Photoactivated mechanical responses that resulted from exposure to 3-NO2-1,4-dihydropyridines (3-NO2-DHP5) or NO-donors were examined in rat isolated oesophageal smooth muscle with a view to determining the role of calcium and cyclic GMP. 2. Isometric contractile force was recorded in preparations bathed in normal Tyrode or 110 mM K(+)-depolarizing solution. Exposure to (+)-PN 202791, (+/-)-Bay K 8644 and (-)-PN 2020791 or the photodegradable NO-donors, sodium nitroprusside (SNP), streptozotocin (STZ) and sodium nitrite photosensitized precontracted tunica muscularis mucosae preparations in a concentration-dependent fashion. Photosensitizing potency followed the order: (+/-)-PN 202791 > (+/-)-Bay K 8644 > (-)-PN 202791 > SNP > STZ > NaNO2. 3. A low amplitude, slow photorelaxation (slope: 1 mg s-1) was obtained with the L-channel antagonists (-)-PN 202791 and (+)-Bay K 4407. Photosensitization by the agonist enantiomers (+)-PN 202 791 and (-)-Bay K 5407, as well as racemic Bay K 8644, was mimicked by NO donors and showed at least three different components, consisting of (i) a fast relaxation (slope: 140 mg s-1), (ii) a fast "off-contraction', and (iii) a delayed slow relaxation. The fast components, but not the delayed slow relaxation, were abolished by blockade of L-type voltage-operated calcium channels, chelation of extracellular calcium and skinning of the plasmalemma, suggesting their mediation by a process linked to calcium entry through L-channels. 4. Both cyclopiazonic acid (3-30 microM) and ryanodine (30 microM) inhibited the fast response. This inhibition was accelerated in the presence of extracellular calcium and resembled that seen in tissues exposed to the calcium ionophore A 23187 (1 microM). In calcium depleted tissues, cyclopiazonic acid (3 microM) prevented restoration of the cis-dioxolane-induced contraction following re-exposure to a calcium containing high K+ buffer, but failed to inhibit the photoresponse. 5. Both the fast and slow relaxations were potentiated by zaprinast (10 microM) and inhibited by LY B3583 (10 microM). However, in calcium-depleted, calyculin A-precontracted preparations only the slow relaxation was evident. 6. The present results support the conclusion that: (i) functional L-channels are required for the expression of the fast components of the 3-NO2-DHP- or NO-donor-induced photoresponse, (ii) NO photorelease followed by activation of soluble guanylyl cyclase is responsible for the photosensitizing activity of 3-NO2-DHPs and (iii) regulation of the contractile proteins via cyclic GMP-dependent phosphorylation may underlie the slow relaxation.
摘要
为了确定钙和环鸟苷酸（cGMP）的作用，我们在大鼠离体食管平滑肌中检测了暴露于3 - 硝基 - 1,4 - 二氢吡啶（3 - NO₂ - DHP5）或一氧化氮供体后产生的光激活机械反应。2. 在浸泡于正常台氏液或110 mM K⁺去极化溶液的标本中记录等长收缩力。暴露于（+） - PN 202791、（±） - Bay K 8644和（ - ） - PN 2020791或可光降解的一氧化氮供体硝普钠（SNP）、链脲佐菌素（STZ）和亚硝酸钠后，预先收缩的黏膜肌层标本出现浓度依赖性的光致敏现象。光致敏效力顺序为：（±） - PN 202791 ＞（±） - Bay K 8644 ＞（ - ） - PN 202791 ＞ SNP ＞ STZ ＞ NaNO₂。3. L型通道拮抗剂（ - ） - PN 202791和（+） - Bay K 4407可产生低幅度、缓慢的光舒张反应（斜率：1 mg s⁻¹）。激动剂对映体（+） - PN 202 791和（ - ） - Bay K 5407以及消旋体Bay K 8644的光致敏作用可被一氧化氮供体模拟，且至少表现出三种不同成分，包括（i）快速舒张（斜率：140 mg s⁻¹），（ii）快速“脱收缩”，以及（iii）延迟的缓慢舒张。快速成分，而非延迟的缓慢舒张，可通过阻断L型电压门控钙通道、螯合细胞外钙以及去除质膜而被消除，这表明它们是由与通过L型通道进入的钙相关的过程介导的。4. 环匹阿尼酸（3 - 30 μM）和ryanodine（30 μM）均抑制快速反应。在细胞外钙存在的情况下，这种抑制作用加速，且类似于在暴露于钙离子载体A 23187（1 μM）的组织中所见的情况。在缺钙组织中，环匹阿尼酸（3 μM）可阻止重新暴露于含钙高钾缓冲液后顺式二氧戊环诱导的收缩恢复，但未能抑制光反应。5. 快速和缓慢舒张均被扎普司特（10 μM）增强，并被LY B3583（10 μM）抑制。然而，在缺钙、calyculin A预先收缩的标本中，仅缓慢舒张明显。6. 目前的结果支持以下结论：（i）功能性L型通道是3 - NO₂ - DHP或一氧化氮供体诱导的光反应快速成分表达所必需的；（ii）一氧化氮光释放后激活可溶性鸟苷酸环化酶是3 - NO₂ - DHP光致敏活性的原因；（iii）通过环鸟苷酸依赖性磷酸化对收缩蛋白的调节可能是缓慢舒张的基础。
