Ding Yanfeng, Schwartz Dean, Posner Philip, Zhong Juming
Dept. of Anatomy, Physiology, and Pharmacology, Auburn University College of Veterinary Medicine, Auburn, AL 36849, USA.
Am J Physiol Cell Physiol. 2004 Aug;287(2):C413-21. doi: 10.1152/ajpcell.00537.2003. Epub 2004 Apr 7.
It has been suggested that L-type Ca(2+) channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca(2+) channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca(2+) channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba(2+) currents (I(Ba)) through L-type Ca(2+) channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca(2+) channel activity but did not alter the voltage-dependent characteristics of Ca(2+) channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-31-8425 or Go-6983, prevented I(Ba) enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased I(Ba) under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on I(Ba) when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca(2+) channel activity in rabbit portal vein smooth muscle cells through activation of PKC.
有人提出，L型钙通道在细胞肿胀诱导的血管收缩中起重要作用。然而，目前尚无直接证据表明血管平滑肌中的钙通道受细胞肿胀调节。我们检验了这样一个假设，即兔门静脉肌细胞中的L型钙通道通过蛋白激酶激活受低渗性细胞肿胀调节。采用传统的全细胞膜片钳技术，在从兔门静脉新鲜分离的平滑肌细胞中记录通过L型钙通道的钡电流（I(Ba)）。用低渗溶液灌流细胞可使钙通道活性可逆性增强，但不改变钙通道的电压依赖性特征。浴槽中加入蛋白激酶C（PKC）的选择性抑制剂Ro-31-8425或Go-6983，可阻止低渗肿胀引起的I(Ba)增强，而特异性蛋白激酶A（PKA）抑制剂KT-5720则无此作用。浴槽中加入佛波醇12,13-二丁酸酯（PDBu）在等渗条件下可显著增加I(Ba)，并阻止低渗肿胀引起的电流刺激。然而，当细胞首先暴露于低渗溶液时，PDBu对I(Ba)没有任何影响。此外，用PDBu对细胞进行过夜处理使内源性PKC下调，可阻止低渗肿胀引起的电流增强。这些数据表明，低渗性细胞肿胀可通过激活PKC增强兔门静脉平滑肌细胞中的钙通道活性。
