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     

Some properties of the calcium pump of the sarcoplasmic reticulum of the skeletal muscles of rabbits with hypercholesteremia

Fragments of sarcoplasmic reticulum (SR) isolated from the skeletal muscles of rabbits with marked atherosclerosis have impaired ability to accumulate Ca⁺⁺. A decrease in the efficiency of the transport process, expressed as a decrease in the Ca/ATP ratio, is accompanied by activation of Ca-ATPase and by a simultaneous increase in the rate of outflow of Ca⁺⁺ from SR. The temperature dependence of Ca-ATPase, plotted between Arrhenius coordinates, normally has an inflection at 20–21°C, but in hypercholesteremia the graph becomes a straight line. Meanwhile the cholesterol/protein ratio in membrane preparations of SR rises sharply in atherosclerosis. The Ca-ATPase of native membranes has an activation energy (E_a) of 15.6 and 28.7 kcal/mole in regions above and below the inflection respectively. The Ca-ATPase of membranes containing an increased amount of cholesterol has an activation energy of 19 kcal/mole over the whole range of temperatures investigated. It is suggested that the cholesterol level in membrane preparations changes not only the physicochemical characteristics of the membranes, but also the enzymic properties of transport ATPase.Laboratory of Molecular Pathology and Biochemistry, Institute of General Pathology and Pathophysiology, Academy of Medical Sciences of the USSR, Moscow. Department of Biochemistry, M. V. Lomonosov Moscow University. (Presented by Academician of the Academy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 7, pp. 32–35, July, 1978.     

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.     

Effects of age on sarcoplasmic reticulum properties and histochemical composition of fa stand slow-twitch rat muscles

Calcium release activity of sarcoplasmic reticulum and enzyme-histochemical properties were investigated in extensor digitorum longus (e.d.l.) and soleus muscles in young (4 months) and old (24 months) male rats. With age, the caffeine threshold concentration for calcium release from the sarcoplasmic reticulum of soleus skinned muscle fibres showed only minor modifications. On the other hand, in e.d.l. skinned muscle fibres, the caffeine threshold concentration decreased significantly (P < 0.05). The histochemical fibre type composition changed with age both in soleus and in e.d.l. muscles, showing a common transformation toward a more oxidative histochemical profile. In fact, in aged soleus, a significant (P < 0.05) increase was observed of type 1 fibres to represent almost the totality of the muscle fibres (more than 98%), while types 2C and 2A were reduced in proportion. In aged e.d.l. the percentage of type 1 (P < 0.05), 2A and 2X (a recently identified fourth component of the fast-twitch muscle types) fibres increased, with a reduction of type 2B (P < 0.01) fibres. The present results suggest that the changes in contractile properties of aged muscles may be related to the changes not only in fibre composition but also in the mechanism of calcium release from sarcoplasmic reticulum.     

Effect of cyclopiazonic acid,an inhibitor of the sarcoplasmic reticulum Ca‐ATPase,on skeletal muscles from normal and mdx mice

Aim: In this study, we investigated Ca²⁺ loading by the sarcoplasmic reticulum in skeletal muscle from mdx mice, an animal model of human Duchenne's muscular dystrophy, at two stages of development: 4 and 11 weeks. Method: Experiments were conducted on fast‐ (extensor digitorum longus, EDL) and slow‐ (soleus) twitch muscles expressing different isoforms of Ca²⁺‐ATPase, which is responsible for the uptake of Ca²⁺ by the sarcoplasmic reticulum. Results: In sarcoplasmic reticulum vesicles, the ATP‐dependent activity and sensitivity to cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum Ca²⁺‐ATPase, were similar in mdx and normal EDL muscle. Furthermore, in chemically‐skinned fibres from both normal and mdx muscles, the presence of CPA induced a decrease in Ca²⁺ uptake by the sarcoplasmic reticulum. However, the sensitivity to CPA was lower in mdx EDL muscle than in normal muscle. In addition, in EDL muscle from 4‐week‐old mdx mice, the expression of the slow Ca²⁺‐pump isoform (SERCA2a) was significantly increased, without any accompanying change in slow myosin expression. In contrast, the expression and function of the Ca²⁺‐ATPase in mdx soleus muscles at 4‐ and 11‐weeks of development did not differ from those in age‐matched controls. Conclusion: These findings show that in dystrophic muscle, where the Ca²⁺ homeostasis was perturbed, the Ca²⁺ handling by the sarcoplasmic reticulum was altered in fast‐twitch muscle, and this was associated with the expression of the slow isoform of SERCA. In these muscles, reduced Ca²⁺ uptake could then contribute to an elevated concentration of Ca²⁺ in the cytosol, and also to Ca²⁺ depletion of the sarcoplasmic reticulum.     

