Increase In aiNa Research Articles

Changes in intracellular Na+ during Na,K pump inhibition in sheep cardiac tissues.

The differences in inotropic and toxic sensitivities of cardiac ventricular tissue to cardiac glycosides were investigated in order to determine whether or not the reportedly greater sensitivity of Purkinje fibers compared to myocardium is the result of a fundamentally different response to Na,K pump inhibition. I measured the changes in intracellular Na+ activity (aiNa) in the two tissue types simultaneously during exposure to actodigin (4.0 microM) and ouabain (0.5 microM) under quiescent conditions. A sheep papillary muscle and a Purkinje fiber from the same heart were placed in an experimental chamber and measurements of aiNa from both were obtained with Na+ -sensitive microelectrodes. In five experiments in which all electrode impalements were successfully maintained, actodigin caused similar changes in aiNa in the two tissues (from 7.2 +/- 1.0 mM in control to 12.9 +/- 1.9 mM in the presence of drug in papillary muscles compared to 7.3 +/- 0.3 mM in control and 13.2 +/- 1.0 mM in Purkinje fibers; means +/- S.E.M.). After washout, exposure to ouabain increased aiNa in both papillary muscles and in Purkinje fibers (from 7.2 +/- 0.7 mM in control and 16.2 +/- 1.4 mM during exposure to drug in papillary muscles compared to 7.4 +/- 0.3 mM and 14.9 +/- 0.8 mM in Purkinje fibers). In fact, ouabain caused a greater increase of aiNa in papillary muscles than in Purkinje fibers (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+ and Na+ in rat myocytes showing different force-frequency relationships

Intracellular [Ca2+] ([Ca2+]i), intracellular Na+ activity (aiNa), and contraction have been monitored in single myocytes isolated from the ventricles of rat hearts. Some of these cells showed an increase in the size of the twitch as stimulation frequency was increased (positive force-frequency relationship), while others showed a decrease in the strength of contraction as the frequency of stimulation was increased (negative force-frequency relationship). In cells that showed a positive force-frequency relationship, increasing stimulation frequency resulted in increases in aiNa, diastolic [Ca2+]i, systolic [Ca2+]i, and the amount of Ca2+ that could be released from the sarcoplasmic reticulum by caffeine. The rate of decline of the [Ca2+]i transient and the twitch also increased as stimulation frequency was increased. In cells that showed a negative force-frequency relationship, increasing stimulation frequency had less effect on aiNa and had either no effect or decreased systolic [Ca2+]i with no change in the amount of Ca2+ that could be released from the sarcoplasmic reticulum using caffeine. The rate of relaxation of the [Ca2+]i transient and the twitch again increased as stimulation frequency increased. The pattern and time course of mechanical restitution was the same in both cell types. Although these data are essentially descriptive, it is consistent with the hypothesis that the final contractile response observed during changes of stimulation frequency may be dependent on how the Ca2+ loading of the preparation varies with stimulation frequency.

Effect of norepinephrine and heart rate on intracellular sodium activity and membrane potential in beating guinea pig ventricular muscle.

The effect of 3 microM norepinephrine (NE) on intracellular sodium activity (aiNa) and resting membrane potential was studied by continuous intracellular recordings with a conventional and an ion-selective microelectrode. The electrodes were impaled simultaneously in small (diameter, 0.3 mm) superfused trabeculae of the beating guinea pig ventricle at 37 degrees C. In the absence of NE, changes of the beating rate produced an increase of aiNa by 1.5 +/- 0.17 mM (from 0 to 1 Hz) and 1.9 +/- 0.47 mM (from 0 to 2 Hz). In the presence of NE, there was a very small significant increase of aiNa during constant stimulation (1 Hz) and at at [K+]o of 4.7 and 11.5 mM. After 7 minutes of exposure, aiNa increased by 0.5 +/- 0.19 mM (mean +/- SEM, n = 4) at [K+]o of 4.7 mM and by 0.5 +/- 0.22 (n = 6) at [K+]o of 11.5 mM. Resting membrane potential became more positive by 1 mV at both levels of [K+]o. The effect of NE became also clearly manifest from the configurational changes of action potentials (profound increase in plateau height and duration). Stimulation of the Na(+)-K+ pump by NE became manifest from the changes of resting membrane potential and aiNa after abrupt cessation of stimulation. The magnitude and the rate of the decrease in aiNa and the initial rate of hyperpolarization were significantly greater in the presence of NE than in its absence.(ABSTRACT TRUNCATED AT 250 WORDS)

