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Conflicting results have been reported in literature about the influence of beta-adrenergic stimulation on the fast cardiac sodium current (INa+). To elucidate these mechanisms in multicellular preparations we used the loose-patch-clamp technique to evaluate the effect of the beta-adrenergic agonist isoproterenol 1-1000 nmol/l. Isoproterenol enhanced INa+ at all membrane potentials by elevation of the maximal available INa+ . Only at the high concentration of 1 micromol/l was INa+ slightly depressed after depolarizing conditioning clamps. The most marked increase of the maximal available INa+ was 30+/-9% after application of 100 nmol/l isoproterenol. To learn about the mechanisms in view of sodium channel modulation we combined isoproterenol with the sodium channel blocker lidocaine (47 micromol/l). Under these circumstances the effects of both drugs were completely independent. This investigation shows clearly that low concentrations of isoproterenol increase INa+ in multicellular preparations by a gating-independent mechanism.

We measured macroscopic sodium currents (INa) in preparations from adult rat ventricle under four different conditions (I-IV): using the cell attached configuration of the tight-seal patch clamp technique on cells isolated with either trypsin followed by collagenase (I) or with collagenase only (II), and using the loose patch technique on cells isolated with collagenase (II) as well as on multicellular preparations not subjected to enzyme treatment (IV). The voltage dependence of the steady-state activation of INa as well as of the steady-state inactivation differed significantly among condition I and II. Moreover, the recordings were voltage shifted in comparison to the recording condition III and IV. The potentials of half maximal activation and inactivation were: [sequence data: see text] The shift of inactivation was time dependent and continued after 3-5 min after the seal formation in condition I, but not in condition II. No time dependent shift was found in III and IV. We conclude, that the voltage dependence of cardiac sodium current is shifted by gigaseal patch recording. The degree of this shift depends on the type of enzymatic isolation procedure, with trypsin causing more pronounced effects than collagenase. The cell isolation itself seems not to interfere with the voltage dependence of INa, since loose patch recordings from multicellular preparations and from single cells isolated with collagenase show no obvious differences.

The loose-patch-clamp technique was used on intact cardiac papillary muscle of the rat to examine whether the fast sodium inward current (INa+) is influenced by the beta-adrenergic stimulant isoproterenol (ISO) or by 8-bromo-3',5'-cyclic adenosine monophosphate (8-Br-cAMP), respectively. The amplitude of INa+ evoked by test pulses of 5 ms to a transmembrane potential of 0 mV and its time to peak were analyzed. The availability of INa+ was tested with conditioning pulses of 2.5 s to potentials between -130 mV and -50 mV. The potential of half-maximal availability was slightly shifted to more negative values by 1 microM ISO (2.0 mV, n.s.), as well as by 50 microM 8-Br-cAMP (4.0 mV; p less than 0.05). The peak amplitude of INa+ elicited from strongly negative potentials was increased by ISO (18%, n.s.), while 8-Br-cAMP exerted no directional effect. Depolarizing conditioning pulses (-60 mV) decreased INa+ to 13.3% of the maximal attainable current under control conditions, while ISO decreased INa+ to 9.1% of control (p less than 0.1). Corresponding values under the influence of 8-Br-cAMP were 11.4% and 8.3% (p less than 0.05). Moreover, in the presence of ISO there was a significant shortening of the time to peak of INa+ (0.56 ms to 0.50 ms at -80 mV conditioning potential, p less than 0.05) which could not be detected in the presence of 8-Br-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Tetrodoxin (TTX) is known to cause a voltage- and frequency-dependent inhibition of the rapid inward sodium current (INa) of cardiac muscle. This effect was studied by means of the loose-patch-clamp method on intact rat papillary muscle. The availability curve of the fast sodium system, determined by variation of the holding potential, is shifted in the presence of TTX (5.5 mumol x 1(-1] by 17 mV to more negative potentials. With clamp pulses of 5 ms duration to 0 mV, a frequency-dependent reduction of INa by TTX is found above 0.1 Hz that saturates at about 10 Hz. This frequency-dependent block was further analysed using trains of pulses (10 Hz) of various durations (minimum 50 microseconds), which allow TTX to equilibrate with channel states reached early during activation. The results show that more than 90% of the frequency-dependent block is attained with pulses of 1 ms duration. An analysis according to the guarded receptor hypothesis reveals that these results are well described by TTX binding to inactivated, activated and probably preactivated channel states.

High-voltage condenser discharges exerting a field strength of up to 1000 V/cm (discharge time constant 0.24-8 msec) applied to isolated sciatic frog nerve lead to disturbances of the propagation of action potentials including transient complete block of conduction. Such conduction disturbances are normally reversible within minutes. Inhibition of the activity of the membrane-bound Na+-K+ATPase prevents the recovery from conduction block. Withdrawal of external Ca2+ also prevents recovery, whereas blockade of protein synthesis by cycloheximide has no influence. The velocity of recovery depends on the temperature, with temperature coefficients (Q10) from 1.31 to 1.84 between 2 degrees and 30 degrees C. Transmission electron microscopy of nerves subjected to strong discharges shows alterations of the myelin sheath (splitting and cleft formation) which are, however, not specific for this mechanism of injury. No alterations are seen in the region of the free axoplasmic membrane of the node of Ranvier or in organelles. The results suggest a breakdown of the transmembrane ionic gradient causing the conduction disturbance.

Frog sciatic nerves were isolated and the middle portion of each exposed to condenser discharges (field strength up to 1000 V/cm; time constants 0.2-8.0 ms) through the bathing fluid. The ability of the nerve to propagate action potentials (AP) was examined by stimulating the proximal end and recording the AP from the distal end of the exposed section. The fraction of the nerve fibers remaining propagative was estimated from the amplitude (or the area) of the compound AP. Strong discharges brought about a total block of propagation lasting for up to 30 minutes, followed by slow, but almost complete, restitution. The restitution was exponential against time and depended on the field strength and duration of the discharge. Discharges equal in energy but different in their voltage--condenser combinations had markedly different actions, with stronger effects being found at higher voltages and vice versa. Hence, the described effects are unlikely to be caused by dissipation of thermal energy only. Other mechanisms (ionic imbalance, dielectric breakdown, punch through) are discussed.

1. Rapid inward sodium current (INa) was studied on intact rat papillary muscles and trabeculae excised from right or left ventricle using the loose-patch-clamp technique. All experiments were carried out at 25 degrees C. 2. Currents were recorded from patches with a large current density of mean 5.9 +/- 0.5 mA/cm2. 3. The current was reduced by tetrodotoxin (TTX) in a dose-dependent manner. The concentration of TTX producing half-maximal blockade of INa was 6.3 +/- 0.8 mumol/l. 4. Na+ current appeared upon depolarization at a threshold potential of about -55 mV and reached its maximum at about -20 mV. 5. Kinetic data were evaluated using the Hodgkin-Huxley model. 6. Time constants of activation (tau m) were estimated using single-pulse and tail-current measurements. They had a maximum of about 0.4 ms near the threshold potential and declined at more positive and at more negative potentials to values near 0.1 ms. 7. Two time constants were necessary to describe inactivation. Both time constants had their maximal values of 135 +/- 8.1 and 29.1 +/- 5.9 ms at about -80 mV and decreased towards 4 and 0.5 ms at potentials positive to -20 mV.