Energetics of relaxation in frog muscle

1. Changes in the levels of phosphocreatine (PCr) and ATP during relaxation after tetanic contraction have been observed and compared with the heat + work production during the same period.2. The results were the same using 2 sec or 15 sec tetani and using 15 or 30 Hz stimulation.3. During relaxation the mean chemical change (of ATP + PCr) was -0.105 +/- 0.078 mumol/g muscle (n = 53). This change is less than would be required to explain the heat + work produced.4. The PCr split during both 2 sec and 15 sec tetani (up to the last stimulus) was less than would be required to explain the heat + work produced. This confirms previous experiments.5. It is concluded that the discrepancy between chemical change and heat + work production does not diminish during relaxation but actually increases.     

Denervated frog skeletal muscle

The effects of denervation on several mechanical and electrical parameters of frog sartorius muscle have been investigated. In denervated muscles, there is no change in the resting potential and a relatively small change in the action potential. The first alteration in the action potential is a reduction of about 30% in the maximum rate of repolarization in muscles that have been denervated for 40 days or longer. Later, the overshoot and maximum rate of depolarization also decline. No tetrodotoxin resistant action potentials could be detected. Fibrillatory potentials were observed infrequently and in most cases in depolarized fibers. Twitch tension is significantly reduced by denervation while the tetanus tension is practically unaffected by denervation. The experiments suggest that the decline in twitch tension produced by denervation reflect a defect in some step of the excitation contraction coupling sequence. On the other hand, post-tetanic potentiation of the twitch is much larger in denervated than in control muscles. This potentiation in denervated muscles is paralleled by an increased action potential duration which returns to its pretetanic duration with a time course indistinguishable from that of the twitch potentiation.     

Thixotropy in frog single muscle fibres

The thixotropic properties of single muscle fibres have been investigated. The effect is larger than in whole muscle and although the time course of stiffness recovery is generally similar, there is an indication that two phases may be involved. Rigor, induced chemically, eliminates thixotropic behaviour.     

Intercellular coupling in frog heart muscle

1. Passive electrical parameters of bullfrog atrial trabeculae were measured in a single gap arrangement. Attention was focussed on the resistance of internal longitudinal pathway. The influence of external Ca²⁺ depletion was tested using EGTA as chelating agent.
2. Morphometry of trabeculae, fine structure of junctional complexes, and distribution of membrane-bound Ca were investigated by light and electron microscopic methods.
3. The specific internal resistance to longitudinal current flow was 523 Ωcm with normal Ringer as perfusing fluid and 1140 Ωcm in EGTA-containing solution. These values are considered to represent the sum of myoplasmic and junctional resistivity.
4. Morphometrical studies indicated an interstitial space of 12%, a mean cell length of 358 μm, and a mean cell diameter of 3.2 μm.
5. In freeze-fractured preparations junctional structures were observed in the form of “atypical gap junctions” consisting of 10 nm particles arranged in a circular or linear array. The number of gap junctions was estimated to range between 20 and 50/cell which is equivalent to a junctional area of 0.01 or 0.03% of total surface area. A mean number of 55 particles/gap junction was calculated. After 20 min of exposure to EGTA the majority of junctional complexes were converted to clusters; the number of particles/gap junction was not significantly altered.
6. The fluorescent dye CTC was used as a probe for membrane-bound Ca of isolated living cells. In normal Ringer a strong fluorescence was seen at the cell surface and in different intracellular compartments. With EGTA both superficial and internal fluorescence disappeared completely.
7. From a combination of electrical and morphometrical data the resistance of intercellular junctions was calculated. Under normal conditions the specific resistance of junctional membrane amounted to 0.4 Ωcm² and the resistance of an individual connexon was of the order of 10¹¹ Ω. With EGTA, the respective values were increased by about 230%. The mechanism underlying this depression of junctional conductance is not clear. It seems not related to a rise of cytoplasmic free Ca²⁺. The EGTA-induced increase in internal resistance was reflected by a decrease of the length constant of a bundle.
8. The nature of “atypical gap junctions” and their relation to tight junctions are discussed. It is concluded that the junctions observed in frog atrial muscle are analogous to gap junctions of insect or mammalian cells in spite of the different size and arrangement of the particles.
9. A theoretical model is presented for the electrical behaviour of a bundle in a single gap arrangement. The bundle is simulated by a partially isolated cable of finite length. Voltage distribution along the cable in response to a d.c. pulse applied externally is described as a function of distance and time.