The Relation Between Contraction and Intracellular Sodium in Voltage‐Clamped Sheep Purkinje Fibers

Contraction and Intracellular Sodium. Intracellular sodium activity (aiNa) and contraction were simultaneously recorded in voltage‐clamped sheep Purkinje fibers while the sodium‐potassium pump was inhibited with strophanthidin or low potassium. Exposures to low (1.5×10−7M) and high (10−5M) doses of strophanthidin resulted in aiNa and contraction increasing together. However, when strophanthidin was washed off, aiNa recovered more slowly than contraction. Due to this lag of aiNa behind contraction, there was hysteresis in the relation between contraction and aiNa. We compared strophanthidin exposures in voltage‐clamped fibers to those in unclamped fibers with action potentials. The lack of association between contraction and aiNa was less when fibers were clamped. We conclude that some of the dissociation previously reported in unclamped fibers was due to changes of membrane potential with strophanthidin. Using voltage clamp, we also compared the effect of different doses of strophanthidin applied to the same fiber. Higher doses of strophanthidin caused a larger rise of contraction for a similar increase of aiNa. Higher doses, which inhibit the Na‐K pump to a greater extent, also resulted in a faster rate of rise of aiNa. Inhibition and reactivation of the Na‐K pump with bathing potassium (or rubidium) also resulted in hysteresis between contraction and aiNa. A greater reduction of potassium caused a larger rise of contraction for a similar rise of aiNa. Lower potassium inhibits the Na‐K pump more completely and results in a faster rate of rise of aiNa. Although the absolute rise of aiNa with strophanthidin or low potassium is related to the increase of contraction, we conclude that this is not the only factor. The inotropic effect of these agents is also related to the rate at which aiNa rises; when the rate of rise is large, a given level of aiNa is associated with a larger contraction. Such behavior may be compatible with the presence of aiNa gradients from the subsarcolemmal space to the bulk cytoplasm during pump inhibition.

Intracellular pH regulation in cultured embryonic chick heart cells. Na(+)-dependent Cl-/HCO3- exchange.

The contribution of Cl-/HCO3- exchange to intracellular pH (pHi) regulation in cultured chick heart cells was evaluated using ion-selective microelectrodes to monitor pHi, Na+ (aiNa), and Cl- (aiCl) activity. In (HCO3- + CO2)-buffered solution steady-state pHi was 7.12. Removing (HCO3- + CO2) buffer caused a SITS (0.1 mM)-sensitive alkalinization and countergradient increase in aiCl along with a transient DIDS-sensitive countergradient decrease in aiNa. SITS had no effect on the rate of pHi recovery from alkalinization. When (HCO3- + CO2) was reintroduced the cells rapidly acidified, aiNa increased, aiCl decreased, and pHi recovered. The decrease in aiCl and the pHi recovery were SITS sensitive. Cells exposed to 10 mM NH4Cl became transiently alkaline concomitant with an increase in aiCl and a decrease in aiNa. The intracellular acidification induced by NH4Cl removal was accompanied by a decrease in aiCl and an increase in aiNa that led to the recovery of pHi. In the presence of (HCO3- + CO2), addition of either amiloride (1 mM) or DIDS (1 mM) partially reduced pHi recovery, whereas application of amiloride plus DIDS completely inhibited the pHi recovery and the decrease in aiCl. Therefore, after an acid load pHi recovery is HCO3o- and Nao- dependent and DIDS sensitive (but not Ca2+o dependent). Furthermore, SITS inhibition of Na(+)-dependent Cl-/HCO3- exchange caused an increase in aiCl and a decrease in the 36Cl efflux rate constant and pHi. In (HCO3- + CO2)-free solution, amiloride completely blocked the pHi recovery from acidification that was induced by removal of NH4Cl. Thus, both Na+/H+ and Na(+)-dependent Cl-/HCO3- exchange are involved in pHi regulation from acidification. When the cells became alkaline upon removal of (HCO3- + CO2), a SITS-sensitive increase in pHi and aiCl was accompanied by a decrease of aiNa, suggesting that the HCO3- efflux, which can attenuate initial alkalinization, is via a Na(+)-dependent Cl-/HCO3- exchange. However, the mechanism involved in pHi regulation from alkalinization is yet to be established. In conclusion, in cultured chick heart cells the Na(+)-dependent Cl-/HCO3- exchange regulates pHi response to acidification and is involved in the steady-state maintenance of pHi.

