Intracellular Sodium Activity Research Articles

EFFECTS OF STROPHANTHIDIN ON THE SLOW INWARD CURRENT IN GUINEA‐PIG ISOLATED VENTRICULAR MYOCYTES

1. The effect of strophanthidin on the slow inward current (Isi) and on contractile force were studied in guinea-pig isolated ventricular myocytes and intact papillary muscles, respectively. In myocytes, both low (10 nmol/L) and high (1-10 mumols/L) concentrations had small or no effects in either direction on Isi whereas norepinephrine (10-100 nmol/L) increased it. To determine whether the same results are obtained after decreasing or increasing intracellular calcium or sodium, the same concentrations of strophanthidin were tested in different procedures that are known to (i) increase [Ca]i and decrease [Na]i (high [Ca]o, 3.6-5.4 mmol/L; low [Na]o, 112 mmol/L; (ii) decrease [Ca]i and increase [Na]i (low [Ca]o, 0.45-1 mmol/L; Sr, 1 mmol/L; (iii) decrease [Ca]i and [Na]i (Cd, 0.1-0.2 mmol/L); and (iv) increase [Ca]i and [Na]i (veratridine, 0.2 mumol/L). High [Ca]o and veratridine increased whereas low [Ca]o and Cd decreased Isi. In contrast, during these various procedures, strophanthidin had small and inconsistent effects at a low or high concentration. In intact papillary muscles, low strophanthidin decreased whereas high strophanthidin increased contractile force. It is concluded that strophanthidin has little direct or indirect effect on Isi and that the decrease in force by low and increase in force by high concentrations in intact muscle are probably related to demonstrated decrease and increase, respectively, in intracellular sodium activity.

Effect of intracellular and extracellular pH on contraction in isolated, mammalian cardiac muscle.

1. Intracellular pH (pHi) and Na+ activity were recorded (ion-selective microelectrodes) in guinea-pig papillary muscle and the sheep cardiac Purkinje fibre while simultaneously recording twitch tension. The effects of intracellular acidosis and alkalosis upon contraction were investigated. 2. A fall of pHi produced by reducing pHo was associated with a fall of twitch tension. Similarly, a rise of pHi produced by raising pHo produced a rise of twitch tension. The time course of the changes in tension correlated with the time course of changes of pHi rather than pHo. These results are consistent with previous work showing that acidosis inhibits contraction and that the inhibition depends upon a fall of pHi. 3. Changes of pHi were produced while maintaining pHo constant at 7.4. Removal of NH4Cl or addition of sodium acetate (pHo 7.4) reduced pHi but this gave either an increase of tension (papillary muscle) or an initial fall followed by a subsequent recovery of tension (Purkinje fibre). The increase or recovery of tension occurred despite the fact that there was an intracellular acid load. Thus, reducing pHi at constant pHo can increase tension whereas reducing pHi at low pHo (6.4, see paragraph 2) inhibits tension. 4. The increase of recovery of tension during intracellular acidosis produced at a constant pHo (7.4) was associated with a rise of intracellular sodium activity (aiNa). Amiloride (1.5 mmol/l), an inhibitor of Na(+)-H+ exchange, prevented the rise of aiNa during intracellular acidosis and also prevented the recovery of tension. It is concluded that the increase or recovery of tension at low pHi is secondary to a rise of aiNa caused by stimulation of Na(+)-H+ exchange. A rise of aiNa will elevate Ca2+ via sarcolemmal Na(+)-Ca2+ exchange and thus will elevate tension. 5. An intracellular acidosis produced by reducing pHo (6.4) does not elevate aiNa in the Purkinje fibre. In papillary muscle, aiNa rises but this occurs slowly and the rise is 50% smaller than that seen when the same intracellular acidosis is induced at normal pHo (7.4). The net depression of tension under these conditions thus correlates with the lack of a large rise of aiNa. 6. Knowing the quantitative dependence of tension upon both aiNa and pHi in the two tissues it is possible to predict the recovery of twitch tension during intracellular acidosis at constant pHo (7.4), using the changes of pHi and aiNa measured under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular pH of canine subendocardial Purkinje cells surviving in 1-day-old myocardial infarcts.

