Acetylcholine Receptors in the Rat Anterior Pituitary Gland*

Abstract

The muscarinic antagonist [³H]quinuclidinyl benzilate (QNB) was used to study muscarinic cholinergic binding sites in a crude membrane fraction prepared from the rat anterior pituitary gland. Specific [³H]QNB binding is saturable with a K_d of 16 pM and a receptor concentration of 46 fmol/mg protein. The rates of association and dissociation were calculated to be 0.0013 pM^-1min^-1 and 0.007 mun^-1, respectively. [³H]QNB binding in the anterior pituitary is linear with tissue protein concentration, and optimal binding occurred within a broad pH range (pH 6.5–7.2) at 37 C. Stereospecificity of [³H]QNB binding was demonstrated by inhibition with acetylcholine (Ki = 10^-5 M), the muscarinic antagonists (scopolamine, atropine, imipramine, and diphenhydramine; Ki = 10^-10 − 10^-9m), and muscarinic agonists (pilocarpine and oxotremorine; Ki = 10^-6m). In contrast, nicotinic agonist (nicotine) and antagonists (d-tubocurarine and dimethylphenylpiperazinium) were less potent (Ki = 5 × 10^-3 − 10^-6m). The concentration of muscarinic receptors in the anterior pituitary is approximately 3-fold less than that in the hypothalamus and 5-fold greater than that in the posterior pituitary. Ovary, kidney, and adrenal contained negligible amounts of QNB-binding sites. Specific a-[¹²⁵I]bungarotoxin binding was used to identify nicotinic receptors. No nicotinic receptor was detected in the anterior or posterior pituitary, whereas the highest concentrations of specific a-bungarotoxin binding were found in the hypothalamus and superior and inferior colliculi. This study is the first demonstration of high affinity, low capacity stereospecific muscarinic cholinergic receptors in the anterior pituitary of rats and provides the basis for further studies on the cholinergic regulation of anterior pituitary function. (Endocrinology106: 1377, 1980)