Binding sites for [125I]-Bolton-Hunter scyliorhinin II in guinea-pig ileum: a radioligand binding, functional and autoradiographic study

Abstract

Binding of the tachykinin NK-3 receptor-preferring radioligand [125I]-Bolton-Hunter scyliorhinin II (BHSCYII) was investigated in membranes from guinea-pig ileum muscularis externa with myenteric plexus (MMP). Specific binding for BHSCYII was saturable and reversible. Curvilinear Scatchard plots and biphasic competition data indicate binding to two classes of sites (0.8 nM, 8% of sites; 340 nM, 92% of sites). Competition-binding data was ambiguous with the rank order of potency neuropeptide K (NPK) > substance P (SP) congruent to [Sar9,Met(O2)11]-SP congruent to [pGlu6,Pro9]SP(6-11) (septide) > neuropeptide gamma (NP gamma) > kassinin congruent to physalaemin > neurokinin A (NKA) > SCYII > neurokinin B (NKB). Senktide, eledoisin, [Lys5,MeLeu9,Nle10]-NKA(4-10), CP 96345, RP 67580, MDL 29913, SR 48968 and Gpp[NH]p were ineffective competitors, suggesting a lack of binding to conventional NK-1, NK-2 or NK-3 receptors. Competition curves for 5 agonists could be resolved into two sites, with no competitor showing outstanding affinity. Autoradiographic studies revealed moderate BHSCYII binding to myenteric plexus ganglia, unaffected by co-incubation with CP 96345 or with senktide. These data suggest a component of BHSCYII binding at unusual NPK/SP-preferring sites on ganglia. [Sar9,Met(O2)11]-SP was the most potent contractile agonist of the isolated ileum, followed by SCYII, NP gamma, senktide and NPK, with [Lys5,MeLeu9,Nle10]-NKA(4-10) least potent. Contractions elicited by senktide were entirely neurogenic. Responses to SCYII were partly sensitive to tetrodotoxin, atropine and CP 96345, indicative of action at both neuronal receptors and smooth muscle NK-1 receptors. The NK-2 antagonist SR 48968 did not alter responses to SCYII, [Sar9,Met(O2)11]-SP or senktide.