A. de Hemptinne

Intracellular pH and surface pH in skeletal and cardiac muscle measured with a double-barrelled pH microelectrode

The construction of a double-barrelled pH sensitive microelectrode for intracellular use is described.Repetitive measurements of intracellular pH were obtained in rat soleus muscle and sheep Purkinje fibres. They yielded pH values ranging between 7.1 and 7.2 in a CO2/HCO3− buffered medium at 37°C.A lower pH value than that of the bulk solution was found at the surface of the cells using either double-barrelled or single-barrelled pH sensitive microelectrodes.

Intracellular pH and contraction of isolated rabbit and cat papillary muscle: Effect of superfusate buffering

The influence of external buffering on surface pH (pHs), intracellular pH (pHi) and developed twitch tension was investigated in rabbit and cat papillary muscle. pHs and pHi were measured using single and double-barreled microelectrodes respectively. In 20 mM HEPES buffered solution, steady state pHi is close to that in control CO2/HCO-3 (25 mM HCO-3, 5% CO2) solution. pHs and developed tension also do not differ greatly from their control values. Decreasing the HEPES concentration to 5 mM, at constant external pH, lowers pHs considerably. The surface acidosis is associated with a small intracellular acidification; steady state pHi in 5 mM HEPES is always more acid than that in control CO2/HCO-3. A significant decrease in developed tension is also seen in 5 mM HEPES. Alteration of the superfusion velocity influences pHs only slightly. Stimulation of the muscle at high frequency is shown to increase surface acidification, the extent of which is dependent on the buffer concentration. The conclusion from the present experiments is that in papillary muscle external buffering influences intracellular pH and contraction via its effect on pHs.

Voltage Clamp Analysis in Isolated Cardiac Fibres as Performed with Two Different Perfusion Chambres for Double Sucrose Gap

SummaryVoltage clamp experiments were performed on isolated frog atrial trabeculae disposed in 2 different perfusion chambres for double sucrose gap. In one of the perfusion chambres, a “liquid partition system” (L.P.S.) was used; in the other, a vaseline sealing (V.P.S.) method was used to separate the various fluid compartments. From the linear electrical properties, the elements of an apparent equivalent electrical circuit were calculated. The apparent nodal capacity was significantly larger in fibres disposed in the “liquid partition system” than those disposed in the “vaseline partition system”. The apparent “error factor” resulting from the presence of the series resistance was larger in the L.P.S. than in the V.P.S. The apparent “error factor” becomes relatively large when the membrane conductance increases, such as during the flow of the inward current.A rapid desactivation of the peak inward current was found on bringing the command potential back to the resting potential in the V.P.S. This was not found in the L.P.S., indicating better voltage control with the V.P.S. than with the L.P.S.Transmembrane microelectrode recordings during voltage clamp experiment in the V.P.S. indicated satisfactory voltage control during the flow of the peak inward current.Adequate voltage control is lost when notches or irregularities appear on the current traces.

Properties of the outward currents in frog atrial muscle

SummaryThe technique of the double sucrose gap was used to perform voltage clamp experiments on small segments of isolated auricular trabeculae of the frog heart. The voltage dependence and kinetics of the delayed rectification current were analyzed. The total outward current could be dissociated into two components: one component is defined as leakage current (Il) and shows inward rectification; a second component is the delayed rectification current (IK). The activation ofIKcan be described by a Hodgkin and Huxley type of kinetical model. The rate constants are found to be smaller by a factor of about 103 than those described in the giant axon of the squid.The results are used to compute the activation of the delayed rectification current and the total outward current during a normal action potential.

pH aspects of transient changes in conduction velocity in isolated heart fibers after partial replacement of chloride with organic anions.

Conduction velocity in isolated rabbit atrial fibers was continuously measured in solutions having a different anionic composition. When 20 mmol/l of chloride was replaced by 20 mmol/l lactate or other anions of weak organic acids at constant pH 6.8, biphasic initial transient changes in conduction velocity were observed. The produced transient changes had a greater amplitude with organic acids which have a greater pK and lipid/water partition ratio. The magnitude of the transients was also greater at pH 6.8 than at pH 7.5, and also when the buffering capacity of the superfusion solution was smaller. Measurements of intracellular pH (pHi) in sheep Purkinje fibers and of pH at the surface (pHs) of sheep Purkinje and rabbit atrial fibers with pH sensitive microelectrodes, showed a transient increase of pHs and a sustained decrease of pHi on replacement of 20 mmol/l chloride by organic anions of weak acids (at constant pH of the superfusion solution). A combined influence of the transient pHs change and the sustained pHi modification seems to be important in the explanation of the biphasic changes in conduction velocity.

