Metabolic aspects of ionic shifts in toad muscle (original) (raw)
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The Relationship Between Sodium, Potassium, and Chloride in Amphibian Muscle
Journal of General Physiology, 1957
The results will be employed (a) to show that the accumulation of intracellular K + is not associated with a Donnan mechanism, and (b) to suggest that the existence of a sodium pump in the resting muscle cell is an unnecessary postulate. Methods Treatment of Musdes.-Muscles which were analysed in ~ivo were carefully dissected from the animal, blotted, weighed, and immediately subjected to the analytical procedure outlined below. Isolated muscles were removed from the animal, blotted and weighed, soaked in the appropriate solution for the Stated time, blotted, weighed again, and then analysed. It will be noted that we did not obtain a dry weight for our muscles, as in previous experiments. This was due to the fact that the analysis of chloride required a wet extraction. The results of our analyses over the preceding 4 years had shown that the water content of muscles in v/vo was constant (27). By means of the above two weighings any volume change could be detected, and hence any change in dry weight. Usually 10 pairs of muscles were used in each experiment. The companion muscle always served as a control for the treated, and unless otherwise stated was soaked in normal Ringer for a comparable time. The results were then subjected to the appropriate statistical analysis. Collection of Blood.-Blood was taken by cardiac puncture into a heparinized syringe. It was placed in a centrifuge tube under an oil seal, and spun immediately. Tke Estimation of Na + K + and C/_-Preliminary experiments had shown that there was an almost complete loss of C1-on asking at 560°C. Eventually it was found that the following technique was satisfactory. The muscle was very finely sliced in a centrifuge tube with scissors, the scissors were then thoroughly washed down with N/50 nitric acid, and the volume made up to l0 ml. in a centrifuge tube. The tubes were allowed to stand at room temperature for 48 hours. They were then stirred thoroughly and centrifuged; 3 ml. of the supernatant was accurately removed for the estimation of Na + and K + by means of a Beckman flame spectrophotometer. The proteins in the remainder of the supernatant were precipitated by the method of Somogyi (31), and the filtrate was used for the estimation of CI.-The efficiency of the extraction method was compared with ashing techniques on companion muscles, and the results showed that the removal of Na + and K + was complete. The chloride ion was estimated by a potentiometfic technique similar to that of Sanderson (22). We are indebted to Mr. Gordon Bennett for the following modification of the above method. The titration apparatus consisted of an inert reference electrode of platinum mounted in the tip of a microburette containing 0.005 N silver nitrate. The burette tip was just immersed in the chloride solution, which was contained in a small glass bucket. A pure silver wire electrode was placed in the bucket, and the contents were stirred by a constant speed electric motor. The main functional modifications of Sanderson's circuit were such as to achieve the greater sensitivity and higher stability necessary when working with chloride
Sodium Currents in Skeletal Muscle Fibres From the Toad Bufo Marinus
Quarterly Journal of Experimental Physiology, 1984
Single fibres from the sartorius muscle of the toad Bufo marinus were used to study ionic currents, using a fast voltage-clamp method. Sodium currents were analysed following Hodgkin-Huxley formulations. Internal caesium fluoride blocks delayed currents but produced shifts in the equilibrium potential of the early channel due to sodium accumulation. Delayed channels are very unstable when fluoride is used inside the fibre. These instabilities and equilibrium potential shifts were eliminated on replacing fluoride by aspartate in the internal solution. Late peaks of inward current, probably associated with activity at the tubular system, were occasionally observed for small depolarizations. The results are compared with those obtained for other amphibian species and used to explain more general electrophysiological properties of muscle fibres of this tropical toad.
