F. Vyskočil

Work-induced potassium changes in skeletal muscle and effluent venous blood assessed by liquid ion-exchanger microelectrodes

SummaryUsing liquid ion-exchanger semimicroelectrodes with a side pore, we measured changes of extracellular potassium concentration (Ke+) in adult rabbit and cat gastrocnemius muscles and in venous effluent blood flowing from the cat gastrocnemius muscle during various bouts of activity induced by sciatic nerve stimulation.1.Isometric tetanic contractions (at 50 Hz) of various durations caused transient accumulation of Ke+ which was non-linearly related to the duration of muscle activity. The peak values of Ke+ in response to muscle stimulation were analogous in rabbits and cats, attaining values, e.g. after a 20-s isometric tetanus, between 8–9 mEq/lK+ in both species.2.Potassium concentration in venous effluent blood (Kven+) was transiently increased after isometric tetani. Since blood flow was measured at the same time, it was possible to calculate the amount of K+ lost by the muscle after tetani of various durations. A 32 g gastrocnemius muscle of the cat, for example, loses 9.36±1.52 μEqK+ after a 20-s isometric tetanus, which corresponds roughly to 0.5% of the total muscle potassium content. The loss of K+ in this muscle was 29.3 pEq K+/impulse/100 g fresh muscle tissue.3.There was no evident difference between the amount of K+ released during isometric tetani, or tetanic contractions performed under isotonic conditions. Single twitches evoked by indirect stimulation at 1 Hz for several minutes also induced a small rise in Kven+.4.If the loss of K+ from the muscle into the blood stream is transiently prevented by arterio-venous occlusion installed immediately before a 10-s isometric tetanus, most K+ is released subsequently when blood flow is renewed, if the occlusion lasts for 20–25 s. It is not until blood flow is occluded for 40–60 s that most K+ is apparently resorbed and only a minor portion is released and is to be found in the venous blood.5.The transient accumulation of muscle extracellular potassium may locally affect nerve endings, skeletal and smooth muscle cells.

Non-quantal release of transmitter at mouse neuromuscular junction and its dependence on the activity of Na+−K+ ATP-ase

SummaryThe postjunctional membrane of mouse diaphragm fibres was hyperpolarized by 1–2 mV during local curarization of the end-plate zone in the presence of anticholinesterase. In a solution containing 5 mM K+, the mean hyperpolarization was 1.0±0.35 mV. Hyperpolarization was 1.43±0.30 mV when the activity of the Na−K pump was blocked by 2×10−5 M ouabain and 1.82±0.43 mV when it was blocked by bathing the muscle in a K+-free medium. Reactivation of membrane ATP-ase by addition of potassium after a period in K+-free medium reduced the hyperpolarization to zero, when measurements were made 10–20 min after the readdition. It is concluded, that spontaneous non-quantal leakage of acetylcholine occurs at the mouse neuromuscular junction, as it does in the frog /2/. Conditions which block the Na+−K+-dependent ATP-ase increased the leakage whereas potentiation of the pump activity decreased it.

The measurement of K e + concentration changes in human muscles during volitional contractions

Changes of extracellular potassium concentration ([K+]e) were measured in human muscles during volitional isometric contractions using liquid ion-exchanger electrodes. In principle, an intramuscular injection needle containing a microelectrode with a side-pore was inserted into the brachioradialis muscle. After insertion of the needle, the glass ion-selective microelectrode (ISM) could be moved out of the protective trocar shield into the muscle tissue. The average values of [K+]e in human muscles during maximal effort rose from 4.5 mmol/l K+ to 9.5 mmol/l K+. These values correspond closely to those previously found in muscles of experimental animals.

Modifications of single and double-barrel potassium specific microelectrodes for physiological experiments

Summary1.The shape parameters of glass microcapillaries for making ion specific microelectrodes were studied. It was found, that the greater the angle set by the walls, the more advantageous is the shape for filling the microelectrode. The time response of the precisely prepared microelectrode does not exceed severall msec.2.The preparation of the double-barrel ion specific microelectrodes is described. One barrel was filled with organic ion exchanger and the other with NaCl solution (reference channel). These electrodes are convenient for measurement of ion movements in the excitable tissues (brain, spinal cord etc) where it is necessary to avoid the electrical activity of the cells.3.A side-pore type of ion specific microelectrode was developed for measurement of extracellular K+ concentration during muscle contraction. This type of electrode does not cut the muscle fibres during contractions and the side orifice of the channel prevents the undesirable movement of the ion exchanger during insertion into the muscle.

