Alpha 1-adrenoreceptors on intact rat anterior pituitary cells: correlation with adrenergic stimulation of thyrotropin secretion.

Abstract

An in vitro study of the alpha-adrenergic control of TSH secretion was carried out on rat anterior pituitary cells in monolayer culture. The ability of adrenergic agonists and antagonists to alter TSH release from the cells was determined. With the use of parallel cell cultures under the same conditions, alpha-adrenergic binding sites were measured and characterized with [3H]dihydroergocryptine (DHE) as the radioligand. Epinephrine (E) and norepinephrine (NE) released TSH over a 2-h period in a dose-dependent and stereospecific manner (ED50 = 1 and 700 nM for the (-) and (+/-) stereoisomers of E; 7 and 600 nM for the active and inactive stereoisomers of NE respectively). Maximum release was 3- to 4-fold greater than basal secretion for both isomers of E but less (2- to 3-fold) for the isomers of NE. Phenylephrine, an alpha 1-agonist, elicited a 2- to 3-fold increase in TSH secretion (ED50 = 13 nM). Clonidine, an alpha 2-agonist, produced only slight stimulation at concentrations greater than 10(-6) M, and isoproterenol was ineffective. Prazosin, an alpha 2-antagonist (IC50 = 0.12 nM), was 500-fold more effective than yohimbine, an alpha 2-antagonist (IC50 = 60 nM), in reversing the TSH stimulation induced by 10(-7) M E. With [3H]DHE and prazosin as competing ligands, alpha-adrenergic receptors could be quantified independently of dopamine receptors present upon the same mixed cell preparations. The kinetics of specific radioligand binding to the cells were rapid (k1 = 1.75 X 10(-7) M-1 min-1, k2 = 0.131 min-1), equilibrium being reached within 15 min at 22 C. Adsorption isotherms and Scatchard analysis revealed a single population of binding sites with high affinity (kd = 7.2 nM) and low capacity (3 fmol/10(5) cells). Competition by adrenergic agonists for [3H]DHE binding was stereospecific. The rank order of potency against binding was identical with that determined functionally against TSH secretion (Ki for prazosin, 0.7 nM greater than thymoxamine, 2.7 nM greater than (-) E, 7 nM greater than phentolamine, 8 nM greater than (-) NE, 11.5 nM greater than phenylephrine, 100 nM greater than yohimbine, 300 nM greater than clonidine, 4500 nM greater than (+/-) E, 5000 nM greater than (+/-) NE, 7000 nM greater than isoproterenol, 3 X 10(5) nM), and typical of binding to an alpha 1-adrenoreceptor. It is concluded that TSH can be specifically released from rat anterior pituitary cells in monolayer culture by the direct effects of adrenergic agonists and that the stimulation is mediated via a single high affinity population of alpha 1-adrenergic receptors.