Inside-positive Potential Research Articles

EFFECTS OF CAFFEINE ON TENSION DEVELOPMENT IN DOG PAPILLARY MUSCLE UNDER VOLTAGE CLAMP

1. Effects of caffeine on tension in the dog papillary muscle were investigated using a voltage clamp with the double sucrose-gap method.2. An S-shaped relationship between membrane potential and developed tension was obtained in Tyrode solution. An increase of the external calcium concentration produced an upward shift of the relationship and a decrease of calcium, a downward shift.3. Caffeine (10 mM/liter) reduced the peak tension at inside positive potentials in Tyrode solution, but scarcely changed the tension at inside negative potentials.4. An administration of caffeine retarded the relaxation phase at first, but the contraction phase was also gradually slowed.5. The magnitude of tension elicited by an action potential after repetitive voltage clamp pulses was dependent on the magnitude of tension during prior clamp pulses in Tyrode solution, but the dependency disappeared in caffeine-treated preparations.6. It was suggested that a tension development in the dog papillary muscle is largely regulated by the sarcoplasmic reticulum and that caffeine reduces the amounts of Ca taken up by and released from the reticulum.

Membrane current and contraction in frog atrial fibres.

1. Membrane current and mechanical activity were recorded from short segments of frog atrial muscle strips using a double sucrose gap voltage clamp arrangement. Experiments were performed at 4-7 degrees C. Two types of contraction were observed dependent upon the duration of the clamp.2. Short-lasting depolarizations caused a flow of Ca inward current, I(Ca), and development of a phasic contraction. Time to peak tension approximated 400 msec. Both I(Ca) and contraction, as functions of membrane potential, had a threshold of about - 40 mV and were maximal at inside positive potentials in normal Ringer fluid. Peak tension decreased at strong depolarizations.3. The minimum time of depolarization required for initiation of a phasic contraction was 40-70 msec. The time necessary for full activation of contraction was 200-300 msec and comparable to the period of time covered by the flow of I(Ca).4. There was no marked change in peak tension upon repetitive depolarization to the same membrane potential.5. Restoration of (phasic) contractility after a preceding contraction was strongly dependent on the level of membrane potential between conditioning and test pulse. Restoration was half complete at potentials around - 45 mV.6. Long-lasting depolarizations generated tonic (sustained) contractions superimposed on the phasic (transient) ones. Threshold potential for initiation of tonic contractions was usually positive to the threshold of phasic contractions. The time taken to attain the final level of tension ranged between 0.7 and 3 sec. Plateau tension, as a function of membrane potential, increased with increasing depolarization and reached a flat maximum at about + 50 mV in normal Ringer fluid.7. At membrane potentials near zero level, plateau tension developed by the tonic mechanism was about twice peak tension due to phasic contraction.8. Removal of Ca ions from the external medium resulted in an almost complete abolition of phasic contraction within 1-2 min and a gradual decrease of tonic contraction during the first 10 min. Application of a ;Ca inhibitor' to normal Ringer fluid caused a strong reduction of both I(Ca) and phasic contraction without affecting tonic contractions.9. It is concluded that phasic contractions are directly activated by the flow of I(Ca). Generation of tonic contractions may be attributed to a Ca transfer mechanism different from I(Ca) or a release of Ca from intracellular stores.

The relation between membrane potential, membrane currents and activation of contraction in ventricular myocardial fibres

