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 techniques to investigate tachykinin receptors in the small intestine of the cane toad Bufo marinus. The radioligand [I-125]Bolton-Hunter [Sar(9);Met(O-2)(11)] sub stance P (selective at mammalian NK-1 receptors) showed no specific binding. Specific binding of [I-125]Bolton-Hunter substance 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 neurokinin A (NKA) greater than or equal to SP(5-11) greater than or equal to neuropeptide gamma greater than or equal to scyliorhinin II > scyliorhinin I greater than or equal to [Sar(9)]-SP greater than or equal to neurokinin B approximate to physalaemin approximate to carassin much greater 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-protein coupled receptor. The order of potency of tachykinins and analogues in contracting the isolated lower small intestine was carassin (EC50 1.4 nM) > eledoisin approximate to SP greater than or equal to physalaemin greater than or equal to ranakinin > SP(6-11) > scyliorhinin II greater than or equal to neuropeptide gamma > neurokinin B approximate to NKA approximate to scyliorhinin I greater than or equal to SP(4-11) 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 senktide were weak or ineffective. There was a strong positive correlation between the pD(2) and pIC(50) values for mammalian tachykinins and analogues (r = 0.907), but not for the non-mammalian tachykinins, which were all full agonists but variable binding competitors. [Sar(9), Met(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. Responses to SP were significantly reduced [n = 8, P<0.001) by the antagonist [D-Arg(1), D-Trp(7,9), Leu(11)]-SP (spantide; 1 mu M). Highly selective NK-1 receptor antagonists including CP 99994 and GR 82334 (both 1 mu M) were ineffective in both functional and binding studies. Tetrodotoxin (1 mu M) did not inhibit contractile responses to SP, NKA and senktide. In summary, this study has shown the presence of one or more tachykinin receptor in the toad intestine. The binding site recognised by [I-125]BHSP prefers SP and ranakinin. This toad "NK-1-like receptor" differs from the mammalian NK-1 receptor in having a low affinity for all mammalian NK-1 selective ligands, including antagonists. For some non-mammalian peptides, their high potency as contractile agonists relative to their poor binding affinity suggests the existence of other tachykinin receptors in the toad small intestine.