| Gender differences in the epidemiology of coronary heart disease have been described in a number of clinical settings, with men having an earlier onset and higher incidence of atherosclerosis than women. Both in vivo and in vitro investigations indicate that androgens play an important role in the arterial vascular system. Androgen receptors (AR) have been identified in vascular smooth muscle cells (VSMCs) and responses to androgens have been linked to AR concentration in some androgen target tissues. In this study, we have evaluated the effects of testosterone exposure on AR gene expression in cultured rat aortic smooth muscle cells, a commonly used model of vascular biology, and the possible mechanisms mediating the effects were also explored.Methods:VSMCs were cultured from thoracic aorta of male Sprague-Dawley rats by using the explant method. Subconfluent VSMCs were incubated with serum-free Dulbecco's modified Eagle's medium (DMEM) for 24 hr to obtain quiescent non-dividing cells, and then treated with various agents in DMEM containing 1% fetal bovine serum (FBS). Cytoplasmic and nuclear extracts were prepared by means of cell lysis and high salt extraction, respectively, and subjected to western blotting analysis for determination of AR protein level. Total RNA was isolated with the single-step guanidine isothiocyanate method, and subjected to Northern blotting analysis fordetermination of AR mRNA expression. Testosterone effects on the viability and growth rate of VSMCs were also determined using cell counting and tritiated thymidine incomporation assay.Trichloroacetic acid extracts of adenosine 3':5'- cyclic monophosphate (cAMP) in VSMCs were prepared and studied with radioimmunoassay. For the determination of protein kinase activity, subcellular fractions (soluble and particulate) of VSMCs were prepared, and purified by application to DEAE-cellulose chromatography. Protein kinase A (PKA) and protein kinase C (PKC) activity were determined by measuring the amount of 32P incorporated into histone substrate.Results:1. Effects of androgen on AR expression in VSMCs1.1 Androgen increases both cytoplasmic and nuclear AR protein expression in VSMCsTreatment of VSMCs with testosterone at different concentrations for 24 hr resulted in a dose-dependent increase in cytoplasmic AR protein, with relative AR protein level 4.90 ?3.05, 2.60 ?1.90, 62.20 ?7.80, 84.00 ?6.00 and 100.00 ?2.10 for testosterone 0, 4 nM, 40 nM, 400 nM and 4 M treatment, respectively. A dose-dependent increase in AR protein level was also observed in nuclear preparations, with relative AR protein level 9.80 ?4.80, 51.50 ?21.50, 80.00 ?13.20, 89.00 ?7.40 and 100.00 ?3.70 for testosterone 0, 4 nM, 40 nM, 400 nM and 4 M treatment, respectively.Next, we characterized the time-response (0 ~ 48 hr) of VSMCs to testosterone at a physiologically relevant concentration of 40 nM. Androgen treatment for 10 min induced a transient down-regulation of cytoplasmic AR-9-protein expression in VSMCs, while a gradual increase in AR level was observed after that, with relative AR protein leve 17.90 ?2.20, 4.70 ?0.65, 17.00 ?5.50, 38.50 ?4.50, 78.50 ?3.50 and 100.00 ?3.00 for incubation time 0, 10 min, 1 hr, 12 hr, 24 hr and 48 hr, respectively. Nuclear AR protein band was evident only after 24 hr of testosterone treatment, with higher expression level after 48 hr of exposure. 1.2Androgen increases AR mRNA expression in VSMCsTreatment of synchronized VSMCs with testosterone for 24 hr increased intracellular AR mRNA expression in a dose-related fashion, with relative mRNA level 53.76 ?4.84, 52.69 ?7.53, 76.88 ?5.91, 95.70 ?8.06 and 100.00 ?10.75 for testosterone 0, 4 nM, 40 nM, 400 nM and 4 M incubation, respectively. Then the time response of AR mRNA to androgen exposure was examined. Treatment of VSMCs with testosterone 40 nM for 24 hr resulted in a 35% (means) up-regulation of AR mRNA level, while no significant differences were observed for 10 min, 1 hr and 12 hr of androgen incubation, compared with control of time ze...
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