A large reduction of intracellular potassium activity in depolarized subendocardial Purkinje fibers 24 hours after coronary artery ligation is accompanied by a much smaller increase in intracellular sodium activity. Similar intracellular ionic changes also occur during acute ischemia in ventricular muscle and are consistent with mechanisms based on intracellular acidification, which is known to occur in acutely ischemic muscle. To determine if canine subendocardial Purkinje cells 24 hours after myocardial infarction are also acidic, their intracellular pH, surface pH, and maximum diastolic potential (MDP) were measured with double-barrel pH-sensitive microelectrodes and compared with control fibers in noninfarcted hearts. In 12 mM bicarbonate Tyrode's solution (5% CO2-95% O2), the average intracellular pH was not significantly different (p greater than 0.25) for normal tissue (6.83 +/- 0.08, SD, MDP = -83.5 +/- 3.2 mV), for depolarized Purkinje fibers in infarct preparations during the first hour of superfusion (6.88 +/- 0.11, MDP = -47.8 +/- 11.8 mV), and for partially recovered Purkinje fibers in infarcts averaged over the third to sixth hours of superfusion (6.85 +/- 0.12, MDP = -74.5 +/- 9.6 mV). In 24 mM bicarbonate Tyrode's solution, infarct intracellular pH during both the first hour of superfusion (7.08 +/- 0.13, MDP = -57.6 +/- 15.7 mV) and during the third to sixth hours of superfusion (7.06 +/- 0.15, MDP = -76.5 +/- 9.6 mV) was significantly alkaline (p less than 0.0005) compared with average control pH (6.92 +/- 0.12, MDP = 82.1 +/- 3.7 mV). In 24 mM bicarbonate Tyrode's solution, the intracellular pH did vary with MDP (0.0032 pH units/mV). During superfusion of normal Purkinje fibers with hypoxic Tyrode's solution, intracellular pH acidified by 0.22 pH units as they depolarized. Therefore, intracellular acidification does not seem to be a cause of the depolarization of subendocardial Purkinje cells 24 hours after myocardial infarction.

Acidosis depresses delayed afterdepolarization in guinea pig myocardium.

We examined the relationship among intracellular sodium activity (aiNa), intracellular pH (pHi), and delayed afterdepolarization (DAD) in guinea pig ventricular papillary muscle fibers exposed to K-free solution in vitro, using Na+- and H+-selective microelectrodes. In unstimulated papillary muscles, exposure to K-free solution caused a rapid increase in aiNa of 16.2-22 mM at the end of a 20-min superfusion period with rates of 0.51-0.73 mM/min. This was paralleled by a decrease in intracellular pH of 0.10-0.15 units. DADs were induced after a train of driven action potentials 5-15 min after superfusion with K-free solution at the elevated aiNa from the steady-state level. Prevention of intracellular acidosis associated with the increased aiNa in K-free solution by external alkalosis at pH 8.0 enhanced the amplitude of the DAD, whereas restoration of external pH to 7.4 and a further acidification of external pH to 6.4 suppressed DADs. We concluded that intracellular acidosis associated with the increased aiNa per se increased the likelihood for inhibition of sarcoplasmic reticulum Ca2+ release in spite of increased cytosolic Ca2+.

Effects of hypoxia and metabolic inhibition on the intracellular sodium activity of mammalian ventricular muscle.