Influence of lactate and other organic ions on conduction velocity in mammalian heart fibers depressed by “metabolic” acidosis

Abstract Conduction velocity in isolated rabbit atrial fibers was continuously measured in several forms of non-respiratory acidosis. Lowering pH by decreasing the bicarbonate concentration, whereby the bicarbonate was replaced either with chloride or lactate, decreases in both cases conduction velocity, but with a different time course. After switching from chloride acidosis directly to lactate acidosis, triphasic transients in conduction velocity occurred. After equilibration, conduction velocity was more rapid in acidosis with lactate, acetate and propionate than in chloride acidosis. Conduction velocity was also greater in l -lactate acidosis than in d -lactate acidosis, which suggests that the reduced depression in lactate acidosis is at least partly the consequence of the utilization of lactate as a substrate. The effect of lactate on conduction velocity was more important in acidosis (pH 6.8) than at normal pH.

The frequency dependence of outward current in frog auricular fibres

SummaryThe voltage-clamp technique has been applied to investigate the frequency-dependence of the membrane currents in isolated frog atria. Two stimulation programs were selected. The experimental results were compared with the predictions of a Hodgkin and Huxley type of empirical mathematical model and a close correspondence was observed.The mechanism responsible for the frequency-dependence of the action potential is discussed in connection with the reported results.

Intracellular pH in depolarized cardiac Purkinje strands

In isolated sheep cardiac Purkinje strands the effect of membrane depolarization on intracellular pH (pHi) and on pHi changes produced by addition and withdrawal of NH4+ and CO2/HCO3− was investigated. pHi was continuously measured with double-barreled glass microelectrodes. Repetitive stimulation at high rate resulted in a moderate intracellular acidification (approximately 0.03 pH unit after a 3 Hz train of 2 min), whereafter pHi returned toward its pre-stimulus level. Prolonged depolarization, evoked either by current injection or by superfusion with high K+ solutions, was accompanied by a small acid shift. In the depolarized cell, addition of NH4+ to the superfusate caused intracellular alkalinization followed by re-acidification which was slower than at normal membrane potential. Following intracellular acidification caused by withdrawal of NH4+, pHi recovery also was slightly slower than in the normally polarized cell. In the depolarized fiber, removal and readdition of CO2/HCO3− produced the expected intracellular alkalinization and acidification respectively. Recovery from CO2-induced acidosis was slowed somewhat in high K+ (low Na+) superfused fibers, not in current depolarized fibers. In the depolarized cell, steady state pHi in CO2/HCO3− containing and in CO2/HCO3− free solution tended to become identical. These experiments support the hypothesis that in the normally polarized Purkinje fiber passive shuttle movement of NH4+/NH3 and CO2/HCO3− occurs and could perhaps at least be partly responsible for the lower steady state pHi as compared to that reached in NH4+-free and CO2/HCO3−-free solutions respectively.

A voltage-clamp study of calcium currents in neurons freshly isolated from the dorsal root ganglion of adult rats

Calcium currents were studied in a preparation of freshly isolated neurons of the dorsal root ganglion (DRG) of adult rats using the whole-cell voltage-clamp technique in conditions of minimal current flow through sodium and potassium channels. A low-threshold (LT) and a high-threshold (HT) current were distinguished on the basis of a different potential of activation. Both currents also showed differences in their peak amplitude, their distribution among the cells, their kinetics, their steady-state inactivation curve and their sensitivity to cadmium. Comparison of the properties of both currents with the known properties of calcium currents in DRG neurons from immature animals indicates a slower rate of inactivation of the LT-current in the adult DRG neuron. The calcium entry blocker flunarizine (10 microM) caused a negative shift of the steady-state inactivation curve of the inactivating component of HT-current, an effect which suggests voltage-dependent inhibition.

Influence of local intracellular pH changes on pH and action potentials in adjacent parts of cardiac purkinje fibers

Isolated sheep cardiac Purkinje fibers were pulled through a latex membrane and both segments were independently superfused with modified Tyrode solutions. Transmembrane potentials and intracellular pH (pHi) were continuously measured in one segment (test compartment, TC), using double-barreled pH sensitive glass microelectrodes, while the internal H+ activity was altered in the adjacent part of the fiber (experimental compartment, EC). In the latter local pHi changes were produced by removal of CO2/HCO3-, by superfusing acidic (pH 6.8) solution, and by addition and subsequent withdrawal of NH4Cl. Withdrawal of CO2/HCO3- in EC was found to have no influence on pHi in TC at 0.35 to 1.0 mm distance. The action potential first shortened and later on prolonged above control duration after switching to HCO3- -free medium. Perfusing EC with an acidic solution had virtually no effect on pHi in TC while action potential duration (APD) increased. Addition and withdrawal of NH4+ in EC decreased, respectively increased, APD. In TC no change in pHi was observed at 0.8 to 1.0 mm distance. At shorter distance a slow acidification was seen, associated with the presence of NH4+ in EC. The presence of amiloride, a blocker of the pHi regulating mechanism, could not unmask a larger pHi change. It is concluded that, in cardiac muscle, large gradients of pHi are possible over a relatively short distance, while electrotonic interaction can produce changes in the time course of the action potential in neighbouring cells having a normal intracellular pH.