Distribution of a persistent sodium current across the ventricular wall in guinea pigs.

A tetrodotoxin-sensitive persistent sodium current, I(pNa), was found in guinea pig ventricular myocytes by whole-cell patch clamping. This current was characterized in cells derived from the basal left ventricular subendocardium, midmyocardium, and subepicardium. Midmyocardial cells show a statistically significant (P<0.05) smaller I(pNa) than subendocardial and subepicardial myocytes. There was no significant difference in I(pNa) current density between subepicardial and subendocardial cells. Computer modeling studies support a role of this current in the dispersion of action potential duration across the ventricular wall.

Background inward current in ventricular and atrial cells of the guinea-pig.

Atrial and ventricular myocytes were exposed to Ca(2+)- and K(+)-free solutions containing blockers of gated channel and exchange currents. Replacement of external sodium by large organic cations revealed a background sodium current ib,Na. In atrial cells, the average conductance was 5.0 pS pF-1. In ventricular cells the conductance was 2.3 pS pF-1. Together with previous results, these figures reveal a strong gradient of background current density: sinus > atrium > ventricle. Replacement of sodium with inorganic cations showed that the channel selectivity behaves like an Eisenman group III/IV sequence, in agreement with previous results. The permeability of the channel to TMA was found to be pH dependent, suggesting that protonation of the channel is a factor determining permeation in addition to ionic size. The values of gb,Na obtained from these experiments are very similar to those assumed in computer modelling of cardiac cell electrical activity.

The effects of prenylamine on single ventricular myocytes of guinea-pig.

1. The action of prenylamine, an antianginal drug, was studied in single ventricular guinea-pig myocytes. In concentrations of 10-50 microM, prenylamine significantly (P less than 0.01) shortened action potentials, and significantly (P less than 0.001) reduced the inward calcium current by 29% to 76% (n = 7). This effect was also present in the presence of adrenoceptor-blockade (with phentolamine and propranolol), and was thus not due to indirect changes in endogenous catecholamine action. 2. Prenylamine did not affect the steady state level of current at the end of long pulses, and does therefore not act by changing time-dependent outward currents. Since the resting potential in the unclamped mode is unchanged during gross changes in action potential duration, it is also unlikely that there are any changes in the background, time-independent potassium conductance. 3. It is concluded that prenylamine has a direct effect on cardiac calcium channels, not mediated by adrenoceptor activation.

Comparison of step and ramp voltage clamp on background currents in guinea-pig ventricular cells.

Isolated cardiac ventricular myocytes from guinea-pig were used to investigate the effect of voltage clamp protocols on background Na+ current (ib.Na) and inward rectifier current (i K1). Using long (4 s) clamp pulses and very long step clamps, the i-V relations showed that removal of Na+ reduces the amplitude and shifts the voltage dependence of i K1 (Spindler et al. 1998). Ramp clamps, however, gave more complicated results, with slower ramps more often giving the same results as steps and pulses. Both i K1 itself and, during faster ramps, other currents show hysteresis, so masking the steady-state changes. Using pulses, TTX had no effect on steady-state current. Small differences occurred in the ramps, but even at fast ramp speeds the effects are very much smaller than in Purkinje tissue. Only part of ib,Na is TTX sensitive and the effect does not occur in all cells.

Thick slurry bevelling: a new technique for bevelling extremely fine microelectrodes and micropipettes.

A simple, inexpensive and rapid bevelling method is described. A settled slurry of 0.05 micrometer alumina powder in saline is used as the grinding surface. The bevelling process is continuous and reproducible over a wide range of electrode resistances.

Inward current related to contraction in guinea-pig ventricular myocytes.

