Establishment and characterization of steroidogenic granulosa cells expressing beta(2)-adrenergic receptor: regulation of adrenodoxin and steroidogenic acute regulatory protein by adrenergic agents.

Primary granulosa cells obtained from PMSG primed immature rats were triple transfected with SV40 DNA, Ha-ras oncogene and an expression vector containing human beta(2)-adrenergic receptors resulting in granulosa cell lines constitutively expressing the beta(2)-adrenergic receptors. Isoproterenol, a potent adrenergic agent, stimulated both cAMP accumulation and progesterone production in these cells in a dose dependent manner. Responsiveness of these cells was specific only to isoproterenol, while hCG (2.4 nM) and hFSH (2.4 nM) had no effect on steroid production. ED(50) for stimulation of cAMP and progesterone in these cells by isoproterenol was 2x10(-6) M and 7x10(-6) M, respectively. Forskolin also showed a dose dependent stimulation of cAMP and progesterone with ED(50) of 1.5 and 0.35 microg/ml, respectively. Epinephrine at a dose of 10(-5) M elicited maximum response to produce cAMP and progesterone. Isoproterenol induced accumulation of cAMP and progesterone in these cells were inhibited by beta(2)-adrenergic blocker, propranolol with an ED(50) of 6x10(-8) and 7x10(-9) M, respectively, whereas the beta(1)-adrenergic blocker, metoprolol was effective only at a very high concentration (ED(50)>10(-4) and 1.9x10(-5) M for inhibiting isoproterenol induced cAMP and progesterone production, respectively). Induction of steroidogenesis by isoproterenol or forskolin involved de novo synthesis of the cytochrome P450 side chain cleavage (SCC) enzyme complex, as assessed by indirect immunofluorescence staining for adrenodoxin. Western analysis indicate that expression of adrenodoxin is upregulated by forskolin, isoproterenol and adrenalin by 7.8-, 6.9- and 10.8-fold, respectively. The presence of StAR protein was identified by Western blotting. StAR expression was elevated by 8.3-, 2.5- and 4.7-fold upon stimulation with forskolin, isoproterenol and adrenalin, respectively. Thus, this cell line could serve as a good model system to study catecholamine mediated regulation of growth and differentiation of granulosa cells and the role of oncogenes in this process.

KEYWORDS: {{ getKeywords(articleDetailText.words) }}