Cyclic nucleotides regulate the activity of L-type calcium channels in smooth muscle cells from rat portal vein.
Cyclic nucleotides regulate the activity of L-type calcium channels in smooth muscle cells from rat portal vein.
复制标题
环核苷酸调节大鼠门静脉平滑肌细胞中 L 型钙通道的活性。
DOI：
10.1006/jmcc.1997.0379
复制
发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Sperelakis,N
中科院分区：
文献类型：
--
作者：
Liu,H;Xiong,Z;Sperelakis,N
为了进一步证实环核苷酸对血管平滑肌细胞宏观慢钙电流的调节作用，采用细胞贴附式膜片钳技术（22-25°C）研究了cAMP和cGMP对慢（I型）钙通道活性的影响。新鲜分离的VSM细胞获自成年雄性大鼠门静脉。对于单通道记录，移液器中加入含有90 mmBa 2+和1μmBay-K-8644溶液的溶液，浴槽中含有140 mmKCl，使膜电位“归零”。从-80 mV的保持电位（HP）到0 mV的去极化脉冲引起内向单一电流。灌流10μmnifedipine可完全阻断上述通道的活性。细胞外灌注的单细胞与膜可渗透的cGMP和cAMP类似物（8Br-cGMP和8Br-cAMP）在1毫米引起轻微的抑制，但更高的剂量（3毫米），清楚地显示出抑制作用的单通道活动。cAMP（100μm）刺激1/5的受试斑片，而100μmcGMP对3个受试斑片无作用。与对照组相比，两种环核苷酸在3 mm处分别降低了26.7±4.1%和37.3± 2.1%。单位振幅和斜率电导无变化。正常Ca ~（2+）通道的电导为20.8± 0.04pS（n=9），cAMP（n=5）和cGMP（n=6）存在时的电导分别为19.3±0.04和20.5± 0.05pS。单通道动力学分析表明cAMP不影响平均开放时间，cGMP使平均开放时间略有缩短。而cAMP和cGMP均使平均关闭时间延长。cAMP使开放概率（NPo）降低1.7倍，从0.26±0.04降至0.15±0.03（P<0.05），cGMP使NPo降低2.5倍，从0.24±0.08降至0.10±0.02（P<0.05）。非特异性蛋白激酶抑制剂H-7可阻断cAMP和cGMP对大鼠门静脉细胞单个Ca ~（2+）通道活性的抑制作用。结果表明，大鼠门静脉平滑肌细胞cAMP和cGMP均具有钙通道活性。这种抑制作用可能是通过β 1型钙通道的cAMP和cGMP依赖性蛋白激酶磷酸化（或相关的调节蛋白）介导的。
In order to advance our previous findings that the macroscopic slow Ca2+currents of vascular smooth muscle (VSM) cells are regulated by cyclic nucleotides, the effects of cAMP and cGMP on the activity of single slow (l-type) Ca2+channels were investigated using cell-attached patch clamp (22–25°C). Freshly isolated VSM cells were obtained from adult male rat portal vein. For the single-channel recordings, the pipette was filled with a solution containing 90 mmBa2+and 1μmBay-K-8644 solution, and the bath contained 140 mmKCl to “zero” the membrane potential. Depolarizing pulses to 0 mV, from a holding potential (HP) of −80 mV, elicited inward unitary currents. The activity of these channels was completely blocked by superfusion of 10μmnifedipine. Extracellular perfusion of the single cells with membrane-permeable cGMP and cAMP analogs (8Br-cGMP and 8Br-cAMP) at 1 mmcaused a slight inhibition, but higher doses (3 mm), clearly showed an inhibitory effect on the single-channel activity. cAMP (100μm) stimulated one out of five patches tested, and 100μmcGMP showed no effect in three patches tested. Compared with control, both cyclic nucleotides at 3 mmdecreased the ensemble-averaged currents by 26.7±4.1% and 37.3±2.1%, respectively. Unit amplitude and slope conductance were not changed. The normal conductance of the Ca2+channel was 20.8±0.04 pS (n=9), and the conductances in the presence of cAMP (n=5) and cGMP (n=6) were 19.3±0.04 and 20.5±0.05 pS, respectively. Single-channel kinetic analysis showed that cAMP did not affect the mean open-time, and cGMP slightly decreased the mean open-time. However, both cAMP and cGMP increased the mean closed-time. In addition, cAMP decreased the open probability (NPo) by a factor of 1.7, from 0.26±0.04 to 0.15±0.03 (P<0.05, Student'st-test) and cGMP decreased NPoby a factor of 2.5, from 0.24±0.08 to 0.10±0.02 (P<0.05). H-7, a non-specific protein kinase inhibitor, prevented the inhibitory effects of both cAMP and cGMP on the activity of single Ca2+channels in rat portal vein cells. The results demonstrate that both cAMP and cGMP inhibitl-type Ca2+channel activities in VSM cells from rat portal vein. This inhibition may be mediated by the cAMP and cGMP-dependent protein kinase phosphorylation of thel-type Ca2+channels (or an associated regulatory protein).