| Cl" channels, extensively existing in mammalian cells, participate in a wide variety of cell functions, including regulation of electrical activity, pH, volume, and may even play a role in cell proliferation, differentiation and apoptosis.Since the identification of Cl- channel in the heart in 1989, functional and molecular studies suggest that three primary types of sarcolemmal anion channels are expressed in cardiac cells: protein kinase A-activated Cl- channel, Ca2+-activated Cl- channel and volume-sensitive Cl" channel. These Cl" channels play some roles in cardiac electrical activities. Our preliminary study suggested that Cl" channel may participate in the regulation of cardiac L-type Ca2+ current. In the present study we investigated further the possibility of regulation of cardiac Na+ channel by Cl- channel.1. Materials and methods(1) Perfusion of isolated hearts and electrocardiographic recordings Sprague-Dawley rats (200~250g) were killed by decapitation,and the hearts were immediately removed and cannulated via the aorta. Retrograde perfusion was performed at 37℃ in a Langendorff apparatus under constant pressure (80 mmHg). Measurement of left ventricular pressure (LVP) was conducted by a water-filled balloon inserted through the mitral valve and secured in the left ventricle. The balloon was connected to a pressure transducer and a physiological recorder to monitor LVP. ECG was recorded via electrodes placed on the apical region of the heart and the aorta. Heart rate (HR) was calculated from the ECG traces.(2) Whole-cell patch-clamp experiments: Whole-cell INa was elicited from a holding potential of -90mV to test potentials ranging from -90 to +30 mV in 10 mV increments. Whole-cell patch-clamp experiments were performed at room temperature (23~25 ℃).2. Results(1) Effects of glutamate- or gluconate-substitution for Cl- and anion channel blockers on heart rate and LVPReplacement of extracellular Cl- with glutamate or gluconate significantly decreased the heart rate by 15.9±5.3% and 7.2±2.1%, and suppressed LVP by 79± 6% and 87±6%, respectively. The same effects were observed by using putative Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid (NFA).(2) Effects of anion channel blockers and union-substitution for Cl- on INaNFA (100 μM) inhibited INa by 65.6 ℃15.6% at -30 mV after administion for 2 min of NFA. Replacement of 95 mM extracellular Cl- by equimolar glutamate reduced the INa by 66.5 ℃ 9.3% at -30 mV. In contrast, Br-, a permeant anion, only induced a slight inhibition of INa (INa was decreased by 23℃9.8%). These results indicate that activities of Na+ channels depend on the permeability of the anions.(3) Effects ofhypotonic stress on cardiac INaExposure of the cells to hypotonic solution (220 mOsm/kg H2O) enhanced INa by 15.1 ℃ 5.3% (n=5) compared with the currents elicited in isotonic condition (300 mOsm/kg H2O).(4) Effects of anion channel blockers on aconitine-induced arrhythmiasAconitine, a Na+ channel agonist that inhibits the inactivation of the channels, is known to induce arrhythmias. 0.1 μ M aconitine produced different ventricular arrhythmias with a latent period of 15-30 s (n=10). Aconitine-induced arrhythmias did not disappear even 15 min after washout of the reagent Aconitine-induced arrhythmias included ventricular premature beats, bigeminy, tachycardia, paroxysmal ventricular tachyarrhythmias (VT). After aconitine-induced arrhythmias appeared, NFA (n=5) added to the both solutions, and caused a sudden termination of aconitine-induced arrhythmias within 1 min. These data indicate that anion channel inhibition prevents the arrhythmias induced by abnormal activities ofNa+ channels.3. DiscussionIn the study of cardiac ion channels, there are some phenomena which are difficult to explain: (1) The non-specific effects of Cl-channel blockers on other channels; (2) The cross-inhibiting effects of distinct channel blockers, for an example, th... |
PDF Full Text Request