1. A component of inward current has been identified in isolated guinea-pig ventricular cells that is closely correlated with the contraction of the cell and not with the rapidly activated calcium current. This is a delayed current most clearly seen as a current 'tail' after 50-200 ms depolarizing pulses. At 22 degrees C the delayed current has a maximum amplitude of approximately 0.5 nA at -40 mV (consistently 10-20% of the peak amplitude of the calcium current) and decays with a half time of approximately 150 ms. 2. Paired-pulse protocols show that at pulse intervals (300-400 ms) at which the calcium current is nearly fully reprimed, the delayed component is very small. It recovers over a time course of several seconds, as does the contraction. Adrenaline speeds the decay of the delayed current (approximately 50%) and similarly accelerates cell relaxation. Adrenaline also shortens the recovery time of both the contraction and the delayed current. 3. During long trains of repetitive pulses, the delayed current amplitude follows that of the contraction 'staircase'. The half-time of the decay of the current 'tail' also matches that of contraction and suggests that both may reflect the time course of the underlying intracellular calcium transient. 4. The half-time of decay of the delayed current is only moderately voltage dependent over the potential range -80 to 0 mV. The amplitude of the delayed current normally reaches a minimum around -20 mV and increases at more negative potentials. 5. The voltage dependence and kinetics of decay of the current show that it should flow and decay largely during the action potential plateau and repolarization rather than during diastole. 6. Diffusion of high concentrations of EGTA into cells abolishes the delayed current and cell contraction. Under these conditions the fast calcium current is increased and its inactivation delayed. 7. When calcium is replaced by strontium, the delayed current amplitude is greatly reduced even though the contraction is larger and slower. 8. The results are consistent with the hypothesis that the delayed inward current is activated by the intracellular calcium transient. It may be carried by the sodium-calcium exchange process and/or by calcium-activated non-specific channels (especially when interal calcium is elevated by reduction of external sodium). 9. In the presence of 1 microM-ryanodine, the calcium current is greatly reduced, whereas the delayed current is not significantly altered.

The control of calcium current reactivation by catecholamines and acetylcholine in single guinea-pig ventricular myocytes.

The time course of reactivation of the calcium current in isolated single cardiac cells is complex. The rising phase is sigmoid and there is an overshoot. Catecholamines increase the initial rate of reactivation but reduce or abolish the overshoot. This combination of effects results in a 'crossover', so that the net effect of adrenaline depends on the pulse interval used. Acetylcholine not only reduces the current amplitude, it also substantially slows recovery. At short intervals the effect of acetylcholine is therefore very large. Agents that increase intracellular cyclic AMP levels affect the amplitude of the current but do not have a large effect on the reactivation time course. It is suggested that the autonomic transmitters exert their effects by controlling the local calcium concentration near the inner surface of the channels. This is supported by the fact that there are natural variations in reactivation time course between different cells and that these are correlated with their calcium loading, as judged by other electrophysiological criteria, such as the speed of calcium current inactivation and the presence of the calcium-dependent slow inward current.

A pace-maker-like current in the sheep atrium and its modulation by catecholamines.

A modified single-sucrose-gap system was used to study sheep atrial trabeculae under voltage-clamp conditions. A time- and voltage-dependent current system is described, which resembles the current if in Purkinje fibres. This current was activated at membrane potentials of between -60 and -70 mV in many fibres. The addition of Ba2+ reduced the instantaneous current (the 'jump') and thus facilitated the study of the current if. Current tails were more prominent in the presence of TTX and Mn2+. Most experiments were done in the presence of Ba2+, Mn2+ and TTX. Standard envelope tests and conductance measurements indicated that this current is an inward current, activated on hyperpolarization. We have also labelled the atrial current if. The instantaneous fully activated current-voltage relationship, i(E) was found to be linear in the activation range. Increasing the level of K+, which increased the current magnitude, also increased the slope of the i(E) curve. The current magnitude was also dependent on the level of Na+ in the medium. The current magnitude was increased by adrenaline or isoprenaline. Only a small part of the increase could be attributed to a shift in the voltage dependence of the gating kinetics. The shifts in activation curves were much smaller (3-4 mV in the depolarizing direction) than those in Purkinje fibres. Large shifts in activation curves were obtained with theophylline, indicating that the presence of Ba2+ or Mn2+ did not occlude any shifts by adrenaline. The magnitude of if was increased by theophylline, with a further increase by adrenaline. There is therefore no mutual occlusion of the two effects on if. The slope of the i(E) curve was increased by isoprenaline, indicating that there was an increase in conductance. The presence of propranolol did not prevent the increase in current amplitude by isoprenaline. A direct effect of catecholamines on the channel is suggested.

