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1.
Thin strips of frog ventricle were isolated and bathed for 15 min in a solution containing 140 mM KCl, 5 mM Na2ATP, 3 mM EDTA, and 10 mM Tris buffer at pH 7.0. The muscle was then exposed to contracture solutions containing 140 mM KCl, 5 mM Na2ATP, 1 mM MgCl2, 10 mM Tris, 3 mM EGTA, and CaCl2 in amounts to produce concentrations of free calcium from 10-4.8 M to 10-9 M. The muscles developed some tension at approximately 10-8 M, and maximum tension was achieved in 10-5 M Ca++. They relaxed in Ca++ concentrations less than 10-8 M. The development of tension by the EDTA-treated muscles was normalized by comparison with twitch tension at a stimulation rate of 9 per min before exposure to EDTA. In 10-5 M Ca++ tension was always several times the twitch tension and was greater than the contracture tension of a frog ventricular strip in KCl low Na-Ringer. Tension equal to half-maximum was produced at approximately 10-6.2 M Ca++. Intracellular recording of membrane potential indicated that after EDTA treatment the resting potential of cells in Ringer solution with 10-5 M Ca or less was between 5 and 20 mv. Contracture solutions did not produce tension without prior treatment with EDTA. The high permeability of the membrane produced by EDTA was reversed and the normal resting and action potentials restored in 1 mM Ca-Ringer. Similar studies of EDTA-treated rabbit right ventricular papillary muscle produced a similar tension vs. Ca++ concentration relation, and the high permeability state reversed with exposure to normal Krebs solution.  相似文献   

2.
Quinine and caffeine effects on 45Ca movements in frog sartorius muscle   总被引:5,自引:1,他引:4  
1 mM caffeine, which produces only twitch potentiation and not contracture in frog sartorius muscle, increases both the uptake and release of 45Ca in this muscle by about 50 %, thus acting like higher, contracture-producing concentrations but less intensely. Quinine increases the rate of release of 45Ca from frog sartorius but not from the Achilles tendon. The thresholds for the quinine effect on 45Ca release and contracture tension are about 0.1 and 0.5 mM, respectively, at pH 7.1. Quinine (2 mM) also doubles the uptake of 45Ca by normally polarized muscle. However, there are variable effects of quinine upon 45Ca uptake in potassium-depolarized muscle. Quinine (2 mM), increases the Ca, Na, and water content of muscle while decreasing the K content. Both caffeine (1 mM) and quinine (2 mM) act to release 45Ca from muscles that have been washed in Ringer''s solution from which Ca was omitted and to which EDTA (5 mM) was added. These results, correlated with those of others, indicate that a basic effect of caffeine and quinine on muscle is to directly release activator Ca2+ from the sarcoplasmic reticulum in proportion to the drug concentration. The drugs may also enhance the depolarization-induced Ca release caused by extra K+ or an action potential. In respect to the myoplasmic Ca2+ released by direct action of the drugs, a relatively high concentration is required to activate even only threshold contracture, but a much lower concentration, added to that released during excitation-contraction coupling, is associated with the condition causing considerable twitch potentiation.  相似文献   

3.
Calcium retained at binding sites of the sarcoplasmic reticulum membranes isolated from rabbit skeletal muscle requires 10-5 - 10-4 M ATP to exchange with 45Ca added to the medium. The ATP requirement for Ca exchangeability was observed with respect to the "intrinsic" Ca of the reticulum membranes and the fraction of Ca that is "actively" bound in the presence of ATP. Furthermore, a concentration of free Ca in the medium higher than 10-8 M is required for ATP to promote Ca exchangeability. This exchangeability is not influenced by caffeine, quinine, procaine, and tetracaine, and Ca that is either nonexchangeable (in the absence of ATP) or exchangeable (in the presence of ATP) is released by 1–5 mM quinine or tetracaine, but neither caffeine (6 mM) nor procaine (2–5 mM) has this effect. Quinine or tetracaine also releases Ca and Mg bound passively to the reticulum membranes. A possible role of ATP in maintaining the integrity of cellular membranes is discussed, and the effects of caffeine, quinine, and of local anesthetics on the binding of Ca by the isolated reticulum are related to the effects of these agents on 45Ca fluxes and on the twitch output observed in whole muscles.  相似文献   

