Journal of Neurochemistry I.ippincott—Raven Publishers, Philadelphia © 1996 International Society for Neurochemistry Characterisation and Molecular Identification of Adrenomedullin Binding Sites in the Rat Spinal Cord: A Comparison with Calcitonin Gene—Related Peptide Receptors Au A. Owji, James V. Gardiner, Paul D. Upton, Mehdi Mahmoodi, Mohammad A. Ghatei, Stephen R. Bloom, and David M. Smith Division of Endocrinology and Metabolic Medicine, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, England Abstract: Calcitonin gene—related peptide (CGRP) and its receptors are found in mammalian spinal cord. We show, for the first time, binding sites for the novel related peptide adrenomedullin in rat spinal cord microsomes. 1251-Adrenomedullin binding showed high affinity (KD = 0.45 i 0.06 nM) and sites were abundant (Bmax = 723 ± 71 fmol/mg of protein). CGRP, amylin, and calcitonin did not compete at these sites (K > 10 tiM). High-aftinity CGRP binding sites (KD = 0.18 ± 0.01 nM) were much less numerous (Bmax = 17.7 ± 2.4 fmol/mg of protein) and showed competition by unlabeled adrenomedullin (K 1 = 34.6 ± 2.4 nM). Chemical cross-linking revealed a major band for 1251-adrenomedullin of Mr = 84,400 ± 1,200 and a minor band of Mr = 122,000 ± 8,700. 1251.. CGRP cross-linking showed bands of lower molecular weight (Mr = 74,500 ± 5,000 and 61,000 ± 2,200). En- zymic deglycosylation of the adrenomedullin binding site showed a considerable carbohydrate content. Neither adrenomedullin nor CGRP was able to increase cyclic AMP in spinal cord. Adrenomedullin mRNA was present in spinal cord, at one-third of its level in lung, and adre- nomedullin immunoreactivity was present, at a low con- centration (40 fmol/g of tissue). Thus, the presence of abundant binding sites and adrenomedullin mRNA and immunoreactivity anticipate an as yet undefined function for this peptide in spinal cord. Key Words: Adrenomedul- lin—Calcitonin gene—related peptide—Spinal cord— Binding sites—Receptors. J. Neurochem. 67, 2172—21 79 (1996). been cloned. The rat peptide has 50 amino acids with two amino acid deletions and six substitutions com- pared with human adrenomedullin. Adrenomedullin mRNA is highly expnssed in adrenal medulla, lung, heart, and kidney in both rats and humans (Kitamura et al., 1993b; Sakata et al., 1993). Using a specific radioimmunoassay, the same group has shown adre- nomedullin to be ubiquitous in human and rat tissues, and present in the circulation (Ichiki et al., 1994; Sa- kata et al., 1994). In common with CGRP, adrenomed- ullin elicits a potent vasodilator effect in the rat (Gardi- ner et al., 1995), which is thought to be mediated in both cases by an increase in vascular smooth muscle cyclic AMP (cAMP) (Hirata et al., 1988; Ishizaka et al., 1994). CGRP is a widely distributed neuropeptide, abun- dant in the spinal cord, especially in the central branches of primary sensory neurones but also in moto- neurones and intrinsic neurones of the dorsal horn (Poyner, 1992). For example, CGRP is present in >50% of dorsal root ganglia neurones in many species (Hokfelt et al., 1992). Although the principal transmit- ter in these sensory neurones is glutamate, CGRP may facilitate dorsal horn transmission postsynaptically. CGRP is also up-regulated in injured motoneurones (Arvidsson et al., 1993) and may play a role in the expression of acetylcholine receptors in skeletal mus- Adrenomedullin is a 52 amino acid peptide, isolated in 1993 from human pheochromocytomas (Kitamura et at., 1993a). The peptide has a six-residue ring structure formed by an intramolecular disulfide bridge and a carboxy terminal—amidated residue, indicating simi- larities with the calcitonin family of peptides, i.e., cal- citonin, calcitonin gene—related peptide (CGRP), and amylin (Kitamura et al., 1993b). Recently, cDNAs encoding both human (Kitamura et al., 1993b) and rat (Sakata et al., 1993) adrenomedullin precursors have Received April II, 1996; revised manuscript received July 1, 1996; accepted July 1, 1996. Address correspondence and reprint requests to Dr. D. M. Smith at Division of Endocrinology and Metabolic Medicine, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, W12 OHS, U.K. The present address of Dr. A. A. Owji is Department of Biochem- istry, Shiraz University of Medical Sciences, Shiraz, Iran. Abbreviations used: BSA, bovine serum albumin; BSOCOES, bis [2-( succinimidooxycarbonyloxy ) ethyl] sulfone; cAMP, cyclic AMP; CGRP, calcitonin gene—related peptide; FPLC, fast protein liquid chromatography; IR, immunoreactivity; PAGE, polyacryl- amide gel electrophoresis; SDS, sodium dodecyl sulphate; TFA, tn- fluoroacetic acid. 2172