Use-dependent reduction and facilitation of Ca2+ current in guinea-pig myocytes.

1. Action potentials, calcium currents (iCa) and cell contraction have been recorded from single guinea-pig myocytes during periods of stimulation from rest. Voltage clamp was carried out using a single microelectrode. Cell contraction was measured optically. All experiments were performed at 18-22 degrees C. 2. An inverse relationship was observed between cell contraction and action potential duration or iCa. Mixed trains of action potentials and voltage clamp pulses preserved this relationship. Long voltage clamp pulses induced negative 'staircases' of iCa and positive 'staircases' of cell contraction. A facilitation of iCa was observed during repetitive stimulation with clamp pulses of 100 ms duration or less and was accompanied by a decrease in cell contraction. 3. The voltage dependence of inward current staircases was found to depend on Ca2+ entry rather than membrane voltage for long voltage clamp pulses and was not affected by 30 mM-TEA or 50 microM-TTX. Current reduction was greatest at 0 mV (P less than 0.05) when iCa was largest. Changes in cell contraction during pulse trains showed a similar voltage dependence. The time constant of current staircases was only mildly voltage dependent. 4. Interference with normal cellular mechanisms for Ca2+ uptake and release by strontium, 1-5 mM-caffeine and 1 microM-ryanodine increased current staircases and could abolish iCa facilitation with short clamp pulses. 5. Variations in the level of Ca2+-dependent inactivation of iCa can explain many features of the changes in iCa during stimulation after rest. Long clamp pulses (or action potentials) may increase cell Ca2+ loading and inhibit iCa. Short clamp pulses reduce available Ca2+ for cell contraction and this may reflect a lowered myoplasmic Ca2+ level which allows facilitation of iCa.

Mechanism of the use dependence of Ca2+ current in guinea-pig myocytes.

1. The mechanism of the use-dependent reduction and facilitation of the calcium current (iCa) in single guinea-pig myocytes described by Fedida, Noble & Spindler (1988) has been examined by varying [Ca2+]o, [Ca2+]i and iCa. 2. Moderate enhancement of [Ca2+]i and [Ca2+]i changes produced by increasing [Ca2+]o reduces iCa and enhances the use-dependent reduction. 3. Intracellular calcium overload, produced by reducing [Na+]o, greatly reduces iCa and almost totally eliminates the use-dependent variations. 4. Use-dependent reduction of iCa is also smaller after substituting external Ba2+ ions for Ca2+ ions. 5. When [Ca2+]i is buffered by intracellular EGTA sufficient to eliminate other [Ca2+]i-dependent processes, such as contraction and Na+-Ca2+ exchange, some use-dependent reduction of iCa remains, although the effect is smaller. Use-dependent facilitation of iCa is more prominent in the presence of internal EGTA. 6. The facilitation of iCa is abolished by Ba2+ replacement of Ca2+ and by the beta-adrenoceptor agonist isoprenaline. This suggests that the facilitation is mediated by Ca2+ entry itself rather than membrane voltage. Facilitation is evident as a delay of current relaxation, even in the presence of internal EGTA.

Ionic mechanisms of action potential prolongation at low temperature in guinea-pig ventricular myocytes.