4.
The sites of lead phosphate precipitation in mouse bladder smooth muscle incubated with adenosine triphosphate and lead nitrate were studied by electron microscopy. The media constituents and incubating conditions were independently varied so that we could determine optimal conditions for histochemical demonstration of ATPase activity in agranular endoplasmic reticulum. Specimens of glutaraldehyde-fixed bladder muscle, frozen, cut into 10–40-µ sections, and incubated for 1 hr at 25°C in medium containing 0.025 M ATP, 0.0025 M lead nitrate, 0.05 M magnesium chloride, and 0.09 M sodium acetate buffer at pH 6.2, exhibited microcrystalline deposits in agranular endoplasmic reticulum and pinocytotic vesicles. Lead salt deposition was also noted in terminal cisternae of sarcoplasmic reticulum in skeletal muscle similarly treated, suggesting that the organelle systems in the two types of muscle cells subserve a common function.  相似文献   

5.
The membrane excitability and contraction were examined in single barnacle muscle fibers with different internal Ca++ concentrations by using buffer solutions made up with EGTA and Ca-gluconate in various proportions. During the passage of dc currents the membrane shows all-or-none spike potentials for internal Ca++ concentrations below about 8 x 10-8 M, oscillatory potential changes in the range between 8 x 10-8 to 5 x 10-7 M, but neither oscillatory nor spike potentials were seen for concentrations above 5 x 10-7 M. All-or-none spike potentials were suppressed when the internal Mg++ concentration exceeded 5 mM. The suppression threshold of the internal Ca++ concentration for the Sr spike is much higher than that for the Ca spike. The threshold concentration of internal Ca++ for contraction was about 8 x 10-7 M.  相似文献   

6.
45Ca efflux was studied in resting anterior byssal retractor muscle. The data are described by a three-compartment system. The most rapidly exchanging compartment, with an average time constant of 7 min, contains about 0.9 mM Ca/liter muscle, and probably represents extracellular space. A second compartment, with a time constant of 83 ± 5 min, contains 1.2 mM Ca/liter, and may represent a membrane calcium store. The presence of a third, or more, compartments, probably representing sarcoplasmic reticulum and contractile proteins, is indicated by the fact that the final time constant is 10 times the 83 min time constant of the second compartment. Serotonin (5HT), on initial application, increases 45Ca efflux from this third compartment(s). This effect has a typical dose-response relationship with a maximum response appearing at 10-7 M5HT. In addition, removal of 5HT causes a secondary increase in 45Ca efflux which has a maximum at a 5HT concentration of 10-7 M and declines at both higher and lower doses.  相似文献   

7.
Fragmented sarcoplasmic reticulum (FSR) membranes isolated from rabbit skeletal muscle are impermeable to inulin-14C (mol wt 5,000), and dextran-14C (mol wt 15,000–90,000) at pH 7.0–9.0, yielding an excluded space of 4–5 µl/mg microsomal protein. In the same pH range urea and sucrose readily penetrate the FSR membrane. EDTA or EGTA (1 mM) increased the permeability of microsomes to inulin-14C or dextran-14C at pH 8–9, parallel with the lowering of the FSR-bound Ca++ content from initial levels of 20 nmoles/mg protein to 1–3 nmoles/mg protein. EGTA was as effective as EDTA, although causing little change in the Mg++ content of FSR. The permeability increase caused by chelating agents results from the combined effects of high pH and cation depletion. As inulin began to penetrate the membrane there was an abrupt fall in the rate of Ca++ uptake and a simultaneous rise in ATPase activity. At 40°C inulin penetration occurred at pH 7.0 with 1 mM EDTA and at pH 9.0 without EDTA, suggesting increased permeability of FSR membranes. This accords with the higher rate of Ca++ release from FSR at temperatures over 30°C. The penetration of microsomal membranes by anions is markedly influenced by charge effects. At low ionic strength and alkaline pH acetate and Cl are partially excluded from microsomes when applied in concentrations not exceeding 1 mM, presumably due to the Donnan effect. Penetration of microsomal water space by acetate and Cl occurs at ionic strengths sufficiently high to minimize charge repulsions.  相似文献   