Na-Ca Exchange Studies in Frog Phasic Muscle Cells
Annals of the New York Academy of Sciences, 1991
A net Ca2+ influx exists during contraction and at rest in skeletal muscle cells,'-3 but since the sarcoplasmic reticulum (SR) has a limited capacity to store Ca2+, a homeostatic mechanism must exist that regulates intracellular calcium concentration ([Ca"],), otherwise contractures may occur. Intra-and extracellular Na+ concentrations affect CaZ+ f l u x e~,~,~ which has been interpreted as evidence of a Na-Ca exchange mechanism.6 Recently, transverse tubule vesicles isolated from frog skeletal muscle were found to exhibit substantial Na-Ca exchange activity7.* and a Na-Ca exchanger has been proposed to explain contractility changes due to extracellular Na+ concentrations ("a' ], ,) modifications9~10 or mechanical triggering. I ' We have found that postfatigue caffeine contractures were larger than pre-fatigue ones and proposed that this is due to activity of this exchanger.I2 But the Na-Ca exchanger has been poorly described and no clear functional role has been attributed to it. The present experiments were undertaken to further characterize this mechanism and its functional role in skeletal muscle. The expeiiments were performed on single muscle fibers isolated from the semitendinosus muscle of the frog Rana pipiens. The dissection, isolation of the fibers, and the experimental setup have been described previou~ly.'~ TWITCH TENSION The fibers were stimulated at 0.008 Hz to avoid the staircase effect. A decrease in "a+], enhanced twitch tension (up to 43%) with an increase of time to peak (up to
The Journal of General Physiology, 1990
We examined the effect of depolarization on intracellular pH (pH.~ of normal (pH i ~7.37) and acidified (pHi 5.90-6.70) frog semitendinosus muscle using microelectrodes. A small bundle was superfused with a Na+-free buffered solution (10 mM HEPES, 100% O~, pH 7.35) containing either 2.5 or 25 mM K +. An NH4CI prepLdse was used to lower pH i. At normal phi, depolarization usually produced a slight (0.04) alkalinization, followed by a fall in pHi. of ~0.2. In contrast, in all 25 acidified bundles pH i rose by 0.1-0.7. The rise was greater the lower the initial pH i. It could be imitated by caffeine and blocked by tetracaine and thus was, most likely, initiated by release of calcium. We ascribed the alkalinization to hydrolysis of phosphocreatine (PCr); 2,4-dinitrofluorobenzene abolished it. Biochemical analysis on fibers at the peak of alkalinization showed PCr to be reduced by one-half, while PCr in normal fibers that had been depolarized for the same period (4-6 rain) showed no change. We postulated that low pH i slows glycolysis with its associated ATP formation by reducing glycogenolysis and particularly by reducing conversion of fructose-6-phosphate to fructose-l,6-diphosphate through inhibition of phosphofructokinase (PFK), an enzyme which is known to be highly pH sensitive. Thus PCr hydrolysis would be required to replace much of the hydrolyzed ATP. This postulated effect on PFK is in agreement with the finding that glucose-6-phosphate (in near-equilibrium with fructose-6-phosphate) was increased nearly fivefold in the depolarized acid fibers, but not in the depolarized normal fibers. However, fructose-l,6-diphosphate also increased significantly; 3-phosphoglycerate was not affected. This suggests an additional acid-induced botdeneck between the latter two substrates. We measured the intrinsic buffering power, fl, of frog semitendinosus muscle with small pulses of NH4C1. It was found to vary with pH i according to fl = 144.6-17.2 (pH 0.
Sodium extrusion induced by ammonium ions in toad skeletal muscle
Journal of Experimental Zoology, 1970
A study was made of the ability of ammonium ions to stimulate active sodium extrusion in toad skeletal muscle. In sodium-rich muscles, 15 mM ammonium induced a net loss of sodium against a concentration gradient slightly larger than 5 mM potassium. Similar results were obtained when a sodium efflux was considered. The activation curve of ammonium-stimulated sodium efflux was strongly S-shaped, reaching a plateau at 10-15 mM ammonium; this could be due to more than one activation site on the outside membrane, or to some cooperative effect of ions. The increment in the rate constant for sodium efflux produced by both ammonium and potassium was completely abolished by 10-4M ouabain. The extent of the inhibition by a submaximal dose of this glycoside was independent of the external ammonium concentration up to 30mM. This behavior corresponds to a non-competitive type of inhibition.
Effect of external sodium and calcium on calcium efflux in frog striated muscle
The Journal of Membrane Biology, 1978
The effect of media with different ionic composition on calcium efflux from the dorsal head of semitendinosus muscles of Rana pipiens was studied. The reduction in the fractional loss of 45Ca, when going from normal Ringer's solution to an ONa-OCa medium, was 60 ~. Withdrawal of only Na or Ca from the external medium also caused a significant drop in the fractional loss (33 ~o and 34 ~, respectively). The effect of different concentrations of Ca (studied in the absence of the external Na) was also studied. It was found that a linear function could describe the relationship between the calciumdependent calcium efflux and the external calcium concentration. These results indicate that calcium efflux from frog muscle fibers consists of three major components: one that is dependent on the presence of calcium in the external medium, one that is dependent on the presence of sodium in the external medium, and one that persists in the absence of these two cations.
