci = 37 GBq) was included in the quiescing medium. Four hours after the addition of [3H]thymidine, experiments were terminated by aspirating the medium and subjecting the cultures to sequential washes with Dulbecco's phosphate-buffered saline (pH 7.4) containing 1 mM CaCl2, 1 mM MgCl2, 10% trichloroacetic acid, and ethanol/ether (2:1). Phase-contrast microscopy was used to inspect the microwells for evidence of cell detachment or changes in cell morphology. Acid-insoluble [3H]thymidine was extracted into 0.5 M NaOH and quantified by using a liquid scintillation counter.Materials. Serotonin, tryptamine, L-tryptophan, Dtryptophan, N-acetyl-5-hydroxytryptamine, 5-hydroxy-Ltryptophan, melatonin, 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, propranolol, atropine, cimetidine, 1-methyl-3-isobutylxanthine, ovalbumin, and Cohn fraction IV were obtained from Sigma; quipazine (2-[1-piperazi- 674The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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