Endothelin is a potent.vasoconstrictor/pressor peptide, which we recently characterized from the conditioned culture medium of porcine aortic endothelial cells. We report here the cloning and partial sequencing of the rat endothelin gene. The nucleotide sequence predicted a 21-residue peptide similar to, but distinct from, porcine endothelin; 15 residues of rat endothelin were identical and 3 residues were substitutions by chemically similar ano acid residues to those in 'the porcine peptide. Synthetic rat endothelin was then prepared according to its deduced amino acid sequence. This synthetic peptide had (i) potent vasoconstrictor activit in the rat aortic strip and in perfused rat heart and (ii) a characteristically long-lasting in vivo pressor activity by intraaortic bolus injection in the conscious rat.Vascular tonus is regulated by various' neural and hormonal stimuli'together with regional regulatory mechanisms of the blood, vessel wall, including the smooth' muscle ,and endothelial layers. The discovery of acetylcholine-induced, endothelium-dependent vasodilatation by Furchgott (1) has, stimulated intense interest in the role of the endothelium in modulating vascular responsiveness. Endothelin is an endothelial cell-derived vasoconstrictor/pressor peptide, which we; orignally isolated and sequenced from the culture medium of porcine aortic endothelial cells (2). Consisting of 21 amino acid residues with two sets of intrachain disulfide linkages, porcine endothelin is one of the most' potent vasoconstrictors known. Sequence analysis of cloned porcine endothelin cDNA showed that porcine endothelin is produced in endothelial cells from a 203-residue prepropeptide much-like many peptide. hormones and neuropeptides. Further,. we cloned a cDNA encoding human endothelin and showed that the amino acid sequehce of humian and porcine endothelin is identical (3). Porcine preprogndothelin mRNA is exptessed not only in the cultured endothelial cells but also in aortic endothelium in vivo, and the level of miRNA expression is markedly influenced by vasoactive agents such as thrombin and adrenaline (2). These observations suggest the existence in the mammalian cardiovascular system of a distinct endothelium-mediated regulatory mechanism for blood pressure and/or local blood distribution.As a basis for investigation on the physiological and resin was treated with anhydrous hydrogen fluoride in the presence of 5% (vol/vol) p-cresol/15% (vol/vol) butanedithiol at -20C for 60 min. After evaporation of excess hydrogen fluoride, the crude peptide was precipitated with ether and extracted with CF3COOH to filter off the solid support. The crude product containing four sulfhydryl groups was then subjected to air oxidation at a concentration of 0.1 mM in aqueous NH40H at room temperature for 16 hr and purified by preparative reverse-phase HPLC (Waters PrepLC System 500) with a 20-45% linear gradient of CH3CN in 0.1% CF3COOH. Overall yield from the peptide resin was 15%. Homogeneity 'bf the final product was confirmed by ...

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The effects of synthetic rat adrenomedullin (rAM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma. on receptor binding and CAMP generation were studied in cultured rat vascular smooth muscle cells (VSMC). A binding study using ["51]rAM revealed the presence of a single class of high-affinity (& 1.3 x lo-* M) binding sites for rAM m VSMC. The apparent K, of rat calcitonin gene-related peptide (rCGRP) was 3 x lo-' M. Affinity labeling of VSMC membranes with ["'I]rAM revealed two distinct labeled bands with apparent molecular weights of 120 and 70 kDa, both of which were abolished by excess unlabeled rAM or rCGRP. rAM stimulated CAMP formation with an approximate EC,, of lo-* M, the effect of which was additive with isoproterenol, but not with rCGRP. The rAM-induced CAMP response was unaffected by propranalol. indomethacin, or quinacrine, but inhibited by a CGRP receptor antagonist, human CGRP[8-371. These data suggest that VSMC possesses specific AM receptors functionally coupled to adenylate cyclase with which CGRP interacts.

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