1. Intracellular Na+ activity (aiNa) has been measured in Purkinje fibres from sheep heart and in ventricular muscle from rabbit heart during hypoxia and metabolic inhibition. The aiNa was measured using liquid sensor ion-sensitive microelectrodes. 2. Hypoxia, produced by replacement of O2 with N2 in the superfusate, produced an increase in aiNa. This increase was larger if sucrose replaced glucose in the superfusing Tyrode solution. The increase in aiNa was accompanied by a small depolarization. Upon reoxygenation aiNa decreased and cells rapidly repolarized. 3. When oxidative phosphorylation was inhibited by application of 2 mM-cyanide, aiNa increased. This increase was also accompanied by a small depolarization. Upon removal of cyanide, aiNa and membrane potential recovered to control levels. 4. After inhibiting glycolysis, by replacing glucose with 2-deoxy-D-glucose, inhibition of oxidative phosphorylation (by addition of cyanide or exposure to hypoxia) produced a much more rapid increase in aiNa and a large contracture. The rise in aiNa and the occurrence of a contracture could not be inhibited by application of amiloride (1 mM) or tetrodotoxin (1 microgram ml-1). Removal of cyanide or reoxygenation and replacement of glucose resulted in a rapid relaxation of the contracture and a slower decrease in aiNa. 5. The relative rates of increase in aiNa during metabolic inhibition were compared with the rate observed when Na+-K+-ATPase was inhibited by application of 10 mumols l-1 of the cardio-active steroid strophanthidin. The rate of increase of aiNa when both oxidative phosphorylation and glycolysis were inhibited was approximately twice that observed with only oxidative phosphorylation inhibited and approximately half that observed in the presence of 10 microM-strophanthidin. 6. Cyanide, applied when aiNa had been elevated (i.e. during exposure to 10 microM-strophanthidin to inhibit Na+-K+-ATPase), did not produce a contracture. The contracture observed in the presence of cyanide and 2-deoxy-D-glucose still occurred when Ca2+ was removed from the superfusate.

Sodium‐dependent membrane current induced by carbachol in single guinea‐pig ventricular myocytes.

1. In the presence of either barium (0.2 mM) or caesium (20 mM), carbachol (3-300 microM) depolarized isolated guinea-pig ventricular myocytes. Carbachol induced an inward current under voltage clamp at a holding potential equal to the resting potential (-75 mV). 2. Acetylcholine and oxotremorine also evoked an inward current but were less effective than carbachol. Atropine (0.3 microM) prevented the depolarization and inward current induced by carbachol and acetylcholine but not by oxotremorine. Moreover, oxotremorine, but not carbachol, induced an inward current in the absence of extracellular sodium. 3. Carbachol increased membrane chord conductance when it induced an inward current. These effects were recorded under experimental conditions that suppressed the voltage- and time-dependent sodium current (tetrodotoxin) and calcium current (cadmium), the inwardly rectifying potassium current, iK1 (caesium, barium and tetraethylammonium) and the current generated by the sodium-potassium pump (zero external potassium). 4. Under these same experimental conditions, the steady-state I-V relationship in the presence of carbachol was subtracted from that in its absence. The apparent reversal potential (Erev) was 25 mV with extracellular Na+ ([ Na+]o) at 143 mM and intracellular Na+ ([Na+]i) at 11 mM. Replacement of [Na+]o by N-methyl-D-glucamine was associated with a shift of the apparent Erev to more negative voltages by approximately 61 mV per tenfold change of [Na+]o. 5. Isoprenaline induced an inward current in ventricular myocytes that depended upon sodium entry, required the accumulation of cyclic AMP and which was partially suppressed by acetylcholine (Egan, Noble, Noble, Powell, Twist & Yamaoka, 1988). In contrast to the current evoked by beta-adrenoceptor agonist, the current induced by muscarinic agonist was smaller and sustained. Moreover, the carbachol-induced current was not suppressed by prior addition of isoprenaline. 6. The findings are consistent with the mechanism that carbachol activates a plasma membrane ion channel that admits sodium and thereby increases intracellular sodium activity. The estimated increase of intracellular sodium activity from electrophysiological data agrees quantitatively with that obtained from measurements with sodium-sensitive microelectrodes (Korth & Kühlkamp, 1985). 7. The ability of carbachol to increase sodium influx may be the first step in a series of reactions that eventually alters sodium-calcium exchange and could account for catecholamine-independent stimulation of developed force in mammalian ventricle.

Acetylcholine increases intracellular sodium activity in sheep cardiac Purkinje fibers.

