兔膈肌肌浆网Ca2+-ATPase活性及钙离子的转运

本文分别用定磷法测定兔膈肌SR Ca 2 -APTase 活性、Fura-2 荧光法测定SR Ca 2 释放、摄取动力学和[ 3 H] -Ryanodine 与RyR 结合实验测定SR RyR 的量,分析其功能特性。 结果显示兔隔肌、心肌和骨骼肌SR Ca 2 -APTase 活性分别为70.13 ±8.25、 41.25 ±6.25 和120.17± 17.03 m mol/L pi/mg 蛋白/h1 。膈肌的SRCa 2 -APTase 活性显著高于心肌P<0.01 。但明显低于骨骼肌P<0.01; 膈肌SR Ca 2 释放量和摄取速度显著快于心肌（P<0.01）,但明显低于骨骼肌（P<0.01）;膈肌SR RyR 同[ 3 H] Ryanodine 的最大结合值（Bmax）是0.78 ±0.05pmol/mg 蛋白,其解离常数（KD）是6.93 1.13nmol/L,分别位于心肌和骨骼肌范围内。本文认为膈肌Ca 2 释放单位、SR Ca 2 -APTase 和SR Ca 2 释放摄取动力学分别具有心肌和骨骼肌的一些特征,其Ca 2 释放可能具有变构偶联和CICR 偶联两种形式,心肌型DHPR 亚型,RyR 3 和SERCA 2 a 的存在可能是膈肌ECC 依赖于细胞外Ca 2 的主要原因。     

Intracellular localization of the caffeinesensitive form of Ca++-dependent ATPase of the sarcoplasmic reticulum

Bulletin of Experimental Biology and Medicine -     

Ca2+ uptake by cardiac sarcoplasmic reticulum ATPase in situ strongly depends on bound creatine kinase

The role of creatine kinase (CK) bound to sarcoplasmic reticulum (SR), in the energy supply of SR ATPase in situ, was studied in saponin-permeabilised rat ventricular fibres by loading SR at pCa 6.5 for different times and under different energy supply conditions. Release of Ca²⁺ was induced by 5 mM caffeine and the peak of relative tension (T/T _max) and the area under isometric tension curves, S _T, were measured. Taking advantage of close localisation of myofibrils and SR, free [Ca²⁺] in the fibres during the release was estimated using steady state [Ca²⁺]/tension relationship. Peak [Ca²⁺] and integral of free Ca²⁺ transients (S[Ca²⁺]_f) were then calculated. At all times, loading with 0.25 mM adenosine diphosphate, Mg²⁺ salt (MgADP) and 12 mM phosphocreatine (PCr) [when adenosine triphosphate (ATP) was generated via bound CK] was as efficient as loading with both 3.16 mM MgATP and 12 mM PCr (control conditions). However, when loading was supported by MgATP alone (3.16 mM), T/T _max was only 40% and S[Ca²⁺]_f 31% of control (P < 0.001). Under these conditions, addition of a soluble ATP-regenerating system (pyruvate kinase and phosphoenolpyruvate), did not increase loading substantially. Both S _T and S[Ca²⁺]_f were more sensitive to the loading conditions than T/T _max and peak [Ca²⁺]. The data suggest that Ca²⁺ uptake by the SR in situ depends on local ATP/ADP ratio which is effectively controlled by bound CK. Received: 23 January 1996/Received after revision: 19 April 1996/Accepted: 3 May 1996     

Calcium transport and ATPase activity of the sarcoplasmic reticulum of normal and denervated rabbit muscles

The properties of the calcium pump of the sarcoplasmic reticulum (SR) from normal and denervated rabbit muscles were studied. The kinetics of transport of Ca⁺⁺ ions in SR from denervated muscles obeys the Michaelis-Menten law. After denervation the rate of fast outflow of Ca⁺⁺ from the vesicles is increased, leading to a decrease in the efficiency of transport and an increase in the activity of basal ATPase. Meanwhile the rate of Ca⁺⁺ accumulation and the activity of transport Ca-ATPase are increased by 1.5 times. The kinetic properties of the reticulum from denervated muscles correspond to the pattern of the contraction-relaxation cycle in those muscles.Department of Biochemistry, Biological Faculty, State University, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 5, pp. 536–539, May, 1976     

The contribution of the sarcoplasmic reticulum Ca2+-transport ATPase to caffeine-induced Ca2+ transients of murine skinned skeletal muscle fibres

