Pharmacological and biochemical investigation of receptors for the toad gut tachykinin peptide, bufokinin, in its species of origin

This is the first report of the development of a new radioligand [I-125]Bol ton-Hunter bufokinin ([I-125]BH- bufokinin) and its use in the characterisa tion of tachykinin receptors in the small intestine of the cane toad, Bufo mar inus. The binding of [I-125]BH-bufokinin to toad intestinal membranes w as rapid, saturable, of high affinity and to a single population of binding sites with K-D 0.57 nM and B-max 3.1 fmol mg wet weight tissue(-1). The ra nk order of affinity of tachykinins to compete for [I-125]-BH bufokinin bin ding revealed similarities with that of the mammalian NK1 receptor, being b ufokinin (IC50, 1.7 nM)>physalaemin (6.7 nM)>substance P (SP, 10.7 nM)great er than or equal to neuropeptide gamma (NP gamma, 12.4 nM)greater than or e qual to kassinin (17.8 nM)>scyliorhinin I (35.3 nM)greater than or equal to eledoisin (40.6 nM)greater than or equal to carassin (43.2 nM)greater than or equal to neurokinin A (NKA, 57.8 nM)greater than or equal to neurokinin B (NKB, 77.5 nM)>scyliorhinin II (338 nM). The mammalian NK3-selective ago nist senktide was a very weak competitor. The radioligand [I-125]neurokinin A showed no specific binding to toad intestinal membranes. In the toad iso lated small intestine, the maximum contractile response to bufokinin was ov er 150% greater than that to acetylcholine in longitudinal muscle, whereas responses to bufokinin and acetylcholine were similar in circular muscle. B ufokinin was the most potent agonist (EC50 0.34 nM) and produced a long-las ting contraction. Other tachykinins such as physalaemin, SP and kassinin we re also potent contractile agents. The potency values of mammalian and amph ibian tachykinins derived from functional studies (pD(2)) correlated signif icantly with those from binding assays (pK(i)). The data for fish and mollu scan tachykinins, however, showed poor correlation. Contractions to bufokin in and SP were unaffected by atropine, indomethacin and tetrodotoxin. The h ighly selective NK1 receptor antagonists CP 99994, GR 82334 and RP 67580 we re ineffective in both binding and functional studies. Bufokinin increased inositol monophosphate formation in a concentration-dependent manner with a n EC50 value of 10.7 nM, suggesting that the tachykinin receptor may be cou pled to phosphoinositol hydrolysis. In summary, this study provides evidenc e for a high-affinity, bufokinin-preferring, NK1-like tachykinin receptor i n the toad small intestine. This is probably not the receptor which mediate s contraction to carassin, scyliorhinin II and eledoisin. The study also pr ovides evidence that bufokinin and its receptor play an important physiolog ical role in regulating intestinal motility.