The mechanism underlying the vasoconstriction induced by endothelin-1 (ET-1) was investigated by measuring the intracellular concentration of Ca2+ ([Ca2+]i), isometric force and phosphorylation of the myosin light chain (MLC) in endothelium-denuded unskinned and beta-escin-treated skinned smooth muscle from resistance vessels of the rabbit mesentery. The role of protein kinase C (PKC) in the action of ET-1 was studied in skinned smooth muscle using a synthetic peptide, PKC19-36, which corresponds to the autoinhibitory domain of PKC. 2. ET-1 (> 0.1 nM) induced slowly developing, maintained increases in [Ca2+]i and force. Nicardipine completely blocked the ET-1-induced increase in [Ca2+]i. BQ-123 (an inhibitor of the ETA receptor) blocked the ET-1-induced contraction but IRL 1620 (Suc-[Glu9,Ala11,15]-ET-1(8-21), an agonist of the ETB receptor) failed to induce contraction. 3. In ionomycin- and 70 mM K(+)-treated strips, ET-1 shifted the [Ca2+]i-force relationship to the left and enhanced the maximum amplitude of contraction induced by 2.6 mM Ca2+. In skinned smooth muscle treated with ionomycin, Ca2+ (0.1-3 microM) increased both force and MLC phosphorylation, in a concentration-dependent manner. ET-1 with GTP shifted both the Ca(2+)-force and Ca(2+)-MLC phosphorylation relationships to the left without significant changes in the maximum responses. ET-1 with GTP did not change the relationship between force and MLC phosphorylation. Similar effects were observed with phorbol 12,13-dibutyrate (PDBu, an activator of PKC). These results indicate that the sensitivity of MLC phosphorylation to Ca2+ is enhanced both by ET-1 with GTP and by PDBu. 4. PKC19-36 (an inhibitor of PKC) modified neither the contraction nor MLC phosphorylation induced by 0.3 microM Ca2+ but blocked the PDBu-induced enhancement of both these Ca(2+)-induced responses. However, PKC19-36 only partly inhibited the enhancement produced by ET-1 with GTP on the Ca(2+)-induced responses. PKC19-36 did not modify the relationship between force and MLC phosphorylation in the presence either of ET-1 with GTP or of PDBu. By contrast, BQ-123, neomycin and guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) each abolished the ET-1-induced enhancement of the contraction induced by 0.3 microM Ca2+. 5. These results suggest that ET-1 acts on the ETA receptor and increases Ca2+ influxes through an activation of the dihydropyridine-sensitive Ca2+ channel, causing a long-lasting and maintained contraction in resistance vessels of the rabbit mesentery.(ABSTRACT TRUNCATED AT 400 WORDS)
摘要
通过测量兔肠系膜阻力血管内皮剥脱的未剥皮及β-七叶皂苷处理的剥皮平滑肌中细胞内钙离子浓度（[Ca2+]i）、等长收缩力和肌球蛋白轻链（MLC）磷酸化水平，研究了内皮素-1（ET-1）诱导血管收缩的机制。使用对应PKC自身抑制结构域的合成肽PKC19-36，在剥皮平滑肌中研究了蛋白激酶C（PKC）在ET-1作用中的角色。2. ET-1（>0.1 nM）诱导[Ca2+]i和收缩力缓慢增加并持续维持。尼卡地平完全阻断ET-1诱导的[Ca2+]i升高。BQ-123（ETA受体抑制剂）阻断ET-1诱导的收缩，但IRL 1620（Suc-[Glu9,Ala11,15]-ET-1(8-21)，ETB受体激动剂）未能诱导收缩。3. 在离子霉素和70 mM K(+)处理的条带中，ET-1使[Ca2+]i-收缩力关系向左移动，并增强了2.6 mM Ca2+诱导的收缩最大幅度。在用离子霉素处理的剥皮平滑肌中，Ca2+（0.1 - 3 microM）以浓度依赖方式增加收缩力和MLC磷酸化。ET-1与GTP一起使Ca(2+)-收缩力和Ca(2+)-MLC磷酸化关系均向左移动，最大反应无显著变化。ET-1与GTP一起未改变收缩力与MLC磷酸化之间的关系。佛波醇12,13-二丁酸酯（PDBu，PKC激活剂）也观察到类似效应。这些结果表明，ET-1与GTP一起以及PDBu均增强了MLC磷酸化对Ca2+的敏感性。4. PKC19-36（PKC抑制剂）既未改变0.3 microM Ca2+诱导的收缩，也未改变MLC磷酸化，但阻断了PDBu诱导的这两种Ca(2+)-诱导反应的增强。然而，PKC19-36仅部分抑制ET-1与GTP一起对Ca(2+)-诱导反应的增强作用。在存在ET-1与GTP一起或PDBu的情况下，PKC19-36均未改变收缩力与MLC磷酸化之间的关系。相比之下，BQ-123、新霉素和鸟苷5'-O-(2-硫代二磷酸)（GDPβS）均消除了ET-1诱导的0.3 microM Ca2+诱导收缩的增强作用。5. 这些结果表明，ET-1作用于ETA受体，并通过激活二氢吡啶敏感的Ca2+通道增加Ca2+内流，导致兔肠系膜阻力血管产生持久且维持的收缩。（摘要截断于400字）
