Hellstrom P M, Murthy K S, Grider J R, Makhlouf G M
Department of Medicine, Medical College of Virginia, Richmond.
J Pharmacol Exp Ther. 1994 Jul;270(1):236-43.
Receptors for tachykinins and the signaling pathway to which they are coupled were characterized in dispersed muscle cells from the longitudinal muscle layer of the rat intestine. A technique of receptor protection whereby selective agonists and antagonists were used to protect one receptor while other receptors were inactivated with N-ethylmaleimide enabled each tachykinin receptor type to be identified separately. Protection of neurokinin (NK)-1 receptors with the selective NK-1 agonist, substance P methylester, or antagonist, GR-82,334 (Glp-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-D-Pro[spiro-gamma-lactam]Leu-Trp-NH2), preserved the contractile response and increase in cytosolic-free Ca++ ([Ca++]i) induced by substance P methylester only; protection of NK-2 receptors with the selective NK-2 agonist, beta-[Ala8]NKA(4-10), or the selective NK-2b antagonist, L-659,877 [cyclo(Leu-Met-Gln-Trp-Phe-Gly)], preserved the contractile response and increase in [Ca++]i induced by beta-[Ala8]NKA(4-10) only; and protection of NK-3 receptors with the selective NK-3 agonist, senktide succinyl-[Asp6,MePhe8]substance P(6-11), preserved the contractile response and increase in [Ca++]i induced by succinyl-[Asp6,MePhe8]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 tachykinin agonist. Protection of NK-1, NK-2 and NK-3 receptors preserved fully the responses to the preferential endogenous agonists, substance 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 tissue, the pattern implied the existence of considerable spareness in the contractile response of muscle cells to tachykinins. Studies on dispersed circular muscle cells using selective tachykinin agonists as protective agents confirmed the presence of three tachykinin receptor types. The results demonstrate the coexistence of NK-1, NK-2b and NK-3 receptors on muscle cells of rat intestine that are preferentially activated by substance P, NKA and NKB, respectively, and are coupled separately to one signaling pathway mediating contraction.
在大鼠肠道纵肌层的分散肌肉细胞中，对速激肽受体及其偶联的信号通路进行了表征。采用一种受体保护技术，即使用选择性激动剂和拮抗剂来保护一种受体，同时用N - 乙基马来酰亚胺使其他受体失活，从而能够分别鉴定每种速激肽受体类型。用选择性NK - 1激动剂P物质甲酯或拮抗剂GR - 82,334（Glp - Ala - Asp - Pro - Asn - Lys - Phe - Tyr - D - Pro[螺环 - γ - 内酰胺]Leu - Trp - NH2）保护NK - 1受体，仅保留了由P物质甲酯诱导的收缩反应和胞质游离Ca++（[Ca++]i）增加；用选择性NK - 2激动剂β - [Ala8]NKA(4 - 10)或选择性NK - 2b拮抗剂L - 659,877 [环（Leu - Met - Gln - Trp - Phe - Gly）]保护NK - 2受体，仅保留了由β - [Ala8]NKA(4 - 10)诱导的收缩反应和[Ca++]i增加；用选择性NK - 3激动剂senktide琥珀酰 - [Asp6,MePhe8]P物质(6 - 11)保护NK - 3受体，仅保留了由琥珀酰 - [Asp6,MePhe8]P物质(6 - 11)诱导的收缩反应和[Ca++]i增加。当用作保护剂时，NK - 2a拮抗剂MEN - 10,376（H - Asp - Tyr - D - Trp - Val - D - Trp - D - Trp - Lys - NH2）不能保留对任何速激肽激动剂的反应。保护NK - 1、NK - 2和NK - 3受体分别完全保留了对优先内源性激动剂P物质、NKA和NKB的反应，但它们也部分（30 - 40%）保留了对非优先激动剂的反应。由于P物质和NKA在肠道肌肉组织中从同一前体共同释放，这种模式表明肌肉细胞对速激肽的收缩反应存在相当大的备用性。使用选择性速激肽激动剂作为保护剂对分散的环肌细胞进行的研究证实了三种速激肽受体类型的存在。结果表明，大鼠肠道肌肉细胞上共存NK - 1、NK - 2b和NK - 3受体，它们分别优先被P物质、NKA和NKB激活，并分别偶联到一条介导收缩的信号通路上。