Consequences of CO2 acidosis for transmembrane Na+ transport and membrane current in rabbit cardiac Purkinje fibres.

1. The influence of sarcolemmal Na(+)-H+ exchange on intracellular Na+ activity (aiNa), intracellular pH (pHi) and membrane holding current (Ih) was investigated in rabbit cardiac Purkinje fibres. pHi and aiNa were measured with liquid sensor ion-selective microelectrodes. A two-microelectrode voltage clamp was used while recording pHi or aiNa.pHi was varied by alternating a nominally CO2-free HEPES buffer and a CO2-HCO3-buffer. 2. The intrinsic buffer capacity was calculated from the decrease in pHi after addition of CO2. The most accurate estimate was obtained when transmembrane pHi regulation was blocked and equalled 18.4 +/- 1.0 mequiv H+/pH unit (mean +/- S.E.M.). 3. aiNa started to rise when pHi fell below 7.0. A hyperpolarization paralleled the increase in aiNa. The magnitude of the rise in aiNa and the hyperpolarization were steeply dependent on pHi. 4. Inhibition of the Na(+)-K+ pump by K(+)-free superfusion increased aiNa. The rate of rise in aiNa was highly dependent on pHi. The rates of rise in 5% (pHi = 7.00 +/- 0.03), 7% (pHi = 6.89 +/- 0.04) and 15% CO2 (pHi = 6.74 +/- 0.02) at constant external pH (pHo) relative to the rate in HEPES solution (pHi = 7.24 +/- 0.02) were: 1.5 +/- 0.2, 2.4 +/- 0.1 and 3.1 +/- 0.2. The acid-induced rise in aiNa was abolished by 2 mM-amiloride. 5. Extracellular acidosis slowed down the recovery of pHi and depressed the rate of rise in aiNa upon intracellular acidification. When both pHi and pHo were decreased to 6.7 the acid-dependent rate increase fell to about 10% of the value found at pHo 7.4. 6. The tetrodotoxin (TTX)-sensitive Na+ current was not influenced by the change in pHi in the range 6.7-7.2. 7. Intracellular acidosis was associated with an early aiNa-independent depolarization and an inward shift in Ih. Current-voltage plots revealed that the initial inward current shift reversed at -81.5 mV on average, showed inward rectification and was largely depressed in the presence of 1 mM-Ba2+. These observations indicate a decrease in K+ conductance when pHi falls. 8. The increase in aiNa elicited a Na(+)-K+ pump-dependent outward current which could override the initial aiNa-independent current shift. At a pHo of 6.7 the initial fall in Ih remained, while the secondary outward current was largely depressed. 9. The rate of active Na+ extrusion and Na(+)-K+ pump current were suppressed by about 30% at pHi 6.7 compared to pHi 7.2.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular Na+ activity in cultured mouse oligodendrocytes

Na(+)-selective double-barrelled microelectrodes were used to measure the intracellular Na+ activity (aiNa) and membrane potential (Em) in oligodendrocytes from cultures of embryonic mouse spinal cord. In Na(+)-free solutions aiNa rapidly fell from its baseline of about 15 mM to values below 1 mM. Elevation of the K+ concentration in the bath ([K+]o) from 5.4 to 15 or 50 mM elicited an aiNa decrease of 4.7 or 9.0 mM, respectively. Ouabain blocked the aiNa decrease in response to 50 mM K+ by 37%. Bath application of 1 mM glutamate resulted in a membrane depolarization of 4.5 mV and a concomitant rise of aiNa by 8.6 mM. aiNa increased by approximately 11 mM after washout of a solution containing 20 mM NH4+. This aiNa increase was not blocked by amiloride, excluding a major contribution of a Na+/H+ antiporter. We conclude that, in cultured oligodendrocytes, transmembraneous Na+ movements are involved in pH regulation, glutamate causes an influx of Na+, and that the Na+/K+ pump and passive KCl uptake contribute to K+ accumulation.

