[Method for isolating single cardiac cells in the guinea pig using pronase. The role of calcium ions]. / Metod vydeleniia odinochnyklh serdechnykh kletok morskoii svinki s pomoshch'iu pronazy. Rol' ionov kal'tsiia.

A method for isolation of guinea-pig cardiomyocytes with pronase has been developed. The method has been assessed in hearts perfused with solutions containing pronase (1 U/ml) and 200 microM Ca2+. Eighty per cent of the cells released were rod-shaped and 1.2 mM Ca2+ tolerant. Enriched medium 199 was used for all solutions. Sodium and slow inward currents recorded from cells dispersed with pronase were similar to those recorded from cells isolated after prolonged exposure to collagenase. Two principal factors are to be marked: (a) presence of high enough amounts of Ca2+ in enzyme solution (up to 200 microM); (b) use of the enriched medium in all the stages of the procedure.

Patch-voltage-clamp method for measuring fast inward current in single rat heart muscle cells.

A patch-voltage-clamp method was used to measure fast inward ionic currents in single heart muscle cells. 1. Theoretical analysis including computer simulation has shown that the method provides fast settling of membrane potential (within 10 microseconds) and reliable voltage clamp on the tested membrane patch when the area of the patch is 200-300 times smaller than the area of the whole cell membrane. 2. The transient time at the I-V converter output is increased up to 70-75 microseconds due to the stray capacity in the I-V converter feedback. When fast-response operational amplifiers are used in the set up this transient time may be decreased to 30 microseconds by partial restoration of the high-frequency components of the signal. 3. Experimental data have shown that the ionic channel population in the membrane patch of about 5 micrometers in diameter is on the one hand large enough to directly observe integral ionic current and on the other hand small enough for the fluctuations of ionic current to be appreciable. This permits the method to be applied to ionic current investigations both by classical methods and by statistical analysis.

["Micro-lead" technic for recording ion currents through the membranes of individual myocardial cells]. / Metod "mikrootvedeniia" dlia registratsii ionnykh tokov cherez membranu odinochnykh serdechnykh kletok.

Patch-voltage-clamp method has been used for registration of the fast inward current in single heart muscle cells. Ionic current were measured on a small patch of membrane which is of about 1/200-1/300 of the total cell surface (diameter of patch is of about 5 micrometer). To correct the distortions of raw signal caused by the stray capacity in feedback of current-voltage converter and adjustable increasing of high-frequency gain was used. This allows one to shorten the net time of transient process in response to switching the command potential up to 30 microseconds. The channel population in membrance patch of micrometer in diameter is, on the one hand, large enough to record directly the integral ionic current and is, on the other hand, small enough for fluctuations of ionic current to be pronounced. This circumstance permits the method to be applied for ionic current investigations both by the classical methods and by statistical analysis.