Regulation of cell-volume and intracellular osmolarity by chloride channels in cardiac cells

1.用显微视频图像分析技术研究了氯电流对豚鼠心肌细胞体积的调节作用。在正常[K]_0下加低渗液可引起细胞肿胀，肿胀的细胞出现轻微但明显的自发性细胞萎缩，表明调节性体积减小(RVD)的机制在低[Cl]_o时发生，在低[Cl]_o时E_&lt；CL&gt；远高于E_m。E_m为阴性时，细胞单调肿胀，未见RVD，肿胀明显大于正常[K]_0。正常[K]_0时的RVD和高[K]_0时的‘额外’细胞膨胀均被I&lt；Cl、Swell&Gt；这些发现表明，细胞膨胀被激活了。I_lt；Cl，Swell&>；和激活I_&lt；Cl，Swell&>；诱发RVD或额外的细胞膨胀，依赖…2.采用全细胞膜片钳方法，观察细胞外三磷酸腺苷对[β-肾上腺素能激活CFTRCl电流(I&lt；Cl，PKa&gt；)的影响。细胞首先暴露于异丙肾上腺素(ISO)中~3min，以激活I_1Cl、PKA和Gt；，然后在ISO存在的情况下暴露于1~100μM ATP中。在大多数被检测的细胞中，ATP能增强I_1，pKa和gt；。在50μM的μM作用下，这种增强作用可使单用ISO激活的Cl~-电导平均增加1.3倍(0.02~(-1)AMP M)。腺苷二磷酸和三磷酸腺苷γS的作用与三磷酸腺苷相似，腺苷对三磷酸腺苷和腺苷均无增强作用。因此，这种增强归因于对P2-嘌呤受体的刺激。PdBu(0.5μM)是PKC的激活剂，它能促进I_1，K_(A)；当有PKC抑制剂Bim(0.2ATP M)存在时，μ不能促进I_1、PKA和Gt；这些发现提示PKC参与了观察到的ATP活动。当在ISO存在的情况下去除ATP时，增强的I_lt；Cl，pKa和gt；仅缓慢地降低(恢复)，并且，如果在这个缓慢的恢复阶段重新施加ATP，则随后的电流增强很弱。因此，三磷酸腺苷对P2嘌呤受体的刺激通过激活蛋白激酶C来促进β-肾上腺素能激活i_1，pKa和gt；，并且在去除三磷酸腺苷后这一电位似乎持续了几分钟。较少

1.The cell-volume regulation by chloride currents was studied in guinea-pig cardiac myocytes, using a microscopic video-image analysis. Application ofhypotonic solution to the cells at normal [K]_0 induced a cell swelling, and the so-swollen cells showed a slight but clear spontaneous cell shrinkage, indicating operation of the mechanism of regulatory volume decrease (RVD)._This RVD could be pronounced at low [Cl]_o, at which E_<Cl> was far more positive than E_m. On the contrary, when the hypotonic solution was applied to the cells at high [K]_o, at which E_<Cl> was negative to E_m, the cells swelled monotonically without showing RVD, the swelling being much greater than that seen at normal [K]_0. Both the RVD at normal [K]_0 and the 'extra' cell inflation at high [K]_0 were suppressed by the inhibitors of I_<Cl, swell>. These findings suggest that the cell inflation activated. I_<Cl, swell>, and that the activation of I_<Cl, swell> induced the RVD or the extra cell inflation, dependi … More ng on the direction and magnitude of the driving force of Cl ions across the membrane.2.The effects of extracellular ATP on [β-adrenergic activation of CFTR Cl current (I_<Cl, PKA>) were examined with the whole-cell patch clamp method. The cells were initially exposed to isoproterenol (ISO) for 〜3 min to activate I_<Cl, PKA>, and then to 1-100 μM ATP in the presence of ISO. ATP was found to potentiate I_<Cl, PKA>, in most cells examined. With 50 μM ATP, the potentiation, on average, resulted in a 1.3 fold increase of the Cl^-conductance activated by ISO alone (0.02-1μM). The effects of ADP and ATPγS on I_<Cl, PKA> were similar to those of ATP, while AMP and adenosine never potentiated I_<Cl, PKA>. Thus the potentiation was attributed, to a stimulation of P2-purinoceptors. PDBu (0.5μM), an activator of PKC, facilitated I_<Cl, PKA>, and in the presence of PDBu ATP did not further potentiate I_<Cl, PKA>. When BIM (0.2μM), an inhibitor of PKC, was present, ATP did not facilitate I_<Cl, PKA>. These findings suggested involvement of PKC in the observed ATP action. When ATP was removed in the presence of ISO, the potentiated I_<Cl, PKA> decreased (recovered) only slowly, and, if ATP was reapplied during this slowly recovering phase, the subsequent current potentiation was weak. Thus the stimulation of P2 purinoceptors by ATP facilitates the β-adrenergic activation of I_<Cl, PKA> through PKC activation, and this potential appears to persist for several min after removal of ATP. Less