The action of acetylcholine (ACh) on intracellular sodium activity (alpha iNa) was studied in sheep Purkinje fibers by means of a Na+-selective microelectrode technique while transmembrane potentials and contractile force were simultaneously recorded. In quiescent fibers, 10(-4) to 10(-5) M ACh shifted the resting potential to less negative values and increased alpha iNa from 5.57 +/- 0.21 to 6.45 +/- 0.35 mM (+15.8%, P less than 0.005). In other experiments, ACh induced a depolarization that initiated spontaneous activity. In fibers driven at 60 beats/min, ACh prolonged the action potential, increased alpha iNa from 7.98 +/- 0.15 to 9.36 +/- 0.3 mM (+17.29%, P less than 0.005), and increased contractile force. Norepinephrine (10(-5) to 10(-6) M) increased contractile force and decreased alpha iNa, but in its presence ACh still increased force and alpha iNa and vice versa. Strophanthidin (10(-4) M) increased alpha iNa, and 3 x 10(-6) M propranolol and 10(-6) M atropine decreased alpha iNa. Both strophanthidin and atropine (but not propranolol) prevented the increase in alpha iNa by ACh. It is concluded that the ACh increases alpha iNa and contractile force through the inhibition of the sodium pump and that these actions are due to the activation of the muscarinic receptor and not to endogenously released norepinephrine.

Intracellular sodium activity and Bretschneider's cardioplegia: continuous measurement by ion-selective microelectrodes at initial equilibration.

Intracellular sodium activity (aiNa), intracellular pH (pHi) and membrane potential were directly and continuously measured in sheep cardiac Purkinje fibers using neutral carrier liquid membrane ion-selective microelectrodes. Changing the superfusing medium from normal Tyrode's solution to the cardioplegic solution "HTK" according to Bretschneider (6) a depolarization from -73.7 +/- 7.2 mV to -55.0 +/- 9.5 mV (n = 25), a decrease of aiNa from 9.1 +/- 1.9 mM to 4.0 +/- 1.4 mM (n = 25) and an intracellular acidification from pHi 7.18 +/- 0.06 to pHi 7.01 +/- 0.06 (n = 11, mean +/- S.D.) occurred at 35 degrees C. The decrease of intracellular sodium activity was not effected by replacement of K, Mg, or histidine by mannitol in the cardioplegic solution. Addition of 4 mM Ca somewhat enhanced aiNa decline. Inhibition of the sodium pump with the cardiac steroid dihydroouabain (10(-4) M) lowered the effect of "HTK" on intracellular sodium by approximately 35% (n = 5). Sodium decline was also sensitive to equilibration temperature, giving a Q10 of 1.54 for the initial decrease velocity (temperature range 20 to 35 degrees C), which is less than that found by other investigators for pure sodium pump activity. It is suggested that although the electrochemical sodium gradient remains inward throughout, sodium may leave myocardial cells on induction of Bretschneider's cardioplegia because of a reduction of inward fluxes by simultaneously increasing sodium pump activity, thus increasing Na efflux. Na/Ca exchange is assumed to be of minor importance and the Na/H exchange may be involved. With respect to the clinical application of the low Na and nominally Ca-free cardioplegic solution "HTK" lowering of intracellular sodium activity is interpreted as a factor minimizing the risk of a "calcium paradox" on reperfusion with Ca at serum levels, as well as a possible mechanism preventing early development of cellular edema.

Glutamate and kainate increase intracellular sodium activity in leech neuropile glial cells

Na+-selective, double-barrelled microelectrodes were used to measure intracellular Na+ activity (aiNa) and membrane potential (Em) in neuropile glial cells of isolated segmental ganglia in the leech Hirudo medicinalis. Bath application of glutamate (10(-3) M) resulted in membrane depolarizations of about 5 mV and a concomitant increase of aiNa by between 2 and 10 mM. Kainate (10(-4) M) elicited depolarizations of up to 40 mV amplitude followed by a prominent after hyperpolarization. During kainate, aiNa increased by 7 to 25 mM. In contrast to glutamate, an initial decrease of aiNa was detected during the action of kainate. N-methyl-D-aspartate (NMDA, 10(-5)-10(-3) M) had no effect of Em and aiNa. The results indicate that leech glial cells have a kainate-preferring non-NMDA glutamate receptor.