Acidification and intracellular sodium ion activity during stimulated myocardial ischemia

With the use of microelectrodes, intracellular pH (pHi), surface pH (pHs), and intracellular Na+ activity (aiNa) were measured in isolated guinea pig papillary muscles during normal superfusion and during a reversible condition of simulated ischemia. Acid loading by NH+4 prepulse or by CO2-HCO3- addition during superfusion with pH 7.4 solutions caused internal acidification followed by a recovery of pHi, which could be inhibited by amiloride. pHi recovery was associated with an amiloride-sensitive peak rise of aiNa and membrane hyperpolarization, indicative of Na(+)-H+ exchange. Peak increase of aiNa was absent if the pH of the superfusion solution was concomitantly lowered. Imposed ischemia after control superfusion caused membrane depolarization and acidification of pHi and pHs. The change of pHs consistently was larger than that of pHi. aiNa decreased from 5.5 to 4.6 mM after 10-min ischemia. Enlarging the pHi (and pHs) decrease in ischemia by prior reduction of the tissue buffer capacity (CO2-HCO3(-)-free superfusion) was unable to induce a rise of aiNa during the subsequent ischemic period. Amiloride had no significant effect on aiNa during ischemia. It is concluded that the important acidification of pHs reduces the rate of pHi regulatory Na(+)-H+ exchange and thereby contributes to a longer maintenance of the Na+ electrochemical gradient in ischemic cardiac muscle.

Effect of norepinephrine on Na(+)-K+ pump and Na+ influx in sheep cardiac Purkinje fibers

Effects of norepinephrine and Ca+ on Na(+)-K+ pump and pacemaker current were investigated by simultaneous measurement of intracellular Na+ activity (aiNa) and membrane potential in driven (1 Hz) and quiescent sheep cardiac Purkinje fibers. Concurrently, twitch force was measured in driven fibers, in which norepinephrine (NE) produced a decrease in aiNa, a prolongation in action potential duration, and a hyperpolarization in diastolic membrane potential, Vdm. In contrast, in quiescent fibers, NE produced an increase in aiNa and a depolarization in resting membrane potential, Vm. The decrease in aiNa, prolongation in action potential duration, and hyperpolarization in Vdm produced by NE were blocked by 5 x 10(-6) M strophanthidin, presumably through inhibition of the Na(+)-K+ pump. The increase in aiNa and membrane depolarization caused by NE were abolished by high [K+]o or Cs+, presumably through inhibition of the pacemaker current, if. These results indicate that in driven fibers NE stimulates predominantly the Na(+)-K+ pump, producing a decrease in aiNa and that in quiescent fibers it increases predominantly if, producing an increase in aiNa. The effect of NE on driven and quiescent fibers differs because of the voltage dependence of if and perhaps the Na(+)-K+ pump. Consequently, the relative magnitude of the two opposing effects of NE on aiNa appears to be dependent on membrane potential. In quiescent fibers, Cs+ monotonically decreased aiNa to a steady-state value, while Cs+ hyperpolarized membrane potential and then slowly depolarized to a steady-state level, producing a transient hyperpolarization. In driven fibers, Cs+ decreased aiNa, shortened action potential duration, and depolarized Vdm. Cs+ decreased aiNa more in quiescent fibers than in driven fibers. The decrease in aiNa and hyperpolarization in membrane potential produced by Cs+ in quiescent fibers were abolished by depolarization induced by high K+ extracellular concentration (25.4 mM) but were not abolished or reduced by 5 x 10(-6) M strophanthidin. These results suggest that the decrease in aiNa and hyperpolarization in membrane potential by Cs+ are caused by blockage of if but not by stimulation of the Na(+)-K+ pump and that if is an important source of Na+ loading into cells.

