Using 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-sisometric tetanus, between 8-9 mEq/1K+ in both species. 2. Potassium concentration in venous effleunt blood (K+ven) 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 muEqK+ 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 K+ven. 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 occuded 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 extra-cellular potassium may locally affect nerve endings, skeletal and smooth muscle cells.

Experimental Physiology : Translation and IntegrationCells with high and fluctuating energy demands (e.g. muscle tissue) require an effective system for metabolic control and energy transfer. The most effective system, integrating energy metabolism into one efficiently regulated metabolic network, is the creatine kinase (CK) shuttle (for review see Walliman et al. 1992;Saks et al. 1996). Creatine kinase (EC 2.7.3.2) controls the near-equilibrium (Kushmerick, 1983) CK reaction:in heart, skeletal muscle, brain and smooth muscle.The spatial organization of creatine kinase isoenzymes has been long recognized, and striated muscle cells are the best example of energy metabolism compartmentation. The CK isoenzymes are localized into energy-producing and energy-utilizing sites, where they are functionally coupled with ATP synthesis (mitochondria, cytosol) or ATPconsuming processes (myofibrils, sarcoplasmic reticulum). This organization of the CK system ensures the regulation of local concentrations of ADP and ATP, maintenance of the optimal ATP/ADP ratio, regulation of adenylate nucleotide fluxes and protection of the adenine nucleotides cellular pool from degradation.It can be seen from eqn (1) that first, the position of the CK reaction equilibrium should be affected by cytoplasmic pH, and second, the CK reaction evidently deviates from equilibrium, and its regulation should be described in terms of non-equilibrium thermodynamics (Mejsnar et al. 1992;Maršík & Mejsnar, 1994). The regulation can be realized by conformational changes of the CK molecule, when its reactive 'closed' conformation is not achieved merely by the substrate-induced energy-minimizing principle (Mejsnar et al. 2002).Stated in another way, any ATPase system that evokes a unidirectional net reverse CK flux towards ATP, by the splitting of ATP will shift the CK reaction out of equilibrium. The functional coupling of myosin ATPase and myofibrillar CK by substrate channelling (ArrioDupont, 1988;Gregor et al. 1999), which is defined as direct transfer of ATP between active sites of these enzymes, emphasizes the key role of the phosphocreatine/creatine Substrate channelling in a creatine kinase system of rat skeletal muscle under various pH conditions The aim of this study was to evaluate myofibrillar creatine kinase (CK) activity and to quantify the substrate channelling of ATP between CK and myosin ATPase under different pH conditions within the integrity of myofibrils. A pure myofibrillar fraction was prepared using differential centrifugation. The homogeneity of the preparation and the purity of the fraction were confirmed microscopically and by enzymatic assays for contaminant enzyme activities. The specific activity of myofibrillar CK reached 584 ± 33 nmol PCr min _1 mg _1 at pH 6.75. Two methods were used to detect CK activity: (1) measurement of direct ATP production, and (2) measurement of PCr consumption. This method of evaluation has been tested in experiments with isolated creatine kinase. No discrepancy in CK activity between the methods was ...

~~E J S N A R ,J., AND JANSI&, L. 1970. Shivering and nonshivering thermogenesis in the bat (hdyotis myotis Borkh.) during arousal from hibernation. Can. J. Physiol. Pharmacol. 4$, 102-106.Nonshivering thermogenesis exists in the bat (Myotis myotis Borkh.) arousing from hibernation at environmental temperatures of 4-6 " C . Nsnshivering thermogenesis is essential for the start of the arousal, and it is stimulated by noradrenaline since hexamethonium prevents the increase in metabolism and body temperature. Injection of noradrenaline abolishes this inhibition by hexamethonium by inducing nonshivering thermogenesis. After simultaneous administration of hexamethonium and aIderlin no calorigenic effect of noradrenaline occurs. Shivering heat production during arousal appears at body temperatures between 18 and 17 "C predominantly. In normotherrnic bats the calorigenic effect of noradrenaline was observed, which indicates that nonshivering thermogenesis might also be present in awake animals. During arousal at 25 "C a great increase in intensity of shivering was observed. Elimination sf nonshivering thermogenesis by hexamethonium does not prevent the attainment of the homoiothermic level of body temperature, and administration of noradrenaline does not speed up the process of arousal. As is evident from the metabolic capacity of the brown fat, the heat derived from this organ could maximally participate in total metabolism by 25% at the beginning and at the late period sf arousal. In the middle range sf body temperatures its significance for total metabolism is only about 18-1 3 % .