Role of aiNa in positive force-frequency staircase in guinea pig papillary muscle.

In the ventricular papillary muscle of guinea pig heart, membrane potential, intracellular sodium activity (aiNa), and twitch force were measured simultaneously and continuously for many hours at stimulation rates of 0, 0.5, 1, 2, 3, 4, 5, and 6 Hz to investigate the relation of aiNa to twitch force and membrane potential both in the steady state and during the changes in these variables. After an increase in stimulation rate, both aiNa and twitch force increased progressively, reaching steady-state levels. The relation between twitch force and aiNa in the steady state was generally sigmoidal over the range of 0.5-5 Hz and steep in the 1- to 4-Hz range. After either increase or decrease in stimulation rate, the time course of change in aiNa was exponential and similar to that of change in twitch force. Moreover, the force-aiNa relation observed after increase in stimulation rate from 0.5 to 3 Hz resembled that observed after decrease in the rate from 3 to 0.5 Hz, indicating an absence of hysteresis in the relation. The results suggest that an increase in aiNa is an important factor involved in the force staircase. As stimulation rate was increased from 0.5 to higher rates (5 or 6 Hz) and then decreased back to 0.5 Hz, a hysteresis phenomenon was observed in the relation between twitch force and aiNa. This suggests that some secondary factor may alter the relation between twitch force and aiNa. As stimulation rate increased and aiNa rose, the steady-state diastolic membrane potential hyperpolarized. This result is consistent with the view that an increase in aiNa enhances the electrogenic Na+-K+ pump and hyperpolarizes the cell membrane.

Relation between Na+-K+ pump, Na+ activity and force in strophanthidin inotropy in sheep cardiac Purkinje fibres.

1. The effects of different concentrations of strophanthidin on intracellular sodium activity (aiNa), membrane potential and contractile force have been studied in cardiac sheep Purkinje fibres under conditions (overdrive) that stimulate Na+-K+ pump activity. 2. In fibres driven at 1 Hz, a 5 min overdrive at 2 Hz in the steady state increased force by +74.2%, aiNa by +10.9% and the maximum diastolic potential (Emax) by 3.32 +/- 0.52 mV. 3. During the recovery from overdrive (the fibres being driven again at 1 Hz), both contractile force and aiNa transiently undershot the control value by -10.5 and -3.7%, respectively. When the fibres were quiescent during the recovery from overdrive, no aiNa undershoot was present. 4. During overdrive, force and aiNa were closely correlated when plotted either on linear (correlation coefficient, R = 0.98) or logarithmic (R = 0.98) co-ordinates. 5. A low concentration of strophanthidin (0.01 microM) decreased force (-31.7%) and aiNa (-7.2%): overdrive increased force and Emax more and aiNa less than in the absence of strophanthidin. During the recovery, the undershoot in force (-12.9%) and aiNa (-5.4%) was larger and longer than in the absence of strophanthidin. 6. An intermediate concentration of strophanthidin (0.05 microM) increased force (+43.5%) and aiNa (+6.4%): overdrive increased force and aiNa as usual, but during the recovery the force remained above the value prior to overdrive and there was no aiNa undershoot. 7. A high concentration of strophanthidin (0.1 microM) increased force (+91.4%) and aiNa (+11.7%): overdrive further increased force and aiNa more than in control but there was no increase in Emax. During the recovery, both force and aiNa remained well above the values prior to overdrive. 8. Force and aiNa were closely correlated whether aiNa decreased in 0.01 microM-strophanthidin (R = 0.99 both on linear and logarithmic co-ordinates) or increased in 0.05-microM- (R = 1.00 on both co-ordinates) and in 0.1 microM- (R = 0.98 and 0.99, respectively) strophanthidin. The two parameters were well correlated also during overdrive in the three strophanthidin solutions. However, the slope of the relation was less steep in the low- than in the higher-strophanthidin solutions. 9. For a 1 mM change in aiNa, force decreased less in the low- than it increased in the intermediate-strophanthidin solution. Also, in low-strophanthidin solution, at the end of overdrive the aNao/aNai ratio was similar to that in Tyrode solution but force was well above control (+73.2%).(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular pH, Na+- and K+-activities at the onset of St. Thomas' cardioplegia: a study with ionselective microelectrodes.