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.

Electrophysiological effects of mucosal Cl- removal in Necturus gallbladder epithelium

The factors responsible for the cell membrane hyperpolarization elicited in Necturus gallbladder epithelium on Cl- removal from the mucosal bathing solution were evaluated with conventional and ion-sensitive microelectrode techniques. Cl- removal causes reversal of apical Cl- -HCO3- exchange, resulting in a fall in intracellular Cl- activity (aiCl) and an increase in intracellular pH (pHi). Concomitantly, the cell membranes hyperpolarize to values close to the K+ equilibrium potential (EK), aiNa falls, and aiK rises. The observed changes in membrane voltage are not attributable to a pHi-dependent increase in cell membrane K+ permeability (PK), because 1) the cell membrane resistances increased and 2) elevating solution partial pressure of CO2 (PCO2) to counterbalance the cellular alkalinization on mucosal Cl- removal caused a further hyperpolarization of the cell membranes to values greater than EK. This additional hyperpolarization was related to the activity of the Na+ pump, inasmuch as it was accompanied by an increase in aiNa and was ouabain sensitive. These results are consistent with, but do not prove, pump electrogenicity. During the period of Cl- removal from the mucosal bathing solution, the cell membrane depolarization caused by raising serosal K+ concentration was increased, whereas the depolarization caused by lowering serosal Cl- concentration was decreased, compared with substitutions under control conditions. These results indicate that mucosal Cl- removal causes a decrease in basolateral PCl, which we speculate could be due to a decrease in cell volume. We conclude that the hyperpolarization of the cell membranes on mucosal Cl- removal is primarily due to the combined effects of the fall in basolateral PCl and the increase in basolateral ECl.

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+.

An inwardly directed electrogenic sodium‐bicarbonate co‐transport in leech glial cells.

1. We have used double-barrelled ion-sensitive microelectrodes to measure the intracellular pH, pHi, the intracellular Na+ activity, aiNa, and the membrane potential in identified glial cells of the central nervous system of the leech Hirudo medicinalis to study the effect of CO2-HCO3-. 2. When a HEPES-buffered saline was exchanged for a saline buffered with 2% CO2 + 11 mM-HCO3-, keeping the pH constant at 7.4, the mean steady-state pHi of the glial cells increased from 6.85 +/- 0.06 to 7.18 +/- 0.13 (mean +/- S.D., n = 25). 3. This CO2-HCO3- -dependent alkalinization was inhibited in the absence of external Na+ (exchanged by N-methyl-D-glucamine), but was unaffected by the inhibitor of Na+-H+ exchange, amiloride (2 mM). 4. The aiNa of the glial cells increased by 2-4 mM from a mean steady state of 7.2 +/- 2 mM (mean +/- S.D., n = 6) upon introduction of CO2-HCO3- -buffered saline. This CO2-HCO3- -dependent rise in aiNa increased to about double when the pHi had been decreased by acid loading the cells (addition and subsequent removal of NH4+). 5. The CO2-HCO3- -dependent increases of pHi and aiNa were inhibited by the stilbene 4,4-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS, 0.5-1.0 mM). 6. Removal of external Cl- and depletion of intracellular Cl- did not inhibit the CO2-HCO3- -dependent alkalinization. 7. The CO2-HCO3- -dependent alkalinization was unaffected by inhibitors of the carbonic anhydrase, acetazolamide (0.2 mM) or ethoxzolamide (2 microM). 8. The membrane potential became more negative by 3-20 mV upon addition of CO2-HCO3-. This hyperpolarization was even further enlarged in the presence of Ba2+ (which reduces the K+ permeability) or at increased external K+ concentration (which depolarizes the membrane and brings the membrane potential to the K+ equilibrium potential). The CO2-HCO3- -induced membrane hyperpolarization was inhibited in Na+-free saline and in the presence of DIDS. Ouabain (0.5 mM) sometimes reduced, but never abolished, the hyperpolarization. 9. The stoichiometry of the co-transport is suggested to be 2 HCO3-:1 Na+ with an equilibrium potential of -90 mV calculated for this coupling ratio in the steady state. 10. It is concluded that in the presence of CO2-HCO3- an inwardly directed electrogenic Na+-HCO3- co-transport is stimulated across the glial membrane, which greatly determines the pHi and thereby affects the intracellular buffering power of the glial cells.

