Search

The characteristics of atropine-sensitive binding of l-[3H]-quinuclidinyl benzilate ([3H]-QNB) to membrane suspensions of sheep posterior pituitary indicate that the binding sites represent muscarinic cholinergic receptors. Scatchard plots of 6 saturation experiments showed a single class of binding sites, with an equilibrium dissociation constant of 16 +/- 2 pM and a density equivalent to 1.8 +/- 0.2 pmol/g wet weight of tissue. Kinetic analysis of 2 association and 3 dissociation curves yielded mean association and dissociation rate constants of 3.9 x 10(8) M-1 min-1 and 4.3 x 10(-3) min-1, respectively. The binding had a detailed pharmacology for 12 drugs consistent with muscarinic receptor identification. In rat neurointermediate lobes, superior cervical ganglionectomy had no demonstrable effect on [3H]-QNB binding. The location(s) and functional role(s) of neurohypophyseal muscarinic receptors remain to be elucidated.

The existence of typical muscarinic receptors in membranes of sheep anterior pituitary was detected by binding of [3H]quinuclidinyl benzilate ([3H]QNB), a potent and specific muscarinic antagonist. [3H]QNB binding sites in anterior pituitary had an equilibrium dissociation constant of about 20-40 pM, a rate constant for association at 37 degrees C of about 2 x 10(8) M-1 min-1, a rate constant for dissociation at 37 degrees C of about 4 x 10(-3) min-1, and the expected specificities for a variety of cholinergic and other drugs. The concentration of [3H]QNB binding sites in anterior pituitary, about 4-6 pmol/g tissue, was at least twice that in posterior pituitary, but less than a quarter that in the hypothalamus. Since there is no established cholinergic or other innervation of the anterior pituitary, the presence there of typical muscarinic receptors, if we assume that they are functional, suggests that acetylcholine reaches the tissue through the hypophyseal portal circulation, and that it may thus have a role in the regulation of pituitary function.

[3H]-3-methyl-His2]thyrotropin releasing hormone ([3H]MeTRH) binds to sites in the sheep anterior pituitary gland which appear to be the same as those occupied by [3H]TRH and which can therefore be identified as TRH receptors. In competition experiments performed in parallel, both ligands gave the same number of binding sites, 15 pmol/g wet weight, and showed the same pharmacology for 19 TRH analogs ranging over more than 5 orders of magnitude in potency. The apparent dissociation constant of binding of [3H]MeTRH was about 3.5 nM compared to 29 nM for [3H]TRH. Kinetic experiments with [3H]MeTRH yielded a rate constant for association of 1.4 x 10(7) M-1 min-1 and rate constants for the biphasic dissociation of 5 x 10(-2) min-1 (fast phase) and 7 x 10(-3) min-1 (slow phase). Detailed methods are described for preparation of [3H]MeTRH by reduction of the dehydroproline precursor and its purification by ion exchange and antibody affinity chromatography. The major advantage of the new ligand is that its higher affinity of binding gives relatively less non-specific binding than is obtained with [3H]TRH, particularly in central nervous tissue.