Abstract
Recently, we have demonstrated the presence of muscarinic receptors in the rat adenohypophysis and described their biochemical characteristics [I]. Our results showed that in contrast to other brain regions [2] antagonist binding in this area is heterogeneous with the existence of at least two subclasses of sites [l]. Physiological data indicate that muscarinic receptors may be coupled to Ca*+-channels in certain systems (3-5) and that calcium may then act as a second messenger. Extracellular calcium is required for the release of several adenohypophysis hormones [6,7]. [3H]4NMPB (9.2 Ci/mmol) were prepared as in [2,8]. The purity of the compounds was > 97%. Methoxyverapamif (known also as ‘D-600’) was a gift from Dr T. Bartfai. Adult female rats of the CD strain were supplied by Levinstein’s farm (Yokneam, Israel) and maintained in an airconditioned room at 24 f 2°C for 14 h fluorescent illumination and 10 h darkness daily. After an adjustment period of at least 4 weeks, daily vaginal smears were taken and only those females having a regular 4-day estrous cycle were used. Since in [l] we found differences in the biochemical characteristics of muscarinic receptors in the adenohypophysis at various stages of the estrous cycle, we chose a well-defined physiological stage Here, we examined whether Ca*+ affects the biochemical characteristics of muscarinic binding in the adenohypophysis, using the highly specific, tritiated muscarinic antagonist, N-methyld-piperidyl benzilate ([3H]4NMPB). We also examined whether the heterogeneity of the binding sites in this area originates from different components of the muscarinic receptor. For this purpose we used the specific photolabile muscarinic antagonist, N-methyl-4-piperidyl azidobenzilate (azido[3H]4NMPB), which has been shown to provide irreversible labeling of a M, 86 000 polypeptide in rat cortex [8]. the estrous day to investigate the effect of calcium.
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