Characterization of α-Adrenergic Binding Sites on Rodent Leydig Cells1

Abstract

A radioligand binding technique was used to study beta-adrenergic binding sites on rodent Leydig cells. Beta-Adrenergic binding sites were found on Leydig cells in both the rat and mouse. Binding of [3H]CGP-12177 [4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benzimidazole-2-one] to purified rat Leydig cells was found to be saturable, temperature and time dependent, stereospecific, and readily reversible by the beta-adrenergic antagonist propranolol. Scatchard analysis revealed the presence of high-affinity sites with an apparent dissociation constant (Kd) of 0.79 +/- 0.22 nM and maximal binding capacity (Bmax) of 1716 +/- 245 sites per rat Leydig cell. Competition of various beta-adrenergic agonists and antagonists with [3H]CGP indicates an order of potency of L-isoproterenol greater than epinephrine = salbutamol greater than norepinephrine greater than D-isoproterenol and dl-propranolol = ICI 118,551 much greater than atenolol, respectively. These observations suggest that the binding sites are predominantly of the beta 2-receptor subtype. Incubation of freshly isolated rat Leydig cells with luteinizing hormone (100 ng/ml) caused consistent stimulation of androgen production, but only occasional stimulation by the beta-agonist isoproterenol (10 microM) was observed. However, these cells consistently responded to the beta-agonist after 3 h in primary cultures. These findings indicate that rodent Leydig cells possess beta-adrenergic binding sites and point out a possible dissociation between receptor recognition and physiologic response.

Pituitary and testis responsiveness of young male sheep exposed to testosterone excess during fetal development

“…Interstitial cells were then filtered through gauze to remove fragments of seminiferous tubules. Leydig cells were purified from the crude interstitial fraction on a discontinuous gradient of Percoll prepared in HEPES buffer, pH 7.4, as described by Anakwe et al (1985). Briefly, 20 ml of a 90% Percoll (Sigma) solution were layered under 20 ml of a 40% Percoll solution in a 50 ml conical tube.…”

Orally applied doxazosin disturbed testosterone homeostasis and changed the transcriptional profile of steroidogenic machinery, cAMP/cGMP signalling and adrenergic receptors in Leydig cells of adult rats

“…Primary cultures of purified Leydig cells were prepared from suspensions of interstitial cells. Suspensions of interstitial cells were prepared according to Anakwe et al (Anakwe et al, 1985) with some modifications described previously by our group (Andric et al, 2007(Andric et al, , 2010aKostic et al, 2008Kostic et al, , 2010Kostic et al, , 2011. Testes were quickly removed, decapsulated and placed in a 50-mL plastic tube (two testes per tube) containing 3 mL of collagenase solution (1.25 mg/mL collagenase Type I; 1.5% BSA; 20 mM HEPES in DMEM/F12) and incubated for 15 min at 34°C in a shaking water bath oscillating at 120 cycles/min.…”

“…The phosphodiesterases (PDEs) terminate cAMP/ cGMP signalling and have regulatory function in Leydig cells (Catt & Dufau, 1973;Dufau, 1998;Tsai & Beavo, 2011. Although Leydig cell steroidogenesis is mainly activated through LH/hCG receptors, regulation itself is a multi-compartmental process and includes neural (Selvage et al, 2006) and complex endocrine, paracrine and autocrine signalling pathways (reviewed in Saez, 1994;Gnessi et al, 1997;Payne & Hales, 2004) including cGMP (Andric et al, 2010a,b) and adrenergic signalling (Anakwe et al, 1985). In addition, many transcription factors are involved in regulation of the steroidogenic machinery expression (reviewed in Simard et al, 2005;Lavoie & King, 2009;Martinez-Arguelles & Papadopoulos, 2010;Midzak et al, 2011).…”

Propranolol inhibits the compensatory increase in androgen secretion after unilateral orchidectomy in rats

“…It therefore appears that unilateral orchidectomy may activate adrenergic neurones innervating the testis which, via ß-receptors, stimulate an increase in androgen secretion. As both Leydig cells (Poyet & Labrie, 1983;Anakwe et al, 1985a) and Sertoli cells (Heindel et ai, 1980) contain ß-receptors it is unclear whether the effect is a direct one on Leydig cells or an indirect one mediated by Sertoli cells. There is also a report (Gerendai, Shaha, Thau & Bardin, 1984) …”

“…ß-Adrenergic agonists are able to stimulate small increases in androgen pro¬ duction by whole rat and mouse testes in vitro (Anakwe & Moger, 1984) and by the perfused dog testis in situ (Eik-Nes, 1969). Leydig cells contain ß-adrenergic receptors (Poyet & Labrie, 1983;Anakwe, Murphy & Moger, 1985a) and ß-adrenergic agonists can stimulate substantial increases in androgen production by mouse (Moger, Murphy & Casper, 1982;Cooke, Golding, Dix & Hunter, 1982;Moger & Murphy, 1983) and rat (Anakwe, Moger & Nance, 1985b) Leydig cells in culture. Sertoli cells, which may indirectly regulate androgen secretion (Sharpe, 1983), also contain ß-adrenergic receptors (Heindel, Steinberger & Strada, 1980).…”