8.
Zinc depresses the contractile force of electrically driven rat atria logarithmically with time. The threshold concentration is about 5 x 10-6 M zinc and the half-time for contractile depression at 10-4 M is about 25 minutes. Zinc also depresses spontaneous activity of atria and alters the transmembrane potential parameters in a manner similar to quinidine. Unlike quinidine, zinc causes an elevation of the resting potential and an elevation of cellular potassium which varies with time in the same way as the resting potential. Exposure to 10-4 M zinc for 60 minutes causes a statistically significant fall in atrial calcium content and an amount of radioactively labeled zinc is taken up which is quantitatively equal to the calcium lost. Zinc has no effect on rigor caused by iodoacetate but inhibits rigor caused by 1-fluoro-2,4 dinitrobenzene. It is postulated that zinc depression of contractile force is not due to metabolic inhibition, probably not due to quinidine-like action on the cell membrane, but may be due to an interference in the handling of calcium by the cell.  相似文献   

9.
The rate at which the postjunctional membrane of muscle fibers becomes desensitized to the action of carbamylcholine is increased after the muscle has been soaked in solutions containing increased concentrations of calcium. Some further aspects of this effect of calcium were investigated by measuring changes in the input resistance of single fibers of the frog sartorius during local perfusion of the neuromuscular junction with 2.73 x 10-3 M carbamylcholine in isolated muscles immersed in 165 mM potassium acetate. It was found that (a) sudden changes in the local concentration of calcium brought about by perfusing fibers with carbamylcholine solutions containing 20 mM calcium, 40 mM oxalate, or 40 mM EDTA were followed within 20 sec by marked changes in the rate of desensitization; (b) prior to 13 sec after the introduction of carbamylcholine, however, no effect on the input resistance could be detected even though the muscle had been presoaked in 10 mM calcium; (c) the ability of high concentrations of calcium to bring about rapid desensitization disappears when a lower concentration of carbamylcholine (0.137 x 10-3 M) is applied to the muscle fiber. These findings suggest that calcium present in the extracellular fluid can act directly on the postjunctional membrane to promote the desensitization process and that an increased permeability of the membrane to calcium brought about by the presence of carbamylcholine is a factor which contributes to this action.  相似文献   

10.
The distribution of the Mg-dependent ATPase associated with a microsomal fraction of rabbit psoas muscle was studied histochemically and its localization in relation to the vesicles of the fraction and to the structure of intact fixed muscle was determined. Although enzyme activity was retained after fixation in hydroxyadipaldehyde and in glyoxal, it was lost after fixation in glutaraldehyde or after 4 hr fixation in formaldehyde. Activity was optimally demonstrated when incubations were conducted at 17°C, in media containing 125 mM Trismaleate buffer, pH 7.5, 5 mM ATP, 4 mM MgCl2, and 1 mM Pb(NO3)2. After such incubations, activity was present throughout the sarcoplasmic reticulum, but was absent from the T system. Activation by Na or K could not be demonstrated histochemically. However, the other biochemical properties of the enzyme in the isolated vesicles and in intact muscle were similar with respect to Mg dependence, substrate specificity, inhibition by Ca, N-ethyl maleimide, p-hydroxymercuribenzoate, and lack of inhibition by ouabain.  相似文献   