1. We studied the effects of low temperature on the action potentials and membrane currents of guinea-pig ventricular myocytes, using a tight-seal whole-cell clamp technique. 2. The action potential duration at 95% repolarization was prolonged from 146 +/- 33 ms (mean +/- S.D., n = 6) at 33-34 degrees C (control temperature) to 314 +/- 83 ms at 24-25 degrees C (low temperature). 3. In whole-cell clamp experiments, low temperature decreased the calcium current (ICa), the delayed rectifier potassium current (IK), and the inwardly rectifying potassium current (IK1) with 'apparent' Q10 (temperature coefficient) values of 2.3 +/- 0.6 for ICa, 4.4 +/- 1.2 for IK tail current and 1.5 +/- 0.3 for IK1 (n = 7). 4. The effect of low temperature on IK was further studied in the presence of 0.6 microM nicardipine to block ICa. The decay phase of the IK tail consisted of two exponential components. The fast but not the slow component was highly sensitive to the temperature change with an apparent Q10 of 4.5. 5. We found that a component of time-independent current is also sensitive to the temperature. The current had a linear I-V relationship and remained almost unchanged after inhibition of Na(+) -K+ pump in K(+)-free external solution. 6. Using our mathematical model of the ventricular action potential (a modification from the DiFrancesco-Noble model), we simulated the action potential at low temperature by modifying some of the membrane currents, namely IK, IK1, ICa and a component of background current. It was shown that simultaneous changes in these currents could reproduce approximately 75% of the action prolongation induced by low temperature.

Carpal ankylosis in juvenile rheumatoid arthritis.

Forty-seven of 100 consecutive juvenile rheumatoid arthritis patients with wrist arthritis were found to have ankylosis at the carpal joints. this finding was significantly more frequent in juvenile than in adult rheumatoid arthritis patients. The carpometacarpal wrist segment was the most frequently ankylosed. Ankylosis was not related to any particular feature of the disease, but disease duration was longer in patients with ankylosis. A significant association was found between carpal ankylosis and cervical apophyseal joint fusion.

The effects of sodium substitution on currents determining the resting potential in guinea-pig ventricular cells.

It has recently been shown that a sodium background current, ib,Na, exists in cardiac muscle cells whose effect is to depolarize the membrane so that the resting potential, Vm, is positive to the potassium equilibrium potential, EK. In ventricular cells, where ib,Na is smallest, Vm is about 10 mV positive to EK (EK = -87 mV at 37 degrees C). Yet, replacement of Na+ ions by large impermeant cations does not cause the expected hyperpolarization. We have studied this problem in guinea-pig myocytes using a single microelectrode recording technique in combination with a rapid external solution switch. Cells depolarized < or = 0.5 mV from potentials between -80 and -73 mV and hyperpolarized up to 5 mV from potentials between -73 and -64 mV when 70 mM choline chloride or N-methyl-D-glucamine chloride were used to replace 70 mM Na+ in the bathing solution. Replacement by 70 mM lithium chloride, however, only caused hyperpolarization in very depolarized cells when the voltage change was much smaller. The changes were complete almost as soon as the solution change, i.e. within 250 ms, indicating that the actions are attributable to the external solution change rather than to secondary changes in intracellular concentrations. Patch clamp recording was used to investigate the mechanism involved. These experiments showed that the presence or absence of the inward rectifier current iK1 determines in which direction Na+ removal acts. In the absence of iK1 the changes are attributable to removal of ib,Na, whereas in the presence of iK1 the changes resemble the i(V) relation for iK1, implying that Na+ regulates iK1 in a way that can mask the changes in ib,Na. These results explain why removal of Na+ does not lead to hyperpolarization in ventricular cells as would be expected if changes in ib,Na were solely responsible. Computer reconstruction shows that the effects may be attributed to actions of sodium removal on the conductance and gating of iK1.

The arrhythmogenic transient inward current iTI and related contraction in isolated guinea-pig ventricular myocytes.