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.

Mechanism of rate-dependent pH changes in the sheep cardiac Purkinje fibre.

1. The mechanism of the rate-dependent decrease in intracellular pH (pHi) and its recovery were studied in isolated sheep cardiac Purkinje fibres. Intracellular Na+ activity (aiNa) and pHi were measured using ion-selective microelectrodes. Twitches were elicited by field stimulation or by depolarizing pulses applied using a two-microelectrode voltage clamp. 2. A 3 Hz train of short (50 ms) depolarizing voltage-clamp pulses induced a reversible fall in pHi which was accompanied by a reversible increase in aiNa. A train of longer (200 ms) pulses also produced a fall in pHi which was now paralleled by a decrease in aiNa. These observations indicate that the rate-dependent acidosis is not dependent upon a rise in aiNa. 3. Neither the fall in pHi nor the increase in aiNa seen upon an increase in action potential frequency was inhibited by amiloride (1 mmol l-1) which indicates that Na+-H+ exchange is not involved in the generation of the acidosis. Furthermore, the rate-dependent acidosis was not abolished in Na+-free solution (Li+ or N-methyl glucamine substituted) indicating that other Na+-requiring processes (such as Na+-Ca2+ exchange) are not a necessary requirement. Rate-dependent pHi changes were also unaffected by the stilbene compound DIDS indicating no participation by Cl--HCO-3 exchange. 4. The rate-dependent acidosis was inhibited by the organic calcium antagonist D600 (20 mumol l-1) which also inhibited twitch tension. This suggests that the acidosis is related to the activation by Ca2+ of developed tension. D600 also inhibited the rate-dependent rise in aiNa (field stimulation). 5. The rate-dependent acidosis was not inhibited by cyanide (2 mmol l-1) but it was blocked by iodoacetate (0.5 mmol l-1) and by 2-deoxyglucose (DOG) (10 mmol l-1, applied in glucose-free solution). These results suggest that the acidosis is generated metabolically via stimulation of glycolysis, following an increase in contraction. Contributions from aerobic metabolism are likely to be small. 6. Twitch tension was inhibited by ryanodine (10 mumol l-1) but the drug had little inhibitory effect on the rate-dependent acidosis. A tonic component of tension was observed, however, in the presence of ryanodine. The lack of effect of ryanodine upon the rate-induced acidosis is discussed. 7. The half-time of pHi recovery from the frequency-dependent acidosis was consistently shorter than that from an intracellular acid load induced by adding and then removing external NH4Cl (10 mmol l-1).(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)

Regulation of intracellular pH in sheep cardiac Purkinje fibre: interactions among Na+, H+, and Ca2+1.

Experiments were performed on sheep cardiac Purkinje fibres using pH- and sodium-selective microelectrodes, while simultaneously measuring tension, to determine if the fall in intracellular pH (pHi) following a rise in intracellular Na+ activity (aiNa) is caused by inhibition or reversal of acid extrusion on Na+-H+ exchange. A rise in aiNa was induced either by using the cardioactive steroid strophanthidin to inhibit the sarcolemmal Na+-K+ pump or by increasing the frequency of stimulation (0-4 Hz). Both of these manoeuvres led to an increase in aiNa and a decrease in pHi. Following exposure to strophanthidin, amiloride (an inhibitor of sarcolemmal Na+-H+ exchange) produced a decrease in both pHi and aiNa. These effects of amiloride increased with decreasing pHi, indicating that acid extrusion on Na+-H+ exchange is stimulated by the fall in pHi. The changes in intracellular Na+ and H+ caused by amiloride were quantitatively consistent with an electroneutral stoichiometry. The fall in pHi during strophanthidin exposure is therefore not caused by inhibition or reversal of acid extrusion Na+-H+ exchange. It is likely that the fall in pHi during a rate increase is also independent of Na+-H+ exchange. This is because (i) it has been shown previously to occur in the presence of amiloride and (ii) the calcium antagonist D600 completely abolished the stimulation-dependent fall in pHi. It is concluded that the intracellular acidosis following inhibition of the sarcolemmal Na+-K+ pump or following an increase in the rate of stimulation is secondary to a rise in intracellular calcium.