11.
Action potential parameters affecting excitation-contraction coupling   总被引:3,自引:0,他引:3  
In quantifying type B potentiation effects, given earlier merely qualitatively, it is found that Zn2+, 1—50 µM, causes increases in action potential duration, twitch tension, and twitch contraction period time, which are all directly proportional to the log of the concentration. Hence, the duration of the action potential, i.e. the magnitude of its mechanically effective period, is a causal factor quantitatively determining the degree of mechanical activation in the isometric twitch. In higher concentrations of Zn2+ up to 1000 µM, the spike duration and the contraction time continue to increase but the twitch tension is disproportionately smaller, evidently because the high zinc (500—1000 µM) raises the mechanical threshold of excitation-contraction (E—C) coupling and reduces the intrinsic strength of the contractile system. Eserine (1.5 mM) and also high Zn2+ not only cause type B potentiation effects, but also slow the rise of the spike, thus causing retardation of the very onset of tension production, which is even greater for high Zn2+ because of the raised mechanical threshold. This retardation is then succeeded by the faster tension output characteristic of type B potentiation resulting from spike prolongation. Thus, the changes in the consecutive, rising and falling phases of the action potential explicitly register their separate effects in the respective very earliest and directly following periods of twitch output; i.e., each phase of the action potential produces its own mechanical "transform." These transforms, and other effects, suggest that the release of activator Ca2+ from the sarcoplasmic reticulum during E—C coupling can be graded in both the rate and the total amount of the release.  相似文献   

12.
The binding of calcium by isolated sarcoplasmic reticulum from cow uterus was studied. Sarcoplasmic reticulum was prepared by differential centrifugation. Three fractions were obtained: I, sedimented between 2,500–15,000 x g; II at 40,000 x g; and III, at 150,000 x g. Fraction II was further purified on a sucrose density gradient. All three fractions contained considerable amounts of intrinsic calcium, mostly in fraction I. Calcium binding in the presence of ATP1 and Mg also was greatest in fraction I, followed by fraction II, with less in fraction III. Without ATP no calcium was taken up. 5 and 10 mM sodium azide partially inhibited calcium binding in fraction I, but not in fraction II, suggesting the presence of some mitochondria or mitochondrial fragments in fraction I. Calcium binding in fraction II was completely inhibited by 3 mM salyrgan; this fraction thus appears to be sarcoplasmic reticulum. ATPase activity was found in all three fractions, highest in fraction II. It is computed that calcium binding in fractions I and II, on the basis of a 50% yield of protein, is sufficient to elicit contraction by supplying calcium to the contractile proteins of the smooth muscle cell and to regulate relaxation and contraction.  相似文献   

13.
Cesium uptake by sodium-loaded frog sartorius muscles was inhibited 100% by 10-6 M ouabain and 10-6 M cymarin. The doses for 50% inhibition of cesium uptake by five cardiotonic aglycones were 1.5 x 10-6 M for strophanthidin, 2 x 10-7 M for telocinobufagin, 1.6 x 10-6 for digitoxigenin, 2.4 x 10-6 M for periplogenin, and 6.3 x 10-6 M for uzarigenin. Because of the limited solubility of sarmentogenin the maximum concentration studied was 2 x 10-6 M which inhibited cesium uptake about 36%. Inhibition of cesium uptake by cymarin was not reversed during a 3.5 hr incubation in fresh solution while the muscles treated with ouabain and strophanthidin recovered partly during this time. Cymarin was a more potent inhibitor of sodium efflux than strophanthidin and periplogenin was less potent. Increased cesium ion concentration in the external solution decreased the strophanthidin inhibition of cesium uptake but 25 mM cesium did not overcome the inhibition by 10-8-10-6 M strophanthidin. Increased potassium ion concentration in the external solution decreased but did not completely overcome inhibition of sodium efflux by strophanthidin. It is concluded that potassium or cesium ions do not compete with these drugs for a particular site on the ion transport complex. The same structural features of the drugs are necessary for inhibition of ion transport in frog muscle as are required for inhibition of ion transport in other tissues, inhibition of sodium-potassium-stimulated ATPases, and toxicity to animals.  相似文献   

