CHARACTERIZATION OF INTESTINAL SMOOTH-MUSCLE RESPONSES AND BINDING-SITES FOR ENDOTHELIN

The contractile activity of and binding sites for endothelin-1 (ET-1) were investigated in isolated guinea-pig ileal longitudinal smooth muscle (GPILM). ET-1 produced concentration-dependent contractions of GPILM that either slowly subsided in the continued presence of ET-1 or rapidly subsided following washing of the tissue. The ED50 value for ET-1 contractions was 4.2 +/- 1.3 x 10(-9) M. The removal of extracellular calcium or pretreatment with nifedipine produced a complete inhibition of the contractions to ET-1. The IC50 value of nifedipine for inhibition of ET-1 mediated contractions was 3.0 +/- 0.8 x 10(-8) M. ET-1 produced a marked prolonged homologous desensitization of its contractile response but did not affect the responses mediated by carbachol, histamine, serotonin, substance P, and PLA2. High-affinity binding sites for I-125-labelled ET-1 were identified on microsomal membranes prepared from GPILM with K(d) and B(max) values obtained by Scatchard analysis of 3.5 +/- 0.6 x 10(-10) M and 2138 +/- 159 fmol/mg protein, respectively. The binding of I-125-labelled ET-1 to GPILM microsomes was characterized by a rapid association (k(ob) value of 0.077 min-1 at a radioligand concentration of 0.45 nM and an extremely slow dissociation (k(1) value of 0.011 min-1; t1/2 value of 793 min). The binding was unaffected by the calcium channel antagonists nifedipine, verapamil, and diltiazem (10(-6) M); the receptor antagonists phenoxybenzamine, atropine. and naloxone (10(-6) M) and propranolol; and the peripheral benzodiazepine receptor antagonists Ro 5-4864 and PK 11195 and psychotomimetic drug phencyclidine (10(-5) M). Incubation of GPILM with ET-1 (2 x 10(-8) M) for 10 min followed by washing of the tissue for 1 h resulted in a significant (p < 0.05 unpaired Student's t-test) reduction (33%) of I-125-labelled ET-I binding that partially recovered following 2 h of washing the tissue. These results demonstrate that ET-1 is an intestinal smooth muscle spasmogen that produces its pharmacologic effects by a mechanism(s) that is not shared by other major intestinal neurotransmitters. Furthermore, intestinal smooth muscle contains specific high-affinity binding sites that likely mediate the contractile responses to ET-1.