COEXISTENCE OF 3 TACHYKININ RECEPTORS COUPLED TO CA-CELLS(+ SIGNALINGPATHWAYS IN INTESTINAL MUSCLE)

Receptors for tachykinins and the signaling pathway to which they are coupled were characterized in dispersed muscle cells from the longitud inal muscle layer of the rat intestine. A technique of receptor protec tion whereby selective agonists and antagonists were used to protect o ne receptor while other receptors were inactivated with N-ethylmaleimi de enabled each tachykinin receptor type to be identified separately. Protection of neurokinin (NK)-1 receptors with the selective NK-1 agon ist, substance P methylester, or antagonist, GR-82,334 (Glp-Ala-Asp-Pr o-Asn-Lys-Phe-Tyr-D-Pro[spiro -gamma-lactam] Leu-Trp-NH2), preserved t he contractile response and increase in cytosolic-free Ca++ ([Ca++](i) ) induced by substance P methylester only; protection of NK-2 receptor s with the selective NK-2 agonist, beta-[Ala(8)]NKA(4-10), or the sele ctive NK-2b antagonist, L-659,877 [cyclo(Leu-Met-Gln-Trp-Phe-Gly)], pr eserved the contractile response and increase in [Ca++](i) induced by beta-[Ala(8)]NKA(4-10) only; and protection of NK-3 receptors with the selective NK-3 agonist, senktide succinyl-[Asp(6),MePhe(8)]substance P(6-11), preserved the contractile response and increase in [Ca++](i) induced by succinyl-[Asp(6),MePhe(8)]substance P(6-11) only. When used as a protective agent, the NK-2a antagonist, MEN-10,376 (H-Asp-Tyr-D- Trp-Val-D-Trp-D-Trp-Lys-NH2), did not preserve the response to any tac hykinin agonist. Protection of NK-1, NK-2 and NK-3 receptors preserved fully the responses to the preferential endogenous agonists, substanc e P, NKA and NKB, respectively, but they also preserved in part (30-40 %) the responses to the nonpreferential agonists. Because substance P and NKA are coreleased from the same precursor in intestinal muscle ti ssue, the pattern implied the existence of considerable spareness in t he contractile response of muscle cells to tachykinins. Studies on dis persed circular muscle cells using selective tachykinin agonists as pr otective agents confirmed the presence of three tachykinin receptor ty pes. The results demonstrate the coexistence of NK-1, NK-2b and NK-3 r eceptors on muscle cells of rat intestine that are preferentially acti vated by substance P, NKA and NKB, respectively, and are coupled separ ately to one signaling pathway mediating contraction.