Creatine kinase (CK) (E.C. 2.7.3.2) buffers cellular ATP concentration during fluctuating ATP turnover. Muscle cytosolic CK isoform interacts with various subcellular structures where it is functionally coupled with relevant ATPases. However, how this interaction affects its activity is not known. We have therefore studied the interaction of CK with myofibrils and the role of different conformational states of CK molecule induced by ATP, phosphocreatine, ADP and the ATP-creatine pair. Purified rabbit psoas myofibrils with CK specific activity of 0.4+/-0.02 IU/mg were used. The exchange rates between the myofibrillar M-band and its surroundings were measured with fluorofore conjugated CK (IAF) by the Fluorescence Lost in Photobleaching (FLIP) method within a very narrow pH range 7.1-7.15. For CK-IAF without docked substrates, the time derivative of the initial loss of the fluorescent signal within the M-band equalled -3.26 at the fifth second and the decrease reached 82% by the 67th second. For CK-IAF with added substrates, the derivatives fell into the range of -0.95 to -1.30, with respective decreases from 16 to 46% at the 67th second. The results show that the substrates slowed down the exchange rate. This indicates that the strength of the bond between CK and the M-band of myofibrils increased.

No abstract

To estimate oxidative capacity of noncontracting rat skeletal muscle, the isolated gracilis muscle was perfused at various high flow rates with high-PO2 (88 kPa) saline-albumin solution and simultaneously perifused at either low (6.3 kPa) or high PO2 in a calorimeter at 28 degrees C. Under low-PO2 perifusion, specific O2 consumption and heat production rates (MO2 and E, respectively) were flow-rate dependent. E values were all larger than those obtained on blood-perfused preparations at 28 degrees C. MO2 reached 0.47 mumol.min-1.g muscle-1 and E reached 4 mW/g. Normalized to 36 degrees C by means of activation energies determined from 30 and 36 degrees C measurements on nonperfused gracilis strips, these maxima correspond to three times the largest MO2 measured by other authors in blood-autoperfused gracilis. Increasing perifusion PO2 from 6.3 to 88 kPa sharply decreased MO2. These results confirm that MO2 of blood-perfused skeletal muscles in vitro (and a fortiori in vivo) is kept much below its maximum for a noncontracting organ; they also suggest that this maximum MO2 is not necessarily an effect of unphysiologically high PO2 in the tissue cells.

The gracilis muscle was excised from cold-acclimated rats, placed in vitro, and simultaneously perfused via its artery by high pO2 medium and superfused by low pO2 medium. With a doubling of the perfusion rate (from 50 to 100 microliters/min) phosphocreatine and ATP increased by 39% and 44%, respectively.

A general equation for muscle energy balance is derived, describing irreversible macroscopic processes of energy transformation in a muscle as a whole, and the interaction of the muscle with its surroundings. The formulation of the equation stems from the balances of mass, mechanical energy, internal energy and entropy. The equation involves isotonic and isometric contractions, as well as energy dissipation stimulated in a non-contracting state. For an isotonic muscle contraction our formulation approximates the Hill equation. An isometric contraction is approximated by the time-dependence of the external muscle force, involving the fluxes of heat and mass. Using the implicit dissipative component of a pressure tensor (sometimes called the "friction clutch" mechanism), the external force can be expressed in units of power, and this can be quantitatively compared with two other muscle loading states. A noncontracting, energy-dissipative muscle is characterized by an uncoupling between the power of chemical reactions and the dissipative part of the tension tensor; the dissipated energy then manifests itself in fluxes of heat and mass. The quantitative estimation of the dissipative power of a muscle under various physiological conditions should clarify the general role of a muscle, including its dissipative non-contracting state.