Cellular edema and cardiac arrhythmias are often closely related to intracellular ionic alterations and, moreover, are an actual problem of clinical cardioplegia and ischemia of the heart. In order to investigate whether the clinically widely used cardioplegic solution of the St. Thomas' Hospital may predispose for these complications, membrane potential (EM), intracellular pH (pHi), and intracellular sodium and potassium activity (aiNa- and aiK) in sheep heart Purkinje fibres were directly measured by means of neutral-carrier ion-selective microelectrodes during equilibration of the fibres' extracellular space with the cardioplegic solution. The experimental temperature was 35 degrees C throughout. During control conditions under superfusion of a HEPES-buffered Tyrode solution EM was -74.4 +/- 5.1 mV (n = 39), pHi was 7.18 +/- 0.05 (n = 16), and aiNa and aiK were 7.6 +/- 1.4 mmol/l (n = 15) and 118.9 +/- 4.6 mmol/l (n = 15) respectively. Superfusing the Purkinje strand for 10 minutes with the cardioplegic St. Thomas' solution led to a depolarization to -57.3 +/- 4.7 mV (n = 21), a slight aiNa decrease to 6.7 +/- 1.6 mmol/l (n = 15; p less than 0.01; cardioplegic solution without procaine) and an increase of aiK to 127.1 +/- 4.4 mmol/l (n = 6; p less than 0.01). pHi needed 15 minutes to reach a steady state value of 7.25 +/- 0.05 (n = 9). The alterations on post-cardioplegic reperfusion with Tyrode solution were reversible within 15 minutes. Simulation of the clinical situation of ice-cold application of the solution by inhibiting the cellular Na/K pump via 0.1 mmol/l dihydroouabaine added to the St. Thomas' solution resulted in a marked increase of aiNa to 10.7 +/- 1.5 mmol/l (n = 6; p less than 0.01; no procaine) during cardioplegic superfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Decrease in the transmembrane sodium activity gradient in ferret papillary muscle as a prerequisite to the calcium paradox.

Sodium-dependent calcium exchange may be an important mediator of calcium reperfusion damage during the calcium paradox phenomenon. We measured intracellular sodium activity with ion-selective electrodes during a 15-min period of calcium reperfusion in isolated ferret papillary muscles. During the calcium-free period, alpha Nai increased from 9.0 +/- 0.9 to 18.9 +/- 4.3 mM. With reinstitution of calcium there was a significant contracture. The amount of contracture after calcium reinstitution was related to sodium loading during the calcium-free period. We were unable to block sodium entry during the calcium-free period with either nitrendipine, tetrodotoxin, or low concentrations of amiloride. 10(-3) M amiloride or lithium for sodium substitution in the calcium-free period, however, obliterated the increase in alpha Nai activity and the subsequent paradox. These data suggest that sodium loading is a necessary prerequisite for the calcium paradox and that one mechanism of sodium entry is through Na+/Ca2+ exchange. Under these conditions, no increase in the rest force is seen without previous sodium gains, suggesting that sodium-dependent calcium exchange is an important trigger for the calcium reflow, the calcium paradox.

RESOLUTION OF OUABAIN‐SENSITIVE AND EXTRACELLULAR K+‐ACTIVATED Na+ EXTRUSION IN CARDIAC TISSUE

Intracellular sodium activity (aiNa) was measured in sheep cardiac Purkinje fibres exposed intermittently to calcium-free solutions containing 1.3 mM-Na+ with 0-16 mM-K+ and 0-0.2 mM ouabain. In potassium-free solution (O K+) mean maximum Na+ pump activity (i.e. excluding Na+ influx and Na+-Ca2+ exchange) was 61% of that in the 4 mM-K+ control solution; in 0.2mM-ouabain-4 mM-K+ it was still 44% of control. This high ouabain concentration abolished Na+ extrusion only in O K+ while reducing it by the same absolute amount at all other extracellular K+ concentrations. The Na+ pump may therefore have two independent components in cardiac tissue.