The present study was carried out to investigate the contribution of the Ca²⁺-transport ATPase of the sarcoplasmic reticulum (SR) to caffeine-induced Ca²⁺ release in skinned skeletal muscle fibres. Chemically skinned fibres of balb-C-mouse EDL (extensor digitorum longus) were exposed for 1 min to a free Ca²⁺ concentration of 0.36 μM to load the SR with Ca²⁺. Release of Ca²⁺ from the SR was induced by 30 mM caffeine and recorded as an isometric force transient. For every preparation a pCa/force relationship was constructed, where pCa = −log₁₀ [Ca²⁺]. In a new experimental approach, we used the pCa/force relationship to transform each force transient directly into a Ca²⁺ transient. The calculated Ca²⁺ transients were fitted by a double exponential function: Y ₀ + A ₁⋅exp (−t/t ₁) + A ₂⋅exp(t/t ₂), with A ₁ < 0 < A ₂, t ₁ < t ₂ and Y ₀, A ₁, A ₂ in micromolar. Ca²⁺ transients in the presence of the SR Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA) were compared to those obtained in the absence of the drug. We found that inhibition of the SR Ca²⁺-ATPase during caffeine-induced Ca²⁺ release causes an increase in the peak Ca²⁺ concentration in comparison to the control transients. Increasing CPA concentrations prolonged the time-to-peak in a dose-dependent manner, following a Hill curve with a half-maximal value of 6.5 ± 3 μM CPA and a Hill slope of 1.1 ± 0.2, saturating at 100 μM. The effects of CPA could be simulated by an extended three-compartment model representing the SR, the myofilament space and the external bathing solution. In terms of this model, the SR Ca²⁺-ATPase influences the Ca²⁺ gradient across the SR membrane in particular during the early stages of the Ca²⁺ transient, whereas the subsequent relaxation is governed by diffusional loss of Ca²⁺ into the bathing solution. Received: 2 February 1996/Accepted: 1 April 1996     

Mechanism of acidic pH‐induced contraction in spontaneously hypertensive rat aorta: role of Ca2+ release from the sarcoplasmic reticulum

Aim: This study was conducted to investigate the mechanism of acidic pH‐induced contraction (APIC) with regard to Ca²⁺ handling using isometric tension recording experiments. Results: Decreasing extracellular pH from 7.4 to 6.5 produced a marked and sustained contraction of spontaneously hypertensive rat (SHR) aorta, that was 128.7 ± 2.0% of the 64.8 mm KCl‐induced contraction. Verapamil, an inhibitor of voltage‐dependent Ca²⁺ channels (VDCC) significantly inhibited the APIC. In Ca²⁺‐deficient solution, sustained contraction induced by acidic pH was abolished completely, while a transient contraction was still observed suggesting the release of Ca²⁺ from intracellular site. Ryanodine (1 μm ), a ryanodine receptor blocker, and 10 μm cyclopiazonic acid (CPA; a sarco/endoplasmic reticulum Ca²⁺ ATPase inhibitor) abolished the transient contraction induced by acidosis. In normal Ca²⁺‐containing solution, ryanodine significantly decreased the rate of rise as well as maximum level of APIC. Interestingly, ryanodine and CPA showed an additive inhibitory effect with verapamil and the combined treatment of ryanodine or CPA with verapamil nearly abolished the APIC. Conclusions: It is concluded that acidic pH induces Ca²⁺ release from ryanodine/CPA‐sensitive store of sarcoplasmic reticulum in SHR aorta. This Ca²⁺ plays an important role in the facilitation of the rate of rise of APIC, as well as contributing to the sustained contraction via a mechanism which is independent of Ca²⁺ influx through VDCC.     

Prolonged exercise reduces Ca2+ release in rat skeletal muscle sarcoplasmic reticulum

Prolonged exercise decreased the rate of Ca⁺ release in sarcoplasmic reticulum (SR) vesicles isolated from rat muscle by 20–30% when release was initiated by 5, 10, and 20 M AgNO₃. [³H]Ryanodine binding was also depressed by 20% in SR vesicles isolated from the exercised animals. In contrast, the maximum amount of Ca²⁺ released by Ag⁺ remained unaffected by exercise. The passive permeability of SR vesicles and the rate of Ca²⁺ release in the presence of ruthenium red, a known inhibitor of the Ca²⁺ release mechanism, was not affected by prolonged exercise. These results suggest that exercise depressed Ca²⁺ release from SR by directly modifying the Ca²⁺ release channel. Current address: Department of Physics, Portland State University, Portland, OR 97207, USA     

Endothelium-dependent rhythmic contractions induced by cyclopiazonic acid,a sarcoplasmic reticulum Ca2+-pump inhibitor,in the rabbit femoral artery