Pflugers Archiv-european Journal of Physiology - PFLUGERS ARCH-EUR J PHYSIOL, 1978
A study has been made of the mechanism underlying stimulation of the Na efflux in barnacle muscle fibers by protonation of a HCO3-containing bathing medium and by microinjecting Ca2+. This became possible as a result of the availability of the cAMP-dependent protein kinase inhibitor in the lyophilized form. The results obtained are as follows: Injection of the protein kinase inhibitor of Walsh (free of EDTA and phosphate) causes a biphasic effect on the Na efflux: inhibition is followed by stimulation. Omission of external Ca2+ before injecting the protein inhibitor results in abolition of the biphasic response. The delayed stimulation observed in the presence of external Ca2+ is largely abolished by injecting EGTA. Injection of the protein inhibitor causes complete abolition of the stimulatory response of the Na efflux to external acidification. Ouabainpoisoned fibers injected with graded amounts of CaCl2 show a stimulatory response to as little as a 10−6 M-solution. Injection of 0.03 M-protein inhibitor completely reverses the stimulation of the ouabain-insensitive Na efflux caused by injection of 0.1 M-CaCl2. Fibers allowed to soak in Ca2+-free ASW for a short period of time show a marked rise in the ouabain-insensitive Na efflux on restoring external Ca2+ provided the fibers are injected with protein inhibitor beforehand. Injection of 0.1 M-CaCl2 fails to modify the stimulated efflux. Fibers bathed in Ca2+-free ASW for a short period of time show a marked rise in the ouabain-insensitive Na efflux, not following restoration of external Ca2+, but following injection of 0.1 M-CaCl2. This effect is completely reversed by injecting 0.03 M-protein inhibitor. The above results are compatible with the view that a fraction of the ouabain-insensitive Na efflux is modulated by myoplasmic cAMP and that external acidification causes stimulation as the result of activation of protein kinase by newly formed cAMP. They are also compatible with the view that the protein inhibitor of Walsh may act not only as an inhibitor of the cAMP-dependent protein kinase reaction but also as a feedback regulator of the membrane adenyl cyclase system or that the preparation of Walsh may contain an additional substance that has the ability to act as an adenyl cyclase inhibitor.
Coupling of sodium transport to respiration in the toad bladder
The Journal of Membrane Biology, 1975
Energy expenditure and transepithelial sodium transport were measured continuously and simultaneously from isolated urinary bladders of the Dominican toad, Bufo marinus. Sodium transport was measured as the short-circuit current and COz produced by the bladder was measured conductometrically by the method of Maffly. The rates of sodium transport and CO2 production were linearly related. The slope of the regression of sodium transport on COz production, dJrqa/dJco2, was found to be quite similar in paired half bladders but to differ significantly between bladders from different toads. Thus, in this preparation there appears to be no unique stoiehiometric ratio characterizing sodium transport and metabolism and past efforts to arrive at such a value by averaging results obtained from different animals do not seem warranted. The COz production by the isolated bladder which is unrelated to sodium transport was determined by two means: 1) extrapolating the regression of JNa on Jco2 to JNa=0, and 2) measuring CO2 production with sodium transport suppressed by removal of all sodium from the mucosal bathing medium. The two methods gave values which were in close agreement in each preparation. This suggests that metabolism which supports nontransport activities in this tissue cannot be recruited to support the energy requirement of sodium transport and vice versa.
Journal of Clinical Investigation, 1984
500 ,uM NaCN dropped the short circuit current to 59, 35, and 24% of control values, respectively. Concurrently, tissue ATP measured at 60 min after the addition of NaCN dropped to 79, 66, and 56% of control values, respectively, and tissue ATP/ADP dropped to 68, 50, and 40%, respectively. The data revealed significant correlation between the change in the rate of sodium transport produced by aldosterone or NaCN as measured by the short circuit current and the concentration of ATP (r = 0.96, P < 0.001), as well as ATP/ADP (r = 0.95, P < 0.001). In conclusion, these results support the view that the stimulatory effects of aldosterone on sodium transport involve an increase in ATP or (ATP)/(ADP) X (Pi).