1. The arrhythmogenic transient inward current, iTI, and contractions were recorded in isolated guinea-pig ventricular myocytes, after exposure to strophanthidin or low external K+ (0.5 mM), using a single-microelectrode voltage-clamp technique and an optical measure of contraction. 2. The inward current, iTI, and after-contraction occurred on repolarization after a depolarizing pre-pulse. Longer pre-pulses to more positive potentials increased the size and reduced the latency of iTI. Oscillatory currents and contractions also occurred during pulses to positive potentials. 3. The voltage dependence of iTI was studied by repolarizing to different potentials after a constant depolarizing pulse. Inward currents preceded after-contractions at all potentials. The iTI was maximal at about -50 mV, diminishing in magnitude at more negative and positive potentials. It remained inward at potentials up to +47 mV. The contraction exhibited a similar voltage dependence. The current-voltage relation varied in the same cell with longer exposure to glycosides. Thus, the voltage dependence of iTI reflected not only that of an underlying ionic mechanism but also the effects of potential on intracellular Ca2+ oscillations which trigger iTI. 4. Uniformity of internal Ca2+ transients was achieved by clamping to different potentials at the peak of an inward current. The iTI remained inward at positive potentials. An inward tail current, seen on repolarizing during iTI at the end of a depolarizing pre-pulse, progressively increased at negative potentials. This voltage dependence may be close to that of the Ca2+-activated inward current responsible for iTI. 5. Replacement of Na+ by Li+ initially increased the magnitude of iTI, but further exposure abolished the inward current, while the after-contractions continued to increase. The potential dependence of iTI was not affected by exposure to zero Na+. Replacement of Ca2+ by Sr2+ also abolished iTI and the after-contraction, but the main effect was to slow their occurrence. 6. The voltage dependence of the Ca2+-activated inward current in guinea-pig ventricular myocytes leads us to favour electrogenic Na-Ca exchange current as a major component of iTI, under our experimental conditions.

A new calcium current underlying the plateau of the cardiac action potential.

A small and very slow inward calcium current has been identified in isolated single ventricular cells using TTX and Cd2+ to block the sodium and fast calcium currents. Activation requires about 300 ms at the threshold potential of -60 mV, decreasing to 80 ms at the peak current voltage of -30 mV. Inactivation is five to ten times longer. Half steady-state activation and inactivation are at -50 and -45 mV respectively. The current is distinctively different in both its kinetics and pharmacology from the conventional calcium current described in single heart cells. It is proposed that it contributes significant current to help maintain a major portion of the long ventricular action potential.

Sodium-calcium exchange during the action potential in guinea-pig ventricular cells.

1. Slow inward tail currents attributable to electrogenic sodium-calcium exchange can be recorded by imposing hyperpolarizing voltage clamp pulses during the normal action potential of isolated guinea-pig ventricular cells. The hyperpolarizations return the membrane to the resting potential (between -65 and -88 m V) allowing an inward current to be recorded. This current usually has peak amplitude when repolarization is imposed during the first 50 ms after the action potential upstroke, but becomes negligible once the final phase of repolarization is reached. The envelope of peak current tail amplitudes strongly resembles that of the intracellular calcium transient recorded in other studies. 2. Repetitive stimulation producing normal action potentials at a frequency of 2 Hz progressively augments the tail current recorded immediately after the stimulus train. Conversely, if each action potential is prematurely terminated at 0.1 Hz, repetitive stimulation produces a tail current much smaller than the control value. The control amplitude of inward current is only maintained if interrupted action potentials are separated by at least one full 'repriming' action potential. These effects mimic those on cell contraction (Arlock & Wohlfart, 1986) and suggest that progressive changes in tail current are controlled by variations in the amplitude and time course of the intracellular calcium transient. 3. When intracellular calcium is buffered sufficiently to abolish contraction, the tail current is abolished. Substitution of calcium with strontium greatly reduces the tail current. 4. The inward tail current can also be recorded at more positive membrane potentials using standard voltage clamp pulse protocols. In this way it was found that temperature has a large effect on the tail current, which can change from net inward at 22 degrees C to net outward at 37 degrees C. The largest inward currents are usually recorded at about 30 degrees C. It is shown that this effect is attributable predominantly to the temperature sensitivity of activation of the delayed potassium current, iK, whose decay can then mask the slow tail current at high temperatures. 5. Studies of the relationship between the tail current and the membrane calcium current, iCa, have been performed using a method of drug application which is capable of perturbing iCa in a very rapid and highly reversible manner. Partial block of iCa with cadmium does not initially alter the size of the associated inward current tail. When iCa is increased by applying isoprenaline, the percentage augmentation of the associated tail current is much greater but occurs more slowly.(ABSTRACT TRUNCATED AT 400 WORDS)