European Journal of Pharmacology, 231 (1993) 459-464 459 © 1993 Elsewer Science Pubhshers B V. All rights reserved 0014-2999/93/$06 00 EJP 52906 In vivo characterization of a phosphoramidon-sensitive endothelin-converting enzyme in the rat David M. Pollock, Barbara J. Divish, Ivan Milicic, Eugene I. Novosad, Neal S. Burres and Terry J. Opgenorth Pharmaceutwal Dtscovery, Abbott Laboratorws, Abbott Park, IL 60064, USA Recewed 25 June 1992, revised MS recewed 10 November 1992, accepted 17 November 1992 Experiments were conducted to characterize the nature of a phosphoramldon-sensltlVe endothelin-convertmg enzyme in VlVO by evaluating the pressor response to a bolus intravenous (l.V) injection of endothelin family peptldes following administration of phosphoramidon in anesthetized Sprague-Dawley rats. Phosphoramidon given l.V. at 10 mg/kg completely prevented the pressor response to human big endothehn-l-(1-38) (big ET-1). The ECs0 for phosphoramidon was determined to be in the range of 1 to 3 mg/kg The pressor response to big ET-1 60 mm after phosphoramldon injection was attenuated by roughly 60% indicating a long inhibitory half-life. Very high doses of big ET-1 (> 20 mg/kg) were capable of over-riding the effect of phosphoramldon and produced characteristic pressor responses suggesting that the inhibition by phosphoramidon can be considered competitive in nature Human big endothelin-3-(1-41) (big ET-3) produced significant increases in arterial pressure although with less potency and efficacy compared to big ET-1. The pressor response to big ET-3 was also inhibited by phosphoramldon. Phosphoramldon does not act indirectly by interfering with ET-1 receptor-mediated actions since the inhibitor has no effect on the in vivo pressor response to ET-1 and does not antagonize [~25I]ET-1 receptor binding or constrictor responses in vitro. These results are consistent with the idea that a phosphoramldon-sensJtlve endothelin-convertmg enzyme is capable of cleaving both big ET-1 and big ET-3 to the actwe peptides in the rat. Endothehn-convertmg enzyme; Big endothehn-1; Big endothelin-3; Phosphoramldon; Endothelin; Hemodynamlcs; (Rat) I. Introduction In their original characterization of the endothelin peptide system, Yanagisawa et al. (1988) proposed that endothelin-1 (ET-1) is created by the specific prote- olytic cleavage of a precursor peptide, named big en- dothelin-l-(1-38) (big ET-1), by a putative endothelin- converting enzyme (ECE). A similar enzymatic path- way is assumed to exist for the conversion of big endothelin-3-(1-41) (big ET-3) to endothelin-3 (ET-3). This idea is further supported by the observations that big ET-1 has relatively low receptor binding affinity and in vitro constrictor activity compared to the mature peptide, but has simdar potencies in terms of pressor effects in vivo (Kashiwabara et al., 1989). D'Orleans- Juste et al. (1990) and Hems6n et a1.(1991) have pro- vided more direct evidence to support the idea that an ECE is capable of very rapid conversion of big ET-I to Correspondence to' D M Pollock, Abbott Laboratories, D-47V, AP 9, Abbott Park, IL 60064 USA Tel l (708) 938-6358, fax 1 (708) 938-2258 ET-1 within the circulation by demonstrating an in- crease in immunoreactive ET-1 in plasma following big ET-1 infusion. The nature and location of the physiologically rele- vant ECE(s) remains to be elucidated. Several labora- tories have shown that ECE activities can be identified from a number of different tissue and cultured cell sources (Opgenorth et al., 1992). In vitro studies have demonstrated that one or more of these ECE activities may be a yet uncharacterized metalloprotease because it is specifically inhibited by phosphoramidon but not by other metalloprotease inhlbitors. These phosphor- amidon-sensitive ECE activities appear to be more selective for big ET-1 than big ET-3 (Opgenorth et al., 1992). Phosphoramidon-sensitive ECE activities ex- tracted from rat brain and vas deferens are capable of converting big ET-1 but not big ET-3 to their respec- tive actwe peptides (T616maque and D'OrlEans-Juste, 1991; Warner et al., 1992). On the other hand, Mat- sumura et al. (1992) have identified an ECE in en- dothehal cells with comparable ability to convert big ET-1 and big ET-3 which is also inhibitable by phos- phoramidon. In intact animals, phosphoramidon also