The dependence of non-quantal acetylcholine release on the choline-uptake system in the mouse diaphragm

The time course of local end-plate hyperpolarization after d-tubocurarine application measured by an intracellular microelectrode was followed in vitro in anticholinesterase-treated mouse diaphragm pinned to the bottom of the perfusion chamber. The d-tubo curarine-induced hyperpolarization, which served as an indicator of non-quantal acetylcholine release, started to decline from 6 mV after 1 h and was negligible after 3 h in continuously perfused preparations. This decline was slowed down by 10 μmol l−1 choline and almost completely prevented by long-term nerve stimulation with a frequency of 3 Hz. The rapid decrease of the d-tubocurarine-induced hyperpolarization was observed within 10–15 min after the application of 1 μmol l−1 hemicholinium-3 and substitution of lithium for sodium. Both these procedures inhibit the fast choline uptake into nerve terminals. Our data suggest that the amount of available acetylcholine for non-quantal release is proportional to the rate of its synthesis and to the number of available carriers in the nerve terminals. Some of our observations might also be explained by postulating that the choline-uptake system as such is responsible for the non-quantal release.

The facilitating effect of gangliosides on the electrogenic (Na+/K+) pump and on the resistance of the membrane potential to hypoxia in neuromuscular preparation

The effects have been investigated of a mixture of gangliosides from beef brain cortex (GM1, GD1a, GD1b and GT1) either added to the bathing medium or injected intraperitoneally on muscle fibres and nerve terminals in mouse diaphragm. The electrogenic (Na+/K+) pump activity of muscle fibres enriched with sodium was increased by 38% after 2-h pretreatment with gangliosides (5×10−8 mol ·l−1). Muscles from animals treated with gangliosides did not show the substantial depolarization of the resting membrane potential (RMP) in K+-free solution (6 h) shown by control muscles. Further, treatment with gangliosides slowed the changes in muscle fibre RMP and frequency of the miniature end-plate potentials in oxygen deprived muscles.

Activation of membrane Na+/K+-ATPase of mouse skeletal muscle by acetylcholine and its inhibition by α-bungarotoxin, curare and atropine

The effect of acetylcholine and of three cholinolytic compounds (α-bungarotoxin, curare and atropine) on electrogenic Na+/K+ pump and activity of the membrane Na+/K+-ATPase of mouse skeletal muscles was studied. It was found that acetylcholine potentiated both the muscle electrogenic ionic pump and the Na+/K+-ATPase activity of crude membrane fractions. The cholinolytic drugs had inhibitory effects on both parameters, with the exception of curare which was ineffective in blocking the electrogenic ionic pump.

The effects of nerve terminal activity on non-quantal release of acetylcholine at the mouse neuromuscular junction.

1. Local endplate depolarization induced by anticholinesterase application to mouse nerve‐diaphragm preparations was taken as a measure of non‐quantal release of acetylcholine. 2. Non‐quantal acetylcholine release occurred within 20‐60 s after anticholinesterase application, either spontaneously or evoked by nerve stimulation. Non‐quantal release declined with time and disappeared after 3‐5 min. 3. The amplitude of stimulation‐evoked non‐quantal release increased with the frequency of stimulation and was maximal at frequencies above 50 Hz. Two stimuli were sufficient to evoke the maximal effect. 4. Micromolar concentrations of atropine, pirenzepine and vesamicol reduced the amplitude and shortened the duration of non‐quantal release. Oxotremorine (10(‐8) M) enhanced the amplitude and ouabain (10(‐4) M) prolonged the duration of non‐quantal release. 5. Our results support the idea that the non‐quantal release is due to the vesicular acetylcholine transport system which becomes transiently a part of the nerve terminal during exocytotic release of quantal acetylcholine.

Electrophysiological and contractile properties of the levator ani muscle after castration and testosterone administration.

Summary1.Electrical and contractile properties of the levator ani muscle were studied in normal rats, in castrated rats and in castrated rats treated with testosterone.2.No significant changes in the frequency of miniature end-plate potentials were found 6 months after castration. The frequency increased already 6 h after testosterone treatment; an increase of about 100% was observed after 7 days of testosterone treatment.3.Castration led to a 2-fold increase of the input resistance of the muscle fibres. After 7 days of testosterone treatment the input resistance was only slightly higher than normal.4.The weight of the muscle was decreased to 18% of the control value after 6 months castration. It increased to 46% after 7 days of testosterone treatment.5.The muscles of castrated animals revealed a prolongation of contraction time and marked changes in maximal rate of tension development and half relaxation time. Partial recovery of these parameters was found after 7 days of testosterone treatment.6.Long-term castration did not induce any denervation-like changes of action potential parameters, and no tetrodotoxin resistance was found in spite of marked muscle atrophy.

Inhibition of non-quantal acetylcholine leakage by 2(4-phenylpiperidine)cyclohexanol in the mouse diaphragm

The drug 2(4-phenylpiperidine)cyclohexanol (AH 5183) caused hyperpolarization by 1.8 +/- 0.6 mV in an end-plate zone of mouse diaphragm fibers without any change in the amplitude of miniature end-plate potentials. This supports the idea that the drug inhibits the non-quantal leakage from motor nerve terminals, probably at those parts of the nerve terminals which were incorporated into the terminal membrane after vesicle exocytosis.