[Use of isolated myocytes and the patch clamp technic in pharmacological studies]

Effects of palmitylcarnitine, a toxic metabolite of ischaemia, on the electrophysiological properties of single ventricular myocytes of guinea pig were studied by means of patch clamp technique. Cells were enzymatically isolated by perfusing the heart with Ca-free Tyrode solution containing collagenase enzyme. Before experimentation, cells were allowed to recover in a regenerating solution for at least one hour at room temperature. An aliquot of the cell suspension was placed in a small (0.5 ml volume) chamber mounted on the stage of an inverted microscope. After a settling period of 10 minutes, the cells were superfused with Tyrode solution at a rate of 2 to 4 ml/min. Bath temperature was maintained at 35 degrees C. Patch electrodes were fabricated from glass capillary tubes. The electrodes having resistances of 2 to 4 M were connected to a LIST EPC-7 voltage clamp amplifier. The passive parameters (membrane resistance, time constant, capacitance), and resting and action potentials of the cells were recorded in current clamp mode, while the ionic currents (inward calcium, outward potassium) were recorded in voltage clamp mode. Palmitylcarnitine at 10(-6) M concentration increased the membrane resistance, time constant, and depolarized the cell membrane which was accompanied by a marked prolongation of the action potential duration. Under the effect of the drug, the outward potassium current responsible for the repolarization was decreased, while the inward calcium current responsible for the plateau phase was significantly increased mainly at positive membrane potentials. The steady-state activation and inactivation curves were shifted to less negative potentials. The results indicate that the changes in the membrane electrical properties play an important role in the development of the well-known arrhythmogenic effect of palmitylcarnitine.