Watts S W, Cox D A, Johnson B G, Schoepp D D, Cohen M L
Department of Pharmacology and Toxicology, Indiana University, Indianapolis.
J Pharmacol Exp Ther. 1994 Nov;271(2):832-44.
Serotonin (5-HT) contracts the guinea pig trachea through stimulation of the 5-HT2A receptor, a receptor generally linked with phosphoinositide (PI) hydrolysis. However, previous limited evidence suggested that 5-HT did not increase PI hydrolysis in guinea pig trachea. The present studies confirmed that the 5-HT2A receptor is not coupled to PI hydrolysis and investigated the calcium source and involvement of protein kinase C (PKC) in 5-HT-induced contraction in guinea pig trachea. In vitro experiments, which used an enriched tracheal muscle preparation, confirmed the inability of 5-HT (10(-10) to 10(-2) M) to increase PI hydrolysis. Short incubations (1-60 min) of the trachea with 5-HT (10(-4) M) to minimize possible 5-HT2A receptor desensitization did not increase PI hydrolysis, whereas carbamylcholine (10(-7) to 10(-3) M) and histamine (10(-7) to 10(-4) M) did. These results demonstrate that, unlike most other 5-HT2A receptors, the 5-HT2A receptor in guinea pig trachea is not coupled to PI hydrolysis. The L-type calcium channel antagonists nitrendipine (10(-6) and 10(-5) M) and diltiazem (5 x 10(-5) M) significantly blocked maximal tracheal contraction to 5-HT (45-60%) inhibition) but not to carbamylcholine. The maximal response to 5-HT in calcium-free buffer (0 calcium, 0.05 mM EGTA) was also inhibited by 56%. The residual contraction to 5-HT in the absence of extracellular calcium suggested that at least a portion of the nitrendipine-insensitive 5-HT contraction was due to the release of intracellular calcium. In support of this idea, ryanodine (3 x 10(-5) M), a compound known to deplete intracellular calcium stores, depressed maximal 5-HT contraction in the presence of either nitrendipine or diltiazem. Neither calphostin C (4 x 10(-8) and 10(-6) M) or staurosporine (10(-8) M), both putative PKC inhibitors, affected tracheal contraction to 5-HT. However, the PKC inhibitor bisindolylmaleimide (5 x 10(-6) M), which abolished contraction to phorbol 12,13-dibutyrate (10(-6) M), unlike calphostin C, inhibited contraction to 5-HT in both the absence and presence of nitrendipine. This finding suggests that PKC activation is involved in 5-HT contraction. Thus, the tracheal 5-HT2A receptor is unique in that activation of the receptor does not result in PI hydrolysis but increases calcium influx through L-type voltage-dependent calcium channels, calcium release from the sarcoplasmic reticulum and activation of a bisindolylmaleimide-sensitive PKC.
血清素（5-羟色胺，5-HT）通过刺激5-HT2A受体使豚鼠气管收缩，该受体通常与磷酸肌醇（PI）水解相关联。然而，先前有限的证据表明5-HT不会增加豚鼠气管中的PI水解。本研究证实5-HT2A受体与PI水解不偶联，并研究了钙源以及蛋白激酶C（PKC）在5-HT诱导的豚鼠气管收缩中的作用。体外实验使用了富集的气管肌肉制剂，证实5-HT（10^(-10)至10^(-2) M）无法增加PI水解。用5-HT（10^(-4) M）对气管进行短时间孵育（1 - 60分钟）以尽量减少可能的5-HT2A受体脱敏，并未增加PI水解，而氨甲酰胆碱（10^(-7)至10^(-3) M）和组胺（10^(-7)至10^(-4) M）则会增加。这些结果表明，与大多数其他5-HT2A受体不同，豚鼠气管中的5-HT2A受体与PI水解不偶联。L型钙通道拮抗剂尼群地平（10^(-6)和10^(-5) M）和地尔硫䓬（5×10^(-5) M）显著阻断了气管对5-HT的最大收缩（抑制45 - 60%），但对氨甲酰胆碱无此作用。在无钙缓冲液（0钙，0.05 mM乙二醇双四乙酸）中对5-HT的最大反应也被抑制了56%。在无细胞外钙的情况下对5-HT的残余收缩表明，至少一部分对尼群地平不敏感的5-HT收缩是由于细胞内钙的释放。支持这一观点的是，已知能耗尽细胞内钙储存的ryanodine（3×10^(-5) M）在存在尼群地平或地尔硫䓬的情况下降低了5-HT的最大收缩。两种假定的PKC抑制剂钙磷蛋白C（4×10^(-8)和10^(-6) M）或星形孢菌素（10^(-8) M）均未影响气管对5-HT的收缩。然而，PKC抑制剂双吲哚马来酰胺（5×10^(-6) M）消除了对佛波醇12,13 - 二丁酸酯（10^(-6) M）的收缩，与钙磷蛋白C不同，在不存在和存在尼群地平的情况下均抑制了对5-HT的收缩。这一发现表明PKC激活参与了5-HT收缩。因此，气管5-HT2A受体的独特之处在于，该受体的激活不会导致PI水解，但会增加通过L型电压依赖性钙通道的钙内流、肌浆网的钙释放以及双吲哚马来酰胺敏感的PKC的激活。