     

Physostigmine-induced contractures in frog skeletal muscle

Summary Physostigmine in 15 mM concentration at pH 8.4 produces reversible contractures of up to 0.3 P₀ tension output in frog's whole toe muscle or in 7–10 fiber bundles of these muscles. At pH 7.2, the 15 mM physostigmine contracture output is only about 0.10 P₀. The 15 mM, pH 8.4 contractures are essentially unaffected by lack of external Ca²⁺, complete depolarization of the fibers, detubulation by glycerol treatment, and 0°C ambient temperature. These results and other evidence indicate that physostigmine produces contracture by directly releasing activator Ca²⁺ from the sarcoplasmic reticulum (SR). Pretreatment of muscles with 4 mM procaine reduces physostigmine's capacity to produce contracture, evidently by means of a competitive inhibition at SR sites. The above results indicate similarities between physostigmine and caffeine contractures. But the physostigmine action differs in that it is reversible, and, especially, it lacks the ability, strongly characteristic of caffeine, to sensitize a muscle to produce a rapid cooling contracture. The internal action of physostigmine requires that it be permeant, and since it is a weak base (pK_a=8.2), this property is provided by its uncharged base. But, once internal, where the pH =6.8, most of the drug will be protonated and it may act on the SR in this form, in contrast with caffeine which, since its pK_a is about 1.0, acts on the SR as uncharged base.     

Caffeine contractures in denervated frog muscle

Caffeine contracture tension, effect of caffeine on the resting membrane potential, and caffeine influx in normal and denervated frog sartorius muscle have been investigated. Peak caffeine contracture tension is increased after denervation at all caffeine concentrations. The percentage increases in tension are highest for lower caffeine concentrations. The caffeine concentration required for half maximum tension is decreased from about 3.6 mM in control muscles to 2.6 mM in denervated muscles. Caffeine at 3.5 mM produces a depolarization of about 6 mV in control muscles and 16 mV in denervated muscles. The large contracture tensions observed in denervated muscles are not due to the greater depolarization produced by the drug in denervated muscles since innervated muscles depolarized to the same level by external K⁺ do not enhance caffeine contracture tension. Both control and denervated muscles are highly permeable to caffeine. The increases in sarcoplasmic reticulum development (Moscatello et al. 1965) and calcium content (Picken and Kirby 1976) promoted by denervation may explain the larger tension elicited by caffeine in denervated muscles.     

Characterization of frog muscle mitochondria.

Studies on oxidative phosphorylation revealed that, in frog skeletal muscle mitochondria (SKMM) from the thigh, the adenosine diphosphate/oxygen ratio (ADP/O) was 2.8 +/- 0.1 SE, and the respiratory control ratio was 9.5 +/- 0.9, with pyruvate/malate as the substrate. Oxygen uptake rate (Qo2) was 225 mumol O2 per minute per gram mitochondrial protein +/- 13; phosphorylation rate (ADP/O X Qo2 X 2) was 1,230 mumol ADP phosphorylation per minute per gram mitochondrial protein +/- 77; and the phosphorylation capacity (phosphorylation rate times tissue mitochondrial protein content) was 3.6 mumol ADP phosphorylated per gram wet weight of muscle +/- 0.2. Tissue mitochondrial protein content was determined by the measurement of nicotinamide adenine dinucleotide (NADH) oxidase activity. Electron microscopy (EM) revealed intact, isolated, energized twisted mitochondria of a condensed form. Frog sartorius muscle mitochondria gave similar oxidative phosphorylation parameters when investigated independently of the rest of the thigh. These values of SKMM respiration from the frog are similar to those values obtained from pigeon and rabbit heart and rat skeletal muscles. However, because of the low NADH-oxidase activity indicating reduced mitochondrial content (this was verified in low-magnification EM pictures), phosphorylation capacity was significantly reduced in frog skeletal muscle mitochondria.     