The vascular responses to cyclopiazonic acid (CPA), an inhibitor of the Ca²⁺-ATPase in the sarcoplasmic reticulum, were investigated in the rabbit femoral artery, suspended in an organ chamber for isometric tension recordings. CPA produced rhythmic contractions in the femoral artery which had been contracted with phenylephrine. CPA, however, did not induce the rhythmic responses in endothelium-denuded arteries. N^G-nitro-L-arginine methyl ester and methylene blue, inhibitors of the formation and the action of nitric oxide, respectively, failed to antagonize the CPA-induced rhythmic contractions in the phenylephrine-contracted artery. In contrast, the CPA-induced rhythmic contractions were abolished by charybdotoxin, a Ca²⁺-activated K⁺ channel antagonist, but not by glibenclamide, a blocker of the ATP-sensitive K⁺ channel. Nifedipine also inhibited the CPA-induced rhythmic contractions in the endothelium-intact artery and relaxed the endothelium-denuded artery treated with CPA. These results indicate that the CPA-induced rhythmic contractions in the phenylephrine-contracted rabbit femoral artery may be attributed to the periodic inactivation of the voltage-dependent Ca²⁺ channel, presumably regulated by the Ca²⁺-activated K⁺ channel. The activation of the K⁺ channel by CPA might occur only when the endothelium is present.     

Calcium transport and ATPase activity in mitochondria and fragments of sarcoplasmic reticulum of the heart and muscles of rabbits with hypercholesteremia

Keeping rabbits on a high-cholesterol diet (1 g/kg) for 3–7 months led to an increase in cholesterol concentration in the mitochondrial membranes and fragments of the sarcoplasmic reticulum (SPR) of the myocardium and skeletal muscles. Saturation of the membranes with cholesterol led to a decrease in efficiency of the Ca-pump of the SPR, as reflected in lowering of the Ca/ATP ratio and an increase in the outflow of Ca⁺⁺ from the SPR. Under these conditions the rate of accumulation of Ca⁺⁺ was higher in SPR than in the mitochondria. Activity of mitochondrial Mg⁺⁺-activated 2,4-DNP-ATPase was reduced in hypercholesteremia.Laboratory of Molecular Pathology and Biochemistry, Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 89, No. 3, pp. 292–294, March, 1980.     

Effect of Ca<Superscript>2+</Superscript> gradient on the structure of sarcoplasmic reticulum membranes

The structure of sarcoplasmic reticulum membranes was studied in the presence of modeled transmembrane Ca²⁺ gradient corresponding to the status of Ca²⁺ depot at different stages of the muscle contraction-relaxation cycle in health and disease. Various sites of the membrane were characterized using spectral analysis of tryptophan, pyrene, and merocyanine-540 fluorescence without evaluating specific changes in the molecules of membrane components (Ca²⁺-ATPase, ryanodine receptor, and lipids). The transmembrane Ca²⁺ gradient modulates the protein-lipid interactions and structural characteristics of the membrane. The proposed model can be used for studies of the effects of pharmacologically active substances and endogenous regulators. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 11, pp. 517–521, November, 2007     

Changes in voltage activation of contraction in frog skeletal muscle fibres as a result of sarcoplasmic reticulum Ca2+-ATPase activity

The effects of cyclopiazonic acid, a specific sarcoplasmic reticulum Ca²⁺-ATPase inhibitor, on isometric tension were studied in response to prolonged steady-state depolarization induced by a rapid change in extracellular potassium concentration (potassium contractures) in frog semitendinosus muscle fibres. Cyclopiazonic acid (1–10 μM ) enhanced the amplitude and time-course of relaxation of 146 m M potassium contracture. In the presence of cyclopiazonic acid 0.5 μM , the relationship between the amplitude of potassium contractures and the membrane potential shifted to more negative potentials, whereas the steady-state inactivation curve was unchanged. These observations suggest that cyclopiazonic acid has no effect on voltage sensors. The difference between potassium contractures in the absence and presence of cyclopiazonic acid in skeletal muscle fibres implies that the amplitude and slow relaxation of tension during prolonged steady-state depolarization may be expected to depend not only on inactivation of the process regulating calcium release from the sarcoplasmic reticulum but also on the ability of the sarcoplasmic reticulum to pump calcium.     

Activation by intracellular calcium of a potassium channel in cardiac sarcoplasmic reticulum

The effects of low (pCa 7.5 to 3) concentrations of intracellular calcium ion on a single potassium channel in the sarcoplasmic reticulum of canine heart ventricular muscle were investigated using a planar lipid bilayer technique. The low concentrations were obtained by mixing EGTA and calcium chloride. By varying the pCa of the cytoplasmic face between 3 to 7.5, two novel effects were observed. First, an increase in the intracellular Ca²⁺ concentration produced an increase in the unit current amplitude of open states; the voltage-current relationship was ohmic at these concentrations. Second, an increase in the Ca²⁺ concentration increased the open probability. Both these effects of Ca²⁺ were dose-dependent, and were consistently observed in all channels tested. Thus, the SR potassium channel observed appears to belong to the class of Ca²⁺ -activated potassium channels.     