14.
Mechanical threshold as a factor in excitation-contraction coupling   总被引:2,自引:0,他引:2       下载免费PDF全文
I-, CH3SO4 -, and ClO4 -, like other previously studied type A twitch potentiators (Br-, NO3 -, SCN-, and caffeine), lower the mechanical threshold in K depolarization contractures of frog skeletal muscle. In potentiated twitches, I-, Br-, CH3SO4 -, ClO4, and SCN, as already reported for NO3 - and caffeine, slightly shorten the latent period (L) and considerably increase the rate of tension development (dP/dt) during the first few milliseconds of the contraction period. Divalent cations (8 mM Ca2+, 0.5–1.0 mM Zn2+ and Cd2+) raise the mechanical threshold of contractures, and correspondingly affect the twitch by depressing the tension output, increasing L, and decreasing the early dP/dt, thus acting oppositely to the type A potentiators. These various results form a broad, consistent pattern indicating that electromechanical coupling in the twitch is conditioned by a mechanical threshold as it is in the contracture, and suggesting that the lower the threshold, in reference to the raised threshold under the action of the divalent cations, the more effective is a given action potential in activating the twitch as regards especially both its early rate and peak magnitude of tension development. The results suggest that the direct action by which the various agents affect the level of the mechanical threshold involves effects on E-C coupling processes of the T tubular and/or the sarcoplasmic reticulum which control the release of Ca for activating contraction.  相似文献   

15.
At concentrations between 1 to 10 mM, caffeine reduced the Ca-accumulating capacity of fragmented reticulum obtained from frog and rabbit muscle. With 8 mM caffeine enough Ca was released from frog reticulum to account for the force of the contracture. Caffeine did not affect all reticulum membranes equally. The fraction which was spun down at 2000 g was more sensitive than the lighter fractions. The percentage of the total accumulated Ca released by caffeine decreased with decreasing Ca content of the reticulum. In parallel with their known effects on the caffeine contracture, a drop in temperature increased the caffeine-induced Ca release while procaine inhibited it. Caffeine also inhibited the rate of Ca uptake, which may in part account for the prolongation of the active state caused by caffeine.  相似文献   

16.
An apparatus is described which collects the effluent from the center 0.7 cm of a single muscle fiber or bundle of muscle fibers. It was used to study the efflux of 45Ca from twitch muscle fibers. The efflux can be described by three time constants 18 ± 2 min, 300 ± 40 min, and 882 ± 172 min. These kinetics have been interpreted as those of a three-compartment system. The fastest is thought to be on the surface membrane of the muscle and of the T system. It contains 0.07 ± 0.03 mM Ca/liter of fiber and the Ca efflux is 0.11 ± 0.04 pM Ca/cm2. sec. The intermediate rate compartment is thought to represent the Ca in the longitudinal reticulum. It contains approximately 0.77 mM Ca/liter. Only the efflux from this compartment increases during stimulation. The most slowly exchanging compartment is poorly defined. Neither Ca-free nor Ni-Ringer solutions alter the rate of loss from the fastest exchanging compartment. Ni apparently alters the rate of loss from the slowest compartment.  相似文献   

17.
The interaction between caffeine and calcium on the rate of desensitization of muscle postjunctional membrane (PJM) receptors during the sustained application of 0.27 mM carbamylcholine (CARB) has been studied in vitro on the sartorius muscle of the frog. The rate of PJM repolarization with CARB added to the solution bathing the muscle or the recovery of the effective transmembrane resistance (EMR) during the microperfusion of CARB directly onto the end-plate region of individual fibers was used as an index of the rate of desensitization. Caffeine (1.5 mM) increased the rate of PJM repolarization with bulk application of CARB in a 1.8 or 10 mM calcium Ringer solution but had no effect on PJM repolarization in a calcium-deficient, 4 mM magnesium Ringer solution. For EMR measurements the preparation was rendered mechanically quiescent by repeated challenges with isotonic KCl during an exposure of several hours to a calcium-free, 4 mM magnesium-1 mM EGTA Ringer solution. In these fibers, the microperfusion of 0.27 mM CARB together with 1.8 mM calcium plus 1.5 mM caffeine significantly increased the rate of EMR recovery above that observed in the absence of caffeine. Raising the calcium concentration to 10 mM had a similar effect; however, no additional increase was noted by the inclusion of 1.5 mM caffeine. It is suggested that the major role of caffeine in PJM desensitization is to increase the calcium permeability of the surface membrane. The transmembrane movement of calcium and the consequent intracellular accumulation of calcium is seen as a critical factor in controlling the rate of PJM desensitization.  相似文献   

