Hiroishi G, Kobayashi S, Nishimura J, Inomata H, Kanaide H
Division of Molecular Cardiology, Faculty of Medicine, Kyushu University, Fukuoha, Japan.
Invest Ophthalmol Vis Sci. 1996 Dec;37(13):2612-23.
To determine the mechanisms of inhibition by diltiazem (Dil) and nitroglycerin (NG) of the contraction induced by serotonin (5-HT) in the ophthalmic artery.
Using front-surface fluorometry of fura-2 and the medial strips of the bovine ophthalmic artery, [Ca2+]i and force were monitored simultaneously. Changes in the force at a constant [Ca2+]i were determined by use of receptor-coupled membrane permeabilization with alpha-toxin.
In the presence of extracellular Ca2+, 5-HT (10(-5) M) induced an initial transient and subsequently lower steady state elevation of [Ca2+]i. The transient elevation of [Ca2+]i was dependent on both intracellular and extracellular [Ca2+]i, whereas the steady state elevation was dependent on only extracellular Ca2+. For a given level of elevation of [Ca2+]i, 5-HT produced a greater force than the depolarization with high external K+ (118 mM) solution. In the permeabilized ophthalmic artery smooth muscle, 5-HT enhanced the contractile response to constant cytosolic Ca2+ (pCa 6.5) in the presence of guanosine triphosphate (GTP, 10 microM), but not in its absence. Therefore, 5-HT induces [Ca2+]i elevation, depending on both extracellular (Ca2+ influx) and intracellular Ca2+ (Ca2+ release), and it potentiates the Ca2+ sensitivity of the contractile apparatus through the activation of G-proteins. 5-HT-induced release of Ca2+ from the store was inhibited by NG, but not by Dil, in a concentration-dependent manner. However, neither NG nor Dil inhibited caffeine (20 mM)-induced release of Ca2+ from the store. Dil (10 microM) and NG (10 microM) inhibited in a concentration-dependent manner the steady state elevations of [Ca2+]i (Ca2+ influx) and force induced by 5-HT (10 microM) in the presence of extracellular Ca2+. Dil equally inhibited the steady state elevations of [Ca2+]i and force induced by 5-HT, whereas NG inhibited the force to a greater extent than expected from the reduction in [Ca2+]i. In the permeabilized ophthalmic artery smooth muscle, NG (10 microM), but not Dil (10 microM), decreased the force development induced by GTP (10 microM) and 5-HT (10 microM) at constant [Ca2+]i (pCa 6.5). These results indicate that NG, but not Dil, decreases the Ca2+ sensitivity of contractile apparatus.
The authors found that 5-HT contracts the ophthalmic artery smooth muscle by the elevation of [Ca2+]i mediated by the release of intracellular Ca2+ and the influx of extracellular Ca2+, as well as by an increase in the Ca2+ sensitivity of the contractile apparatus through the activation of G-proteins, and that Dil relaxes 5-HT-mediated contraction of ophthalmic artery primarily by inhibiting the Ca2+ influx and, hence, by decreasing [Ca2+]i without having any effect on the Ca2+ sensitivity of the contractile apparatus. Nitroglycerin relaxes the ophthalmic artery not only by decreasing [Ca2+]i (inhibition of both the Ca2+ release and Ca2+ influx) but also by decreasing the Ca2+ sensitivity of the contractile apparatus.
确定地尔硫䓬（Dil）和硝酸甘油（NG）对血清素（5-HT）诱导的眼动脉收缩的抑制机制。
使用fura-2的表面荧光测定法和牛眼动脉的内侧条带，同时监测细胞内钙离子浓度（[Ca2+]i）和张力。通过使用α-毒素进行受体偶联的膜通透化来确定在恒定[Ca2+]i下张力的变化。
在细胞外钙离子存在的情况下，5-HT（10^(-5) M）诱导[Ca2+]i最初出现短暂升高，随后是较低的稳态升高。[Ca2+]i的短暂升高依赖于细胞内和细胞外的[Ca2+]i，而稳态升高仅依赖于细胞外钙离子。对于给定的[Ca2+]i升高水平，5-HT产生的张力比用高浓度外部钾离子（118 mM）溶液去极化产生的张力更大。在通透化的眼动脉平滑肌中，在存在三磷酸鸟苷（GTP，10 μM）的情况下，5-HT增强了对恒定胞质钙离子（pCa 6.5）的收缩反应，但在不存在GTP时则没有增强。因此，5-HT诱导[Ca2+]i升高，这依赖于细胞外（钙离子内流）和细胞内钙离子（钙离子释放），并且它通过激活G蛋白增强收缩装置对钙离子的敏感性。5-HT诱导的钙离子从储存库的释放受到NG的浓度依赖性抑制，但不受Dil的抑制。然而，NG和Dil均未抑制咖啡因（20 mM）诱导的钙离子从储存库的释放。Dil（10 μM）和NG（10 μM）在细胞外钙离子存在的情况下，以浓度依赖性方式抑制5-HT（10 μM）诱导的[Ca2+]i稳态升高（钙离子内流）和张力。Dil同样抑制5-HT诱导的[Ca2+]i和张力的稳态升高，而NG对张力的抑制程度大于根据[Ca2+]i降低所预期的程度。在通透化的眼动脉平滑肌中，NG（10 μM）而非Dil（10 μM）在恒定[Ca2+]i（pCa 6.5）下降低了GTP（10 μM）和5-HT（10 μM）诱导的张力发展。这些结果表明，NG而非Dil降低了收缩装置对钙离子的敏感性。
作者发现5-HT通过细胞内钙离子释放和细胞外钙离子内流介导的[Ca2+]i升高以及通过激活G蛋白增加收缩装置对钙离子的敏感性来使眼动脉平滑肌收缩，并且Dil主要通过抑制钙离子内流从而降低[Ca2+]i来松弛5-HT介导的眼动脉收缩，而对收缩装置对钙离子的敏感性没有任何影响。硝酸甘油不仅通过降低[Ca2+]i（抑制钙离子释放和钙离子内流）来松弛眼动脉，还通过降低收缩装置对钙离子的敏感性来实现。