Effect of 10‐day cast immobilization on sarcoplasmic reticulum calcium regulation in humans

This study investigated the effects of 10‐day lower limb cast immobilization on sarcoplasmic reticulum (SR) Ca²⁺ regulation. Muscle biopsies were analysed in eight healthy females for maximal rates of SR Ca²⁺ release, Ca²⁺ uptake and Ca²⁺ ATPase activity at control, during immobilization at day 3 (IM 3), day 6 (IM 6) and day 10 (IM 10). Quadriceps muscle cross‐sectional area (CSA) and 1‐repetition maximum (1RM) leg extension strength were measured to determine the extent of muscle size and strength adaptations. Muscle CSA and strength decreased following 10 days of immobilization (11.8 and 41.6%, respectively, P < 0.01). A decrease in SR Ca²⁺ uptake rate (analysed per g wet wt) was found at IM 3 (13.2%, P=0.05), with a further decrease at IM 10 (19.8% from control, P < 0.01). At IM 10, a decrease in SR Ca²⁺ uptake rate (per mg protein) also occurred (19.9%, P < 0.01). Sarcoplasmic reticulum Ca²⁺ ATPase activity and rate of Ca²⁺ release were not altered with 10 days of immobilization. This study observed a decrease in SR Ca²⁺ uptake rate, muscular atrophy and strength loss over 10 days of immobilization in humans.     

Disturbances of the Ca++ transport enzyme system in membranes of the sarcoplasmic reticulum caused by hydroperoxides of phospholipids and of fatty acids

The hydroperoxide (HP) of phosphatidylethanolamine, if added to a suspension of vesicles of the sarcoplasmic reticulum (SR), was shown to have a weak activating effect on Ca-dependent ATPase and to increase the permeability of SR membranes for Ca⁺⁺, measured during activity of the enzyme. HP of linoleic acid did not affect the parameters of the Ca⁺⁺ transport enzyme system, the activity of Ca⁺⁺-dependent ATPase, the Ca/ATP ratio, or the rate of outflow of Ca⁺⁺ in SR membranes on account of the low level of its incorporation into SR fragments. It is concluded that among the primary molecular peroxidation products (HP of free fatty acids, HP of phospholipids), induced both in vitro (by the Fe⁺⁺+ascorbate system) and in vivo (ischemia, avitaminosis-E), only phospholipid HP is an effective modifier of Ca⁺⁺ transport in SR membranes.This was a combined research project of the M. V. Lomonosov Moscow State University and the I. M. Sechenov First Moscow Medical Institute.Laboratory of Physical Chemistry of Biomembranes, M. V. Lomonosov Moscow State University. Laboratory for Transplantation of Organs and Tissues, Academy of Medical Sciences of the USSR, and Department of Operative Surgery and Topographic Anatomy, I. M. Sechenov First Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Kovanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 2, pp. 145–149, February, 1979.     

Calcium uptake and release modulated by counter-ion conductances in the sarcoplasmic reticulum of skeletal muscle

The sarcoplasmic reticulum (SR) plays the central role in regulating the free myoplasmic Ca²⁺ level for the contractile activation of skeletal muscle. The initial stages of the voltage-controlled Ca²⁺ release mechanism are known in molecular detail. However, there is still very little known about the later stages of Ca²⁺ uptake and total Ca²⁺ turnover in the contraction–relaxation cycle under normal physiological conditions or under conditions influenced by fatigue or disease. Ca²⁺ uptake and release are both accompanied by ‘counter-ion’ movements across the SR membrane which prevent or reduce the generation of SR membrane potentials and balance for electroneutrality in the SR lumen. The SR membrane is permeable for the cations K⁺, Na⁺, H⁺ and Mg²⁺ and the anion Cl^-. Using electron-probe X-ray microanalysis, it has been shown that during tetanic stimulation the Ca²⁺ release was mainly balanced by uptake of K⁺ and Mg²⁺, leaving a charge deficit that was assumed to be neutralized via H⁺ ion or organic counter-ion movement. The low time resolution of electron-probe X-ray microanalysis leaves the possibility of other transient concentration changes in the SR, e.g. for Cl^- ions. Possible physiological roles of the SR counter-ion conductances can be tested using skinned muscle fibre preparations with intact sarcoplasmic reticulum and removed or chemically permeabilized outer sarcolemma. In skinned fibres, the SR K⁺ conductance can be effectively reduced with SR K⁺ channel blockers such as 4-aminopyridine, tetraethylammonium and decamethonium. Interestingly, these blockers increase Ca²⁺ loading as well as Ca²⁺ release, whereas other less specific blockers, such as 1.10-bis-quanidino-n-decane, seem to reduce Ca²⁺ release, possibly also via blocking Ca²⁺ release channels. Thus, it seems very important also to test the effects of counter-currents carried by K⁺, Mg²⁺, H⁺ or Cl^- ions on intact and voltage-clamped single-fibre preparations.