Ischemic damage to the sarcoplasmic reticulum of skeletal muscles: The role of lipid peroxidation

The development of ischemia was shown to be accompanied by inhibition of the Ca²⁺ enzyme transport system (ETS) (a decrease in the Ca²⁺/ATP ratio and in activity of Ca²⁺-dependent ATPase), which correlates with accumulation of the primary and secondary molecular lipid peroxidation products (POL) in vivo and in the membranes of the sarcoplasmic reticulum (SR) of the skeletal muscles. Administration of antioxidants (2,6-di-tert-butyl-4-methylphenol, -tocopherol) prevents activation of POL in the ischemic muscle and partially protects the Ca²⁺ ETS against injury. Restoration of the blood flow after prolonged ischemia leads to further inhibition of the Ca²⁺ ETS while the concentration of POL products remains unchanged.A joint research project of the M. V. Lomonosov Moscow State University and the I. M. Sechenov First Moscow State Medical Institute.Laboratory of Transplantation of Organs and Tissues, Academy of Medical Sciences of the USSR. Department of Operative Surgery and Topographical Anatomy, I. M. Sechenov First Moscow Medical Institute. Laboratory of Physical Chemistry of Biomembranes, M. V. Lomonosov Moscow State University. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Kovanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 6, pp. 683–686, June, 1977.     

Stress-induced modification of the sarcoplasmic reticulum Ca transport system of the heart and its resistance to endogenous damaging factors

Research Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy or Medical Sciences of the USSR G. N. Kryzhanovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 9, pp. 271–274, September, 1989.     

Calcium uptake and release through sarcoplasmic reticulum in the inferior oblique muscles of patients with inferior oblique overaction

We characterized and compared the characteristics of Ca2+ movements through the sarcoplasmic reticulum of inferior oblique muscles in the various conditions including primary inferior oblique overaction (IOOA), secondary IOOA, and controls, so as to further understand the pathogenesis of primary IOOA. Of 15 specimens obtained through inferior oblique myectomy, six were from primary IOOA, 6 from secondary IOOA, and the remaining 3 were controls from enucleated eyes. Ryanodine binding assays were performed, and Ca2+ uptake rates, calsequestrins and SERCA levels were determined. Ryanodine bindings and sarcoplasmic reticulum Ca2+ uptake rates were significantly decreased in primary IOOA (p < 0.05). Western blot analysis conducted to quantify calsequestrins and SERCA, found no significant difference between primary IOOA, secondary IOOA, and the controls. Increased intracellular Ca2+ concentration due to reduced sarcoplasmic reticulum Ca2+ uptake may play a role in primary IOOA.     

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。     

Sarcoplasmic reticulum of human skeletal muscle: age-related changes and effect of trainirig

The effect of ageing on human skeletal muscle was investigated using needle biopsies from young and aged subjects and from aged subjects trained with different activity patterns. Histochemical staining for myofibrillar ATPase of ageing m. vastus lateralis demonstrated an unchanged fibre type distribution but a selective atrophy of type IIa and type IIb fibres. Analysis of myosin heavy chain (MHC) composition showed that type I MHC increased with ageing (P< 0.05). The relative content of the MHC isoforms correlated with the relative area of the respective fibre types. Sarcoplasmic reticulum (SR) proteins were investigated in muscle extracts by electrophoretic and immunoblotting techniques. When compared to a young control group (28 0.1 years old, n = 7) blots of post-myofibrillar supernatant proteins probed with polyclonal antibodies to the rabbit fast SR Ca-ATPase, a marker of extrajunctional SR, showed that the content of Ca-ATPase was significantly lower (P < 0.05) in the old control group (68 ± 0.5 years old, n= 8). On the other hand the content of calsequestrin (CS), the major intraluminal protein of SR terminal cisternae (TC), and of the 350-kDa ryanodine-binding protein, which is localized in the junctional regions of TC, did not show a concomitant decrease. These results suggest that ageing differentially affects extrajunctional and junctional SR of human skeletal muscle. These age-related changes were not observed within a group of old strength-trained subjects.