Mechanism of inhibition by SUN 1165, a new Na channel blocking antiarrhythmic agent, of cardiac glycoside-induced triggered activity

The mechanism of the antiarrhythmic action of SUN 1165, a selective Na channel blocker, in digitalis-induced arrhythmias was investigated by means of conventional microelectrode and suction pipette methods with isolated canine Purkinje fibers and guinea-pig single ventricular cells, respectively. SUN 1165 decreased acetylstrophanthidin-induced delayed afterdepolarization and completely blocked the initiation of triggered activity by acetylstrophantidin in Purkinje fibers. Delayed afterdepolarization, which was completely abolished either by intracellular dialysis with EGTA or by extracellular superfusion with caffeine, was decreased by SUN 1165 in a concentration-dependent manner in single ventricular cells. These results suggest that an increase in intracellular Ca 2+ concentration is a requirement for delayed afterdepolarization. Furthermore, the antiarrhythmic action of SUN 1165 in cardiac glycoside-induced arrhythmias in dogs could be mediated not only by an inhibition of sodium channels and subsequent reduction in the intracellular sodium activity, but also by a reduction of intracellular calcium activity due to the Na +-Ca 2+ exchange mechanism.

Electrical and ionic mechanisms of early reperfusion arrhythmias in sheep cardiac Purkinje's fibers

The mechanisms of induction of early reperfusion arrhythmias were studied in sheep cardiac Purkinje's fibers superfused in vitro. Transmembrane potentials, intracellular sodium activity (a i Na), and contractile force were recorded. Stoppage of the flow of Tyrode's solution (ischemia) for 1 hour initially decreased slightly a i Na (−0.57 mmol −7.2%), increased the action potential amplitude (+6.1%) and duration (+7.8%), and decreased diastolic depolarization slope (−45.2%). As the ischemia continued, a i Na increased progressively (to 12.53 mmol, +56.2%), whereas force peaked (+395%) after about 30 minutes and then began to decrease. By the end of ischemia, there was a decrease in action potential amplitude (−14.9%) and duration (−39.6%), whereas diastolic depolarization slope reincreased again almost to control value (−7%). When the flow of Tyrode's solution was resumed (reperfusion), force markedly increased (+211.1%) and oscillatory potentials initiated arrhythmias (extrasystoles and repetitive fast discharge) in 64% of tests. Force and a i Na decreased relatively rapidly. The arrhythmias initiated after 58.4 ± 1.8 seconds of reperfusion and lasted 101.5 ± 3.2 seconds. When [Na] o was increased by +19.2%, reperfusion arrhythmias occurred after only 30 minutes of ischemia. Thus, in Purkinje's fibers superfused in vitro, early reperfusion arrhythmias are induced by oscillatory potentials caused by calcium overload, which is enhanced by the increase in a i Na during ischemia.

Cell Volume Regulation in Liver

The maintenance of liver cell volume in isotonic extracellular fluid requires the continuous supply of energy: sodium is extruded in exchange for potassium by the sodium/potassium ATPase, conductive potassium efflux creates a cell-negative membrane potential, which expelles chloride through conductive pathways. Thus, the various organic substances accumulated within the cell are osmotically counterbalanced in large part by the large difference of chloride concentration across the cell membrane. Impairment of energy supply leads to dissipation of ion gradients, depolarization and cell swelling. However, even in the presence of ouabain the liver cell can extrude ions by furosemide-sensitive transport in intracellular vesicles and subsequent exocytosis. In isotonic extracellular fluid cell swelling may follow an increase in extracellular potassium concentration, which impairs potassium efflux and depolarizes the cell membrane leading to chloride accumulation. Replacement of extracellular chloride with impermeable anions leads to cell shrinkage. During excessive sodium-coupled entry of amino acids and subsequent stimulation of sodium/potassium-ATPase by increase in intracellular sodium activity, an increase in cell volume is blunted by activation of potassium channels, which maintain cell membrane potential and allow for loss of cellular potassium. Cell swelling induced by exposure of liver cells to hypotonic extracellular fluid is followed by regulatory volume decrease (RVD), cell shrinkage induced by reexposure to isotonic perfusate is followed by regulatory volume increase (RVI). Available evidence suggests that RVD is accomplished by activation of potassium channels, hyperpolarization and subsequent extrusion of chloride along with potassium, and that RVI depends on the activation of sodium hydrogen ion exchange with subsequent activation of sodium/potassium-ATPase leading to the respective accumulation of potassium and bicarbonate. In addition, exposure of liver to anisotonic perfusates alters glycogen degradation, glycolysis and probably urea formation, which are enhanced by exposure to hypertonic perfusates and depressed by hypotonic perfusates.

