TACHYKININ RECEPTORS IN THE SMALL-INTESTINE OF THE CANE TOAD (BUFO-MARINUS) - A RADIOLIGAND BINDING AND FUNCTIONAL-STUDY

In this study, we have used radioligand binding and functional techniq ues to investigate tachykinin receptors in the small intestine of the cane toad Bufo marinus. The radioligand [I-125]Bolton-Hunter [Sar(9);M et(O-2)(11)] sub stance P (selective at mammalian NK-1 receptors) show ed no specific binding. Specific binding of [I-125]Bolton-Hunter subst ance P ([I-125]BHSP) was saturable, of high affinity (K-d 0.3 nM) and was inhibited by SP (IC50 0.64 nM) > ranakinin approximate to neurokin in A (NKA) greater than or equal to SP(5-11) greater than or equal to neuropeptide gamma greater than or equal to scyliorhinin II > scyliorh inin I greater than or equal to [Sar(9)]-SP greater than or equal to n eurokinin B approximate to physalaemin approximate to carassin much gr eater than SP(7-11) approximate to eledoisin greater than or equal to SP(4-11) approximate to SP(6-11). Binding was also inhibited bg Gpp[NH ]p greater than or equal to GTP gamma S > App[NH]p, indicating a G-pro tein coupled receptor. The order of potency of tachykinins and analogu es in contracting the isolated lower small intestine was carassin (EC5 0 1.4 nM) > eledoisin approximate to SP greater than or equal to physa laemin greater than or equal to ranakinin > SP(6-11) > scyliorhinin II greater than or equal to neuropeptide gamma > neurokinin B approximat e to NKA approximate to scyliorhinin I greater than or equal to SP(4-1 1) greater than or equal to SP(5-11) > [Sar(9)]SP > SP(7-11). In both studies, the selective mammalian NK-1, NK-2 and NK-3 receptor agonists [Sar(9), Met(O-2)(11)]SP, [Lys(5), Me-Leu(9), N1e(10)]NKA(4-10] and s enktide were weak or ineffective. There was a strong positive correlat ion between the pD(2) and pIC(50) values for mammalian tachykinins and analogues (r = 0.907), but not for the non-mammalian tachykinins, whi ch were all full agonists but variable binding competitors. [Sar(9), M et(O-2)(11)]-SP(pD(2) 5.7) was approximately 25-fold less potent as an agonist than [Sar(9)]SP, which was itself 25-fold weaker than SP. Res ponses to SP were significantly reduced [n = 8, P<0.001) by the antago nist [D-Arg(1), D-Trp(7,9), Leu(11)]-SP (spantide; 1 mu M). Highly sel ective NK-1 receptor antagonists including CP 99994 and GR 82334 (both 1 mu M) were ineffective in both functional and binding studies. Tetr odotoxin (1 mu M) did not inhibit contractile responses to SP, NKA and senktide. In summary, this study has shown the presence of one or mor e tachykinin receptor in the toad intestine. The binding site recognis ed by [I-125]BHSP prefers SP and ranakinin. This toad ''NK-1-like rece ptor'' differs from the mammalian NK-1 receptor in having a low affini ty for all mammalian NK-1 selective ligands, including antagonists. Fo r some non-mammalian peptides, their high potency as contractile agoni sts relative to their poor binding affinity suggests the existence of other tachykinin receptors in the toad small intestine.