1. Membrane currents and contractile responses have been measured in ventricular myocardial preparations under voltage clamp conditions.2. In Tyrode solution, steady-state contraction was obtained only after 5-8 depolarizations to a given potential level. The threshold of steady-state tension was identical to the potential where the calcium inward current, I(Ca), was activated (about -35 mV). Both thresholds were shifted in the same direction along the voltage axis and by the same amount by changing [Ca](o) or [Na](o). Maximum tension was obtained at inside positive potentials.3. The time courses of steady-state tension and of activation of I(Ca) were different by more than one order of magnitude in Tyrode solution. But in order to achieve any appreciable steady-state tension, I(Ca) had to flow during several identical depolarizations. Tension decreased again at potentials above E(Ca). This suggests that calcium inward current must flow in order to fill intracellular calcium stores from which calcium can be released by an unknown mechanism.4. The ability of a fibre bundle to contract again after a preceding twitch is greatly dependent on the membrane potential between two equal depolarizations. In Tyrode solutions with 1.8 and 7.2 mM-CaCl(2) half restoration of this ability occurred at -45 +/- 3 mV (+/- S.E. of mean) and -23 +/- 4 mV, respectively.5. In sodium-free bathing solutions, steady-state tension was attained upon the first depolarization provided I(Ca) was activated. Furthermore, at different potentials, the time courses of activation of tension and of activation of I(Ca) were identical, i.e. tension reached its maximum when I(Ca) was fully activated. This suggests that in sodium-free solutions the flow of calcium ions into the fibre directly activates contraction.

Voltage clamp experiments on ventricular myocardial fibres

1. A voltage clamp method utilizing a sucrose gap and glass microelectrodes was developed and used to study dog ventricular myocardial fibre bundles. The limitations and the reliability of this method are demonstrated by a series of tests.2. A dynamic sodium current, excited at membrane potentials more positive than -65 mV, was measured. The equilibrium potential for this large, rapid inward current depends directly on [Na](o), shifting 29.0 +/- 2.3 mV (+/- S.E. of mean), as opposed to a theoretically expected value of 30.6 mV, when [Na](o) is reduced to 31% of normal.3. Sodium current is inactivated by conditioning depolarizations. Complete inactivation occurs with conditioning potentials more positive than -45 mV, and 50% inactivation occurs at about -55 mV. The location of the inactivation curve shifts along the voltage axis, when [Ca](o) is varied between 0.2 and 7.2 mM.4. A second, much smaller and slower net inward current, with a threshold around -30 mV, and an equilibrium potential above +40 mV was also observed.5. The ;steady-state' current-voltage relationship (after 300-600 msec) exhibits inward-going (anomalous) rectification with negative slope between -50 and -25 mV.6. A small, very slowly developing component of outward current was observed at inside positive potentials. The equilibrium potential for this current, although slightly dependent on [K](o), is neither identical with the potassium equilibrium potential nor with the resting potential in normal Tyrode solution.7. Anatomical limitations, primarily resistance in the extracellular space within the bundle, prevent complete characterization of the rapid, large sodium current, but do not limit the application of the clamp method to the study of other, smaller and slower currents. The evidence for this is discussed extensively in the Appendix.

A study of synaptic transmission in the absence of nerve impulses.

1. The axo-axonic giant synapse in the stellate ganglion of the squid has been used to study synaptic transmission.2. When nerve impulses have been eliminated with tetrodotoxin, synaptic transfer of potential changes can still be obtained by applying brief depolarizing pulses to the presynaptic terminal.3. Suitably matched pulses are as effective as the normal presynaptic spike in evoking post-synaptic potentials. The synaptic delay and the time course of the post-synaptic potential are very similar to that in the normal preparation.4. The synaptic transfer (input/output) characteristic has been studied under different experimental conditions. With brief (1-2 msec) current pulses, post-synaptic response becomes detectable when the presynaptic depolarization exceeds about 30 mV. The post-synaptic potential increases about tenfold with 10 mV increments of presynaptic depolarization.5. Calcium increases, magnesium reduces the slope of the synaptic transfer curve. The influences on this curve of (i) duration of the pulse, (ii) preceding level of membrane potential, (iii) position of recording electrode, (iv) rate of repetitive stimulation are described.6. After loading the synaptic terminal with tetraethylammonium ions, large inside-positive potentials can be produced in the terminal and maintained for many milliseconds.7. By raising the internal potential to a sufficiently high level, synaptic transfer becomes suppressed during the pulse, and the post-synaptic response is delayed until the end of the pulse.8. This observation is in accord with a prediction of the ;calcium hypothesis', viz. that inward movement of a positively charged Ca compound, or of the calcium ion itself, constitutes one of the essential links in the ;electro-secretory' coupling process of the axon terminal.