Tachykinin receptors in the small intestine of the cane toad (Bufo marinus): a radioligand binding and functional study

Abstract

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 [125I]Bolton-Hunter [Sar9,Met(O2)11]substance P (selective at mammalian NK-1 receptors) showed no specific binding. Specific binding of [125I]Bolton-Hunter substance P ([125I]BHSP) was saturable, of high affinity (Kd 0.3 nM) and was inhibited by SP (IC50 0.64 nM) > ranakinin approximately neurokinin A (NKA) > or = SP(5-11) > or = neuropeptide gamma > or = scyliorhinin II > scyliorhinin I > or = [Sar9]-SP > or = neurokinin B approximately physalaemin approximately carassin >> SP(7-11) approximately eledoisin > or = SP(4-11) approximately SP(6-11). Binding was also inhibited by Gpp[NH]p > or = GTPgammaS > 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 approximately SP > or = physalaemin > or = ranakinin > SP(6-11) > scyliorhinin II > or = neuropeptide gamma > neurokinin B approximately NKA approximately scyliorhinin I > or = SP(4-11) > or = SP(5-11) > [Sar9]SP > SP(7-11). In both studies, the selective mammalian NK-1, NK-2 and NK-3 receptor agonists [Sar9,Met(O2)11]SP, [Lys5,Me-Leu9,Nle10]NKA(4-10) and senktide were weak or ineffective. There was a strong positive correlation between the pD2 and pIC50 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. [Sar9,Met(O2)11]-SP(pD2 5.7) was approximately 25-fold less potent as an agonist than [Sar9]SP, which was itself 25-fold weaker than SP. Responses to SP were significantly reduced (n = 8, P<0.001) by the antagonist [D-Arg1,D-Trp7,9,Leu11]-SP (spantide; 1 microM). Highly selective NK-1 receptor antagonists including CP 99994 and GR 82334 (both 1 microM) were ineffective in both functional and binding studies. Tetrodotoxin (1 microM) 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 [125I]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.