Effects of forskolin on intracellular sodium activity in resting and stimulated cardiac Purkinje fibers from sheep

In the present investigation, the effects of forskolin on intracellular sodium activity were studied in quiescent and electrically stimulated cardiac Purkinje fibers from sheep using Na+-sensitive microelectrodes. Also assessed, were the effects of this promoter of cytosolic cAMP production on resting membrane potential, action potential and twitch tension. In the quiescent fibers, forskolin (12 microM) caused intracellular sodium activity to decrease in the face of cellular depolarization. This cellular depolarization was occasionally accompanied by spontaneous firing of action potentials. In the stimulated fibers, forskolin (10 microM) also caused intracellular sodium activity to decrease. Moreover, it caused a marked acceleration of phase 4 pacemaker depolarization, an elevation of the plateau of the action potential and an increase in twitch tension. When the Na+ pump was inhibited by either strophanthidin (1 microM) or by 0 mM extracellular K+, forskolin had no effect on intracellular sodium activity. In summary, the results of the present study indicate that forskolin, presumably by increasing intracellular cAMP, causes the following to occur in cardiac Purkinje fibers from sheep: (a) a decrease in intracellular sodium activity when the Na+ pump is functioning normally; (b) a promotion of membrane depolarization in quiescent fibers; (c) an increase in the steepness of the pacemaker potential in electrically stimulated fibers, and (d) an increase in the force of contraction. Therefore, forskolin will be a useful tool for investigating the role of cAMP in physiological function of cardiac cells.

Strophanthidin and force regulation by intracellular sodium activity in cardiac Purkinje fibers

The role of intracellular sodium activity (a Na i) in the inotropy of a low concentration of strophanthidin (5 × 10 −8 M) was studied in sheep cardiac Purkinje fibers by recording contractil force, a Na i and transmembrane potentials under conditions that vary a Na i. High [Na] 0, strophanthidin and tetrodotoxin (TTX) changed force and a Na i in a closely related manner: on logarithmic coordinates, the data were well fitted by a single line obtained through the regression equation F = b (a Na i) s where b represents the intercept and s the slope of the relation. With low strophanthidin, force increases as a linear function of (a Na i) −5) and with high [Na] 0 as a linear function of (a Na i) −6). However, the combined administration of high [Na] 0 and strophanthidin results in a potentiated inotropic effect as force becomes a linear function of (a Na i) −14). This potentiation and its abolition by TTX suggest that factors other than a Na i powerfully modify the inotropy of a low strophanthidin concentration.

Selective updates on mechanisms of action of positive inotropic agents.

Several extensive reviews concerning the actions of new positive inotropic agents in the treatment of congestive heart failure, often with reference to their mechanism of action, have recently been published. Each of them has presented specific points of view. This review will place special emphasis on the significance of intracellular sodium activity for the modulation of myocardial inotropy; the continuing importance, after a 200 year history, of the use of cardiac glycosides as strong inotropic agents; the emerging significance of the phosphoinositide (PIP2) pathway to provide additional second messengers for the modulation and regulation of cardiac inotropy; the contribution of the alpha 1-adrenergic system to cardiac inotropy; the increasing awareness of the significant involvement of adenosine and its antagonists in cardiac function; and the increasing realization that myocardial tissues are not homogeneous, i.e., that in many species the atrial and ventricular tissues are using different, even opposite mechanisms in the generation of their functional responses.