The anaerobic recovery of frog muscle

Summary The muscle may undergo a partial recovery of its high energy phosphate stores in the absence of oxygen by the way of glycolysis (anaerobic recovery). This process has been studied in 41 pairs of frog gastrocnemii at different degrees of exhaustion induced by variable trains of supramaximal stimuli. Anaerobic recovery appears to be inadequate to replenish the fraction of muscle high energy phosphate stores (GP=ATP+PC) split as a consequence of the stimulation. The maximal amount of recovery (on the average about 5 Moles of GP per gram of fresh tissue) occurs when the muscle resting stores have been reduced to about 50%. This limitation in the extent of recovery is not a consequence of a reduced availability of glycogen but it is possibly related to the production of some metabolic intermediate, limiting the rate of the glycolytic sequence, likely the accumulation of lactic acid in the fiber. The time course of the anaerobic recovery process is characterized by at1/2 of about 2 min. The efficiency of the process, i.e. the number of the high energy phosphate bonds resynthesized by one Mole of lactic acid, appears to vary between 1.5 and 1.8, being of the same order of magnitude as the GP/L.A. ratio obtained from muscle extracts.     

The kinetics of mechanical activation in frog muscle

1. The kinetics of mechanical activation were examined in muscle fibres of the frog's sartorius muscle, using a voltage clamp to control membrane potential, tetrodotoxin to eliminate electrical activity and microscopic observations to determine the mechanical threshold.2. The strength-duration curve was determined over a range of membrane potentials varying between -52 mV (rheobase) and +90 mV. At 4 degrees C the critical duration was about 11 msec at -30 mV, 4 msec at 0 mV and 2 msec at +40 mV.3. For pulses where V > -10 mV the threshold criterion at 4 degrees C was that the ;area above -30 mV' must exceed about 120 mV msec.4. The effect of a brief subthreshold pulse declines with a time constant of about 3 msec at -100 mV and about 8 msec at -85 mV at 4 degrees C.5. Although the strength-duration curve is well fitted by assuming a first-order mechanism in which the rate of release of activator increases with membrane potential, other experiments show that the over-all mechanism is probably second order in time.6. A short pulse must be at least 50% threshold if it is to give a visible contraction when added to a long pulse which is just below rheobase.7. Delayed rectification was conspicuous with medium or long pulses which were just below the mechanical threshold, but short pulses could give contraction without turning on any appreciable potassium conductance.8. The Appendix extends Falk's (1968) treatment of the charging of the tubular system under a voltage clamp.     

Distribution of acetylcholine-sensitivity in frog slow muscle fibres

The distribution of acetylcholine-sensitive membrane areas in slow muscle fibres of pyriformis muscles of Rana temporaria was examined by iontophoretic application of acetylcholine from high resistance pipettes. ACh-sensitivity varied considerably along individual slow fibres and from fibre to fibre. In some fibres the sensitivity was restricted to segments of less than 100 microns, in others it was continuous over several millimeter. Segments of variable length, but up to several millimeter, were completely insensitive to acetylcholine. Highly sensitive spots (greater than 1,000 mV/nC) were found occasionally, their diameter being of the order of 10-20 microns only. The occurrence at rather regular intervals of ACh-sensitive areas was a rare observation; no evidence was found for a generalized ACh-sensitivity. There were marked differences in the lengths of ACh-sensitive segments between surface fibres and fibres located in deeper layers of the muscles. It is concluded that the ACh-sensitive membrane areas correspond to individual nerve muscle contacts of the small motor system whose spatial distribution is extremely variable. In superficial slow fibres the synaptic contacts seem to be located predominantly on the internal circumference of the fibres.