Carnosine protection of Ca2+ transport against damage induced by lipid peroxidation

The authors studied the protective action of carnosine on sarcoplasmic reticulum (SR) membranes from frog skeletal muscles destroyed by ascorbic acid-dependent lipid peroxidation (LPO). It was demonstrated that addition of carnosine to the incubation medium at a concentration of 25 mM sharply decelerated inactivation of Ca-ATPase of SR membranes, maintaining at the same time the coupling of hydrolysing and transport functions of the Ca-pump. When given at the same concentration carnosine inhibited the accumulation of LPO products reacting with 2-thiobarbituric acid. This effect of carnosine was followed by its utilization.     

Role of lipid peroxidation in changes in the structure of Ca-ATPase in skeletal muscle sarcoplasmic reticulum during hypercholesterolemia

Using the method of spin labels it was shown that in hypercholesterolemia (HCh), the following parameters decreased: the velocity of maleimide spin label binding to sarcoplasmic reticulum (SR) Ca-ATPase of rabbit skeletal muscles, the accessibility of spin-labeled thiol groups of the enzyme to potassium ferricyanide and sodium ascorbate, and the mobility of the Ca-ATPase molecule fragment to which the spin label was attached. In addition, intensification of lipid peroxidation was demonstrated in SR membranes. Supplementation of the high-cholesterol diet with alpha-tocopherol resulted in the decreased rates of lipid peroxidation in SR membranes and increased values of the above parameters relative to the values found under HCh. It is concluded that the effect of alpha-tocopherol in vivo on the structure of the Ca-ATPase proteolipid complex in HCh is due mainly to antioxidant properties of the diet-supplementing substance.     

Modification of an enzymic system of Ca2+ transport in sarcoplasmic reticulum during lipid peroxidation. In vivo damages in the development of pathological changes

The role of lipid peroxidation (LPO) in the damages of the enzymic system of Ca2+ transport in sarcoplasmic reticulum (SR) membranes of skeletal and cardiac muscles under conditions of vitamin E deficiency, ischemia and limb reoxygenation as well as in emotional-pain stress was investigated. It was shown that these processes are associated with activation of endogenous LPO in SR membranes "in vivo" and with simultaneous inhibition of Ca2+ transport, (i. e. decrease of the Ca2+/ATP ratio) and inactivation of Ca-ATPase. The degree of damage of the Ca2+ transport system was correlated with the concentration of LPO products accumulated in SR membranes "in vivo and during LPO induction by the Fe2+ + ascorbate system 'in vitro". Injection of natural and synthetic free radical scavengers (e. g. 4-methyl-2.6-ditretbutylphenol, alpha-tocopherol) to experimental animals resulted in practically complete suppression of LPO activation "in vivo" and in partial protection of the Ca2+-transporting capacity of SR membranes. A comparison of experimental results allowed to estimate the role of LPO in SR damage under pathological conditions. Model experiments with "contraction-relaxation" cycles including isolated components of muscle fibers (SR fragments and myofibrils) demonstrated that LPO induction in SR membranes by the Fe2+ + ascorbate system results in complete elimination of the relaxation step in myofibrils due to the loss of the SR affinity to decrease the concentration of Ca2+ in the incubation medium. This effect can be removed by free radical scavengers. The role of LPO in pathological changes of muscle contractility is discussed.     

Transport of Ca2+ in the sarcoplasmic reticulum of skeletal muscles in hyperthermia]

Study of functional changes in sarcoplasmic reticulum membranes of the rat skeletal muscles shows that mild hyperthermia is accompanied by the activation of Ca-transporting system function (the increase in accumulation of Ca ions and the rise of Ca(2+)-ATPase activity are observed), a passive release of this ion being unchanged. The revealed changes are regarded as reaction induced by the body temperature increase. The effectiveness of the SR Ca-pump decreases sharply under heat stroke, but the increase of Ca2+ release from SR vesicles is observed. This is considered to be one of possible factors, causing disorder of electromechanical coupling and the disturbance of the skeletal muscle contractility.     

"Contractile" calcium and relation between the sarcoplasmic reticulum and the T-system in the myocardium and skeletal muscle

On the basis of published experimental data a consideration of the role of Ca ions in the myocardial and skeletal muscle contractility of the warmblooded was made. It has been shown that during the relaxation period Ca concentration in the cardiomyocytes sarcoplasmic reticulum (SR) must be of the order 10(-4) M and the corresponding concentrative gradient of Ca "SR-myoplasma" of 10(3) is maximally possible at the work of Ca-pump (the greater gradients of Ca concentration are the products of individual gradients). During the contraction period Ca "SR-myoplasma" gradient is lowered at the level 1 divided by 10(1). The SR membrane Ca-pump power is approximately 10(2) times greater than one of the sarcolemmal membranes. It was shown that because of spacely morphological peculiarities of the muscle cell structure out of the whole external Ca (coupled with the sarcolemma) only the part of Ca located at T-system can take part in the contractile act. The peculiarities of the T-system structure and the interrelation of the volumes and areas of T-system and SR permit to introduce a notion about "the coefficient of using external Ca in the contractile act" (UCa) and also enable to explain the differences in cardiomyocytes and skeletal myocytes resistance to a decrease of Ca concentration in the external environment.     