18.
Sartorius muscle cells from the frog were stored in a K-free Ringer solution at 3°C until their average sodium contents rose to around 23 mM/kg fiber (about 40 mM/liter fiber water). Such muscles, when placed in Ringer''s solution containing 60 mM LiCl and 50 mM NaCl at 20°C, extruded 9.8 mM/kg of sodium and gained an equivalent quantity of lithium in a 2 hr period. The presence of 10-5 M strophanthidin in the 60 mM LiCl/50 mM NaCl Ringer solution prevented the net extrusion of sodium from the muscles. Lithium ions were found to enter muscles with a lowered internal sodium concentration at a rate about half that for entry into sodium-enriched muscles. When sodium-enriched muscles labeled with radioactive sodium ions were transferred from Ringer''s solution to a sodium-free lithium-substituted Ringer solution, an increase in the rate of tracer sodium output was observed. When the lithium-substituted Ringer solution contained 10-5 M strophanthidin, a large decrease in the rate of tracer sodium output was observed upon transferring labeled sodium-enriched muscles from Ringer''s solution to the sodium-free medium. It is concluded that lithium ions have a direct stimulating action on the sodium pump in skeletal muscle cells and that a significantly large external sodium-dependent component of sodium efflux is present in muscles with an elevated sodium content. In the sodium-rich muscles, about 23% of the total sodium efflux was due to strophanthidin-insensitive Na-for-Na interchange, about 67% being due to strophanthidin-sensitive sodium pumping.  相似文献   

19.
Isolated tetrameric particles (166S) derived from the crystalline lattices known to appear in hypothermic chicken embryos consist of mature 80S ribosomes which contain all species of ribosomal RNA and a complete set of ribosomal proteins. Ribosome tetramers are not a special type of polysomes since in solutions of high ionic strengths (500 mM KCl and 50 nM triethanolamine-HCl buffer) containing 5 mM MgCl2 they dissociate into 40S and 60S ribosomal subunits, without the need of puromycin, and at a concentration of Mg++ higher than 3 mM they are not disassembled by mild RNase treatment. Tetramers spontaneously disassemble into 80S monomers when the Mg++ concentration is lowered to 1 mM at relatively low ionic strength. Tetramers failed to couple in vitro puromycin-3H into an acid-insoluble product, indicating the lack of nascent polypeptide chains. Although tetramers have no endogenous messenger RNA activity, they can be programmed in vitro with polyuridylic acid (poly U) to synthesize polyphenylalanine. All ribosomes within a tetramer can accept poly U, without the need of disassembly of the tetramers into monomers or subunits.  相似文献   

20.
Skeletal muscle myofibrils, in the presence of 2 mM MgCl2 at pH 7.0, were found to have two classes of calcium-binding sites with apparent affinity constants of 2.1 x 106 M -1 (class 1) and ∼3 x 104 M -1 (class 2), respectively. At free calcium concentrations essential for the activation of myofibrillar contraction (∼10-6 M) there would be significant calcium binding only to the class 1 sites. These sites could bind about 1.3 µmoles of calcium per g protein. Extraction of myosin from the myofibrils did not alter their calcium-binding parameters. Myosin A, under identical experimental conditions, had little affinity for calcium. The class 1 sites are, therefore, presumed to be located in the I filaments. The class 1 sites could only be detected in F actin and myosin B preparations which were contaminated with the tropomyosin-troponin complex. Tropomyosin bound very little calcium. Troponin, which in conjunction with tropomyosin confers calcium sensitivity on actomyosin systems, could bind 22 µmoles of calcium per g protein with an apparent affinity constant of 2.4 x 106 M -1. In view of the identical affinity constants of the myofibrils and troponin and the much greater number of calcium-binding sites on troponin it is suggested that calcium activates myofibrillar contraction by binding to the troponin molecule.  相似文献   

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