The Regulation And Molecular Identification Of Swelling-activated Chloride Currents In Human Atrial Myocytes

Objective:the swelling-activated chloride current(ICl.swell) may play an important role in cardiac electrophysiology and arrhythmogenesis,but the regulation and molecular identification of the current are not clearly understood. In order to clarify the obscurity, we focused on phenylephrine, anα1-adrenoceptor agonists, and PDBu, a PKC activator, in the regulation of ICl.swell in human atrial myocytes by a whole-cell patch-clamp technique. Furthermore, we investigated the characteristic and correlativity of the expression of CLC-3 in human right atrial tissue via reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry.Method: patch-clamp technique: Atrial myocytes were isolated from specimens of right atrial tissue obtained from patients undergoing coronary artery bypass. Atrial myocytes were enzymatically dissociated. Whole-cell currents were recorded by a whole-cell patch-clamp technique. ICl.swell was obtained by perfusion with hyposmotic bath solution ( 0.6-times isosmotic, 0.6T ). All drug effects were assessed after a superfusion interval long enough to achieve steady state effects.RT-PCR and immunohistochemistry: Obtaining the right atrial tissues from 33 patients undergoing cardiac surgery, Detecting the m-RNA expression of CLC-3 via RT-PCR, Semiquantitating the expression of CLC-3 m-RNA by the ratio of the CLC-3'IOD values/IOD for theβ-actin, Establishing the multiple linear regression equation(dependent variable: IOD CLC-3/IODβ-actin, independent variables: gender, age, atrial fibrillation or not, transverse diameter of right atrium) by using the least squares method, ANOVA (analysis of variance) testing the models, Detecting the expression and the location of CLC-3 via immunohistochemistry.Result: Phenylephrine could enhance ICl.swell which was distinct from the reports in other species, and the effect was concentration-dependent (EC50=9.8uM at +40mV), and reversed by 150uM DIDS or after exposure to the isotonic solution (1.0T ). Phenylephrine (100uM) still enhanced ICl.swell in the preliminary presence of prazosin (1uM), anα1-adrenoceptor antagonist, but the effect was attenuated. ICl.swell could also be enhanced by PDBu which distincted from the current associated with ClC-3, a most possible molecular candidate for ICl.swell.The expression of CLC-3 was deteted via RT–PCR and immunohistochemistry, and the CLC-3 located in intra-cellular. All models of the multiple linear regression equation was tested and there was no statistical significance (ANOVA, P>0.05).Conclusion: phenylephrine can enhance ICl.swell in human atrial myocytes which indicating species-dependent variation in the modulation of cardiac ICl.swell byα1-adrenoceptor agonist. To our knowledge, we report the phenomenon the first time. PDBu has the same effect as phenylephrine, which confirms the involvement of PKC in the function ofα1-adrenoceptor agonist on ICl.swell and indicats species-dependent variations in the modulation of cardiac ICl.swell by PKC. We also found that 1, CLC-3 existed in human atrial myocytes and presented intra-cellular localization. 2, there was no correlation between the m-RNA expression of CLC-3 and atrial fibrillation or transverse diameter of right atrium. 3, the enhancement of ICl.swell by PDBu distincted from the current associated with ClC-3. These indicates the possibility that ICl.swell in human atrial myocytes is not mediated by CLC-3.