Phospholamban decreases the energetic efficiency of the sarcoplasmic reticulum Ca pump

We tested the hypothesis that increased Sarcoplasmic reticulum (SR) Ca content ([Ca](SRT)) in phospholamban knockout mice (PLB-KO) is because of increased SR Ca pump efficiency defined by the steady-state SR [Ca] gradient. The time course of thapsigargin-sensitive ATP-dependent (45)Ca influx into and efflux out of cardiac SR vesicles from PLB-KO and wild-type (WT) mice was measured at 100 nm free [Ca]. We found that PLB decreased the initial SR Ca uptake rate (0.13 versus 0.31 nmol/mg/s) and decreased steady-state (45)Ca content (0.9 versus 4.1 nmol/mg protein). Furthermore, at similar total SR [Ca], the pump-mediated Ca efflux rate was higher in WT (0.065 versus 0.037 nmol/mg/s). The pump-independent leak rate constant (k(leak)) was also measured at 100 nm free [Ca]. The results indicate that k(leak) was < 1% of pump-mediated backflux and was not different among nonpentameric mutant PLB (PLB-C41F), WT pentameric PLB (same expression level), and PLB-KO. Therefore differences in passive SR Ca leak cannot be the cause of the higher thapsigargin-sensitive Ca efflux from the WT membranes. We conclude that the decreased total SR [Ca] in WT mice is caused by decreased SR Ca influx rate, an increased Ca-pump backflux, and unaltered leak. Based upon both thermodynamic and kinetic analysis, we conclude that PLB decreases the energetic efficiency of the SR Ca pump.     

Lipid peroxidation--the factor promoting cholesterol accumulation in cells in atherogenesis

The cholesterol transfer between human erythrocytes and main classes of serum lipoproteins (LP) from healthy donors and artery-coronary disease patients was studied (artery-coronary disease is the main manifestation of atherosclerosis). It is shown that low-density lipoproteins (LDL) are capable of transporting cholesterol to erythrocytes, which lack the specific receptors for LDL. The cell cholesterol content in comparison with erythrocytes incubated without LDL was increased by 11.4%. The effect was even higher in case of LDL, isolated from serum of artery-coronary subjects (the cell cholesterol content was increased by 33.8%). High-density lipoproteins (HDL) accept cholesterol from cell membranes. However, cholesterol-accepting properties of HDL from artery-coronary disease patients were suppressed as compared with normal HDL. Both discovered events must promote the cholesterol accumulation in cell membranes in atherosclerosis. As it is shown by the spin probe method, lipid peroxidation (LPO) causes the disturbance of the structural organization of LP and as the consequence of that--the increase of LDL cholesterol-donating ability and the decrease of HDL cholesterol-accepting ability. The greater LDL are oxidized, the more cholesterol they transport to erythrocytes during incubation. The greater is the level of HDL peroxidation, the stronger their cholesterol-accepting function is suppressed. These results suggest that LPO can play an important role in LP modification, the disturbance of their interaction with cell surface and the cholesterol accumulation in cells in atherosclerosis.     

Modification of an enzymic system of Ca2+ transport in sarcoplasmic reticulum membranes during lipid peroxidation. Induction and regulation systems of lipid peroxidation in skeletal and heart muscles

The enzymic and non-enzymic systems which induce and control lipid peroxidation (LPO) in muscle cells were studied. The maximal activity of enzymic NADH- and NADPH-dependent LPO was observed in sarcoplasmic reticulum (SR) membranes. It was found that an essential role in enzymic LPO induction belongs to superoxide radical anions and to hydroxyl radicals. The maximal concentration of the natural LPO inhibitor, alpha-tocopherol, was detected in SR membranes. The glutathione peroxidase and superoxide dismutase activities were determined in the cytosol fraction of myocytes. The role of compartmentation of enzymic and non-enzymic systems of LPO induction in muscle cells is discussed.     