Factors affecting intracellular sodium during repetitive activity in isolated sheep Purkinje fibres.

1. Intracellular Na+ activity (aiNa) was measured using neutral-carrier Na+-sensitive micro-electrodes in voltage-clamped sheep Purkinje fibres during and after 4 min sequences of depolarizing pulses applied to around 0 mV, at a rate of 2.5 Hz. After trains of pulse duration 50 ms the mean increase in aiNa was 0.65 +/- 0.3 mM (mean +/- S.D., n = 18) whereas with longer pulse durations this rise became progressively smaller. At pulse durations of 300 ms a fall in aiNa was usually found. 2. Recovery of aiNa after a pulse sequence followed a roughly exponential time course. The half-time of decline after a rise in aiNa using 50 ms pulses was 111 +/- 52 s (n = 10), compared with a half-time of 318 +/- 116 s (n = 6) for recovery from a fall in aiNa during a sequence of 300 ms pulses. 3. Application of 2 mM-Cs+ to block the pace-maker current (if) resulted in a decrease in resting aiNa by 0.85 +/- 0.45 mM (n = 6) and an outward current shift. Na+ loading during a depolarizing pulse train was greater in 2 mM-Cs+ than in control solution. The rise in aiNa produced by a train of 50 ms pulses in Cs+ was 1.15 +/- 0.4 mM (n = 10). At short pulse durations in the presence of Cs+, Na+ loading at the end of a pulse train increased as a function of pulse duration, becoming maximal at a duration of approximately 50 ms and then diminishing at longer pulse durations. 4. Application of 2.5 X 10(-5) M-tetrodotoxin (TTX) produced a fall in resting aiNa of 0.55 +/- 0.2 mM (n = 6) and an outward current shift, suggesting that a TTX-sensitive component of steady-state Na+ current exists at potentials in the region -65 to -80 mV. 5. TTX greatly reduced the rise in aiNa during a depolarizing pulse train at all pulse durations tested. A fall in aiNa was now found after trains of shorter pulse duration than in control solution. Similar results were obtained in the absence of TTX if the pulse train was initiated from a holding potential which was positive to the Na+ current (iNa) threshold. When iNa had been blocked, using either TTX or a low holding potential, the mean rise in aiNa after a train of 50 ms pulses was 0.25 +/- 0.2 mM (n = 8).(ABSTRACT TRUNCATED AT 400 WORDS)

Dissociation between force and intracellular sodium activity with strophanthidin in isolated sheep Purkinje fibres.

1. We have recorded membrane potential, intracellular Na activity (aiNa) and force of contraction in sheep Purkinje fibres. Force and aiNa were recorded continuously and simultaneously during exposure to strophanthidin and its subsequent washing off. 2. Exposure to strophanthidin in concentrations of 1.5 X 10(-7), 5 X 10(-7) and 10(-5) M caused an increase in force of contraction which was temporally dissociated from the increase of aiNa. 3. There was hysteresis in the relationship between force and aiNa when the period of increasing force was compared to the period of decreasing force. When force increased on exposure to strophanthidin, the same aiNa was always associated with a higher force than when force was decreasing while washing off the drug. 4. For the same rise of aiNa higher doses of strophanthidin produced larger rises of force than lower doses. 5. When diphenylhydantoin was present in the bathing solution at concentrations of 10(-5) and 10(-4) M, the relation between force and aiNa with 10(-5) M-strophanthidin had a less steep slope, but still displayed hysteresis. 6. The relationship between force and aiNa during changes of the bathing K concentration also displayed a hysteresis, which was in the same direction as that found with strophanthidin. 7. These results are discussed in relation to proposed mechanisms of action of strophanthidin and more generally in relation to the factors linking force of contraction and aiNa. We conclude that in sheep Purkinje fibres the increase of force caused by strophanthidin is not solely due to an increase of aiNa, and that other interventions can also result in hysteresis between force and aiNa.