Expression of Adrenomedullin ( ADM ) and Its Binding Sites in the Rat Uterus : Increased Number of Binding Sites and ADM Messenger Ribonucleic Acid in 20-Day Pregnant Rats Compared with Nonpregnant Rats *

RIA of nonpregnant rat uterus extracts showed 0.68 6 0.08 pmol/g adrenomedullin (ADM) and 3.23 6 0.08 pmol/g calcitonin gene-related peptide (CGRP). In the pregnant (20 days gestation) uterus, the ADM content was 0.90 6 0.17 pmol/g, and CGRP could not be detected. ADM messenger RNA was detected at high levels in the uterus, with a 1.8-fold increase in expression in pregnancy. Pharmacologically distinct binding sites for ADM (Bmax 5 21 6 2 fmol/mg protein, dissociation constant 5 80 6 6 pM), and CGRP (Bmax 5 101 6 18 fmol/mg protein, dissociation constant 5 140 6 20 pM) were identified in nonpregnant uterus. Competition for I[Tyr]aCGRP binding was shown by both ADM and CGRP (8–37), whereas CGRP and CGRP (8–37) did not compete for I-ADM-binding sites. The density of the ADM-binding sites was 10 times greater in pregnant uterus (Bmax 5 211 6 39 fmol/mg protein, P , 0.01) than nonpregnant uterus. CGRP receptor messenger RNA was identified in both nonpregnant and pregnant uteri. In isolated nonpregnant rat uteri, CGRP and ADM attenuated the contractile response to galanin by 77 6 10% and 57 6 10%, respectively. The responses to both CGRP and ADM were abolished by CGRP (8–37). These results demonstrate, for the first time, the presence of ADM and specific binding sites for both ADM and CGRP in the rat uterus. (Endocrinology 138: 2508–2514, 1997) A (ADM) is a 52-amino acid peptide originally isolated from human phaeochromocytomas (1). It is a member of the calcitonin family of peptides with sequence homology to calcitonin gene-related peptide (CGRP) and islet amyloid polypeptide (IAPP). The distribution and biological effects of ADM and CGRP overlap in many tissues. ADM immunoreactivity (ADM-IR) and messenger RNA (mRNA) have been identified in a number of human and rat tissues, including heart and lung (2–5). CGRP-immunoreactive (CGRP-IR) neurons innervate the majority of tissues, with the capsaicin-sensitive aCGRP-containing primary afferent neurones predominating in the cardiovascular system, brain and urinogenital tract but with both aCGRP and bCGRP-containing neurons present in the gastrointestinal tract (6–8). One of the major biological effects of both CGRP and ADM is their ability to relax smooth muscle. In the guinea pig lung, ADM acts as a potent bronchodilator (9), and CGRP has been shown to relax guinea pig trachea and rat respiratory tract smooth muscle (10, 11). ADM and CGRP are potent vasodilators in many species (12–16). The specific CGRP receptor subtype 1 (CGRP1) antagonist, CGRP (8–37), inhibits the vasodilator response to ADM in the isolated perfused mesenteric vascular bed (17) and in the isolated rat heart (18), indicating that the effect of ADM in these tissues is via CGRP1 receptors. However, the systemic hypotensive effect of CGRP, but not that of ADM, is blocked in vivo by CGRP (8–37) (14, 19), showing that their systemic effects are mediated by different receptors. Specific binding sites for ADM that do not bind CGRP have been identified in a number of rat tissues (20), blood vessels (14), and vascular smooth muscle cells (21). We have recently cloned a rat ADM receptor (22) that displays a similar pharmacology and mRNA distribution to the binding sites identified in rat tissues (20). In addition, we have shown that the orphan dog RDC1 receptor, when expressed in COS-7 cells, has the pharmacological characteristics of a CGRP1 receptor (23). CGRP-IR previously has been identified in neurones innervating the female rat urinogenital tract (7). Capsaicin treatment depletes the majority of CGRP-IR in these tissues, indicating that this immunoreactivity is probably aCGRP localized to the primary sensory afferent neurones (7). Immunocytochemistry of rat uterus has shown that CGRP-IR fibres innervate the mesometrium of the uterine horn, the mesometrial smooth muscle, and vascular smooth muscle and also are located in the myometrium, endometrium, and Received November 4, 1996. Address all correspondence and requests for reprints to: D. M. Smith, Ph.D., Division of Endocrinology and Metabolic Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 ONN, United Kingdom. E-mail: dsmith@rpms.ac. uk. * This work was supported by grants from the Medical Research Council and RPMS Small Grants Scheme. † Supported by a Medical Research Council studentship. 0013-7227/97/$03.00/0 Vol. 138, No. 6 Endocrinology Printed in U.S.A. Copyright © 1997 by The Endocrine Society