The effect of transplasmalemmal Ca2+ influx on the [Ca2+]i dependence of smooth muscle contraction was evaluated by measuring intracellular [Ca2+] (as estimated by aequorin), myosin phosphorylation, and isometric stress in swine carotid media. 2. Extracellular Ca2+ was removed by incubation in physiological saline with 1 mM-EGTA and no added CaCl2 for 20 min (termed EGTA treatment). In some preparations, intracellular Ca2+ was released by a brief (5 min) histamine stimulation while in this Ca2(+)-free EGTA solution (termed histamine treatment). 3. Restoration of extracellular CaCl2 to EGTA and histamine-treated preparations in the continued presence of histamine was associated with an initial large aequorin light transient. However, this light transient was not initially associated with an increase in myosin phosphorylation or rapid stress development, suggesting that the contractile apparatus was desensitized to aequorin-estimated myoplasmic [Ca2+]. The desensitization was temporary, and resolved by 10 min after restoration of extracellular CaCl2. 4. The light transient observed upon restoration of extracellular CaCl2 was smaller in preparations only EGTA treated when compared to preparations treated with both EGTA and histamine, suggesting that histamine treatment further desensitized the contractile apparatus. 5. The stress development rate was not slowed when histamine and extracellular CaCl2 were simultaneously added to EGTA-treated preparations, suggesting that the desensitization was only to transplasmalemmal Ca2+ influx (from extracellular CaCl2 readdition), and not intracellular Ca2+ release (from the histamine stimulation). 6. In EGTA and histamine-treated preparations, restoration of extracellular CaCl2 in the presence of 109 mM-KCl was associated with a larger aequorin light signal than was observed upon readdition of CaCl2 in the presence of histamine, suggesting that depolarization also further desensitized the contractile apparatus. 7. Depolarization of EGTA-treated preparations did not increase [Ca2+] or stress, suggesting that depolarization did not release intracellular Ca2+ stores. 8. No significant light transient was observed upon addition of extracellular LaCl3, suggesting that tissue damage or leakage of aequorin into the extracellular space was not the cause of the Ca2(+)-reintroduction light signal. 9. These data suggest that removal of extracellular CaCl2 desensitizes the contractile apparatus of smooth muscle to transplasmalemmal Ca2+ influx. This desensitization is only to readdition of extracellular Ca2+; the contractile apparatus still responds to intracellular Ca2+ release. The desensitization is increased by prior depolarization or brief histamine treatment (potentially by depleting intracellular Ca2+). The source of activator Ca2+ appears to affect the relationship between aequorin light and phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
摘要
通过测量猪颈动脉中层的细胞内[Ca2+]（由水母发光蛋白估计）、肌球蛋白磷酸化和等长张力，评估跨膜Ca2+内流对平滑肌收缩的[Ca2+]i依赖性的影响。2. 通过在含1 mM - EGTA且未添加CaCl2的生理盐水中孵育20分钟（称为EGTA处理）来去除细胞外Ca2+。在一些标本中，在这种无Ca2+的EGTA溶液中通过短暂（5分钟）的组胺刺激释放细胞内Ca2+（称为组胺处理）。3. 在持续存在组胺的情况下，向EGTA和组胺处理的标本中恢复细胞外CaCl2与最初较大的水母发光蛋白光瞬变有关。然而，这种光瞬变最初与肌球蛋白磷酸化增加或快速的张力发展无关，这表明收缩装置对水母发光蛋白估计的肌浆[Ca2+]脱敏。这种脱敏是暂时的，在恢复细胞外CaCl2后10分钟消退。4. 与同时用EGTA和组胺处理的标本相比，仅用EGTA处理的标本在恢复细胞外CaCl2时观察到的光瞬变较小，这表明组胺处理进一步使收缩装置脱敏。5. 当组胺和细胞外CaCl2同时添加到EGTA处理的标本中时，张力发展速率没有减慢，这表明脱敏仅针对跨膜Ca2+内流（来自重新添加细胞外CaCl2），而不是细胞内Ca2+释放（来自组胺刺激）。6. 在EGTA和组胺处理的标本中，在109 mM - KCl存在下恢复细胞外CaCl2与比在组胺存在下重新添加CaCl2时观察到的更大的水母发光蛋白光信号相关，这表明去极化也进一步使收缩装置脱敏。7. EGTA处理的标本去极化并未增加[Ca2+]或张力，这表明去极化并未释放细胞内Ca2+储存。8. 添加细胞外LaCl3时未观察到明显的光瞬变，这表明组织损伤或水母发光蛋白泄漏到细胞外空间不是Ca2+重新引入光信号的原因。9. 这些数据表明，去除细胞外CaCl2使平滑肌的收缩装置对跨膜Ca2+内流脱敏。这种脱敏仅针对重新添加细胞外Ca2+；收缩装置仍对细胞内Ca2+释放有反应。预先的去极化或短暂的组胺处理（可能通过耗尽细胞内Ca2+）会增加脱敏。激活剂Ca2+的来源似乎影响水母发光蛋白光与磷酸化之间的关系。（摘要截断于400字）