The role of cholesterol in activating lipid peroxidation in platelets]

The relationship between the cholesterol (Ch) content and the concentration of lipid peroxidation (LPO) products in activated platelets and the effect of these parameters on the structure-function characteristics of platelet membranes were studied. It was found that esterified Ch activates free radical processes occurring in platelets. Nonesterified Ch does not induce the production of primary products of LPO (dienoic conjugates) but promotes the accumulation of a secondary LPO metabolite, malonic dialdehyde, this reaction being mediated via indirect mechanisms. The higher (in comparison with normal) orderliness and orientation of membranes in platelets reflect the increase in the concentration of dienoic conjugates and nonesterified Ch. The observed differences in the aggregability of platelets are due to the changes in the Ch content as well as to the "rigidity" of blood platelets.     

Cholesterol distribution and structural differentiation in the sarcoplasmic reticulum of rat cardiac muscle cells

Summary The polyene compound, filipin, was used as a probe to localize cholesterol in the membranes of the rat cardiac muscle cell, with particular reference to the sarcoplasmic reticulum (SR). Filipin binds specifically to cholesterol (and related 3--hydroxysterols) in membranes, producing distinct deformations which can be viewed by freeze-fracture and used as markers for the presence of cholesterol-rich regions in the membrane plane. In freeze-fracture replicas of filipin-treated rat myocardium, the muscle cells revealed abundant deformations in their plasma membranes, no deformations in mitochondrial membranes, and an intermediate response in the SR. These results are in agreement with the levels of cholesterol reported in isolated fractions of the different membrane types, and confirm the specificity of filipin action. Within the SR, the filipin-induced deformations were not randomly distributed but occurred more commonly in free SR at or near the Z-region of the sarcomere than in other parts of the free SR or the junctional SR. This finding is interpreted as evidence for a non-homogeneous distribution of cholesterol in cardiac muscle cell SR. The possible significance of cholesterol in relation to structural differentiation and function of the SR is discussed.     

Hepatic low density lipoprotein receptor binding and lipid profile in Mastomys natalensis during Plasmodium berghei infection

Plasmodium berghei infection to Mastomys natalensis showed hyper beta-lipoproteinemia. The increase in serum cholesterol is associated with decreased uptake of low density lipoprotein (LDL) by the liver through receptor mediated endocytosis. The membranes prepared from infected M. natalensis exhibit up to 50% decline in high affinity binding sites for human 125I-LDL. Significant increases in serum lipids, cholesterol, triglyceride and lipid peroxide (LPO) contents of liver membrane were observed. Effects of lipid constituents and LPO content of liver membrane in relation to LDL catabolism and other possible mechanisms have been explained.     

The role of free calmodulin in the regulation of calcium-pump activity in erythrocytes

The activity of Ca-pump in inside-out oriented vesicles obtained from erythrocyte membranes after their 30 min treatment with EGTA at 20 degrees C (membranes A) and 37 degrees C (membranes B) was investigated. It was shown that in membranes A placed into an incubation medium containing 0.1 mM EGTA (pH 7.4) the overall effect of exogenous calmodulin is due to the increase in the maximal activity of the enzyme, its affinity for Ca2+ being unaffected thereby. In membranes B placed into the same medium (pH 6.75) the activation of the Ca-pump by calmodulin is due to the increased affinity for Ca2+ at a constant maximal activity of the enzyme. The dependencies of the value of the calmodulin-stimulated component of membranes A and the Ca2+-binding capacity of calmodulin measured by the intensity of N-phenyl-1-naphthylamine fluorescence on the concentration of free Ca2+ are coincident. In the case of membranes B, the stimulation of Ca-pump by calmodulin occurs at much lower Ca2+ concentrations than the Ca2+ binding-induced conformational shifts in calmodulin. The experimental results suggest that the affinity of the Ca-pump for Ca2+ may affect calmodulin existing in a Ca2+-independent state. The hydrophobic interactions between the Ca-calmodulin complex and the Ca-ATPase molecule are apparently essential for the regulation of the maximal enzyme activity.     

Mechanism of involvement of calmodulin in the regulation of affinity to Ca2+ and maximum activity of Ca-pump in the erythrocyte membrane

The activity of the Ca-pump in inside-out oriented human erythrocyte membrane vesicles was studied with the use of 45Ca and membrane filters. It was found that trifluoroperazine fully inhibits the calmodulin-induced increase in the maximal activity of the Ca-pump without affecting the calmodulin-stimulated increase in the Ca-pump affinity for Ca2+. The dependence of calcium concentrations of calmodulin-stimulated components of the Ca-pump activity, both inhibited and noninhibited by trifluoroperazine, as well as the dependence on calcium concentrations of the fluorescence intensity of N-phenyl-1-naphthylamine were analyzed. This analysis revealed essential differences between the mechanisms of calmodulin action on the maximal activity of the Ca-pump and its affinity for Ca2+. The maximal activity was elevated by addition of the Ca-calmodulin complex, whereas the increase in the affinity for Ca2+ was induced by calmodulin alone. These findings were supported by data on the dependence of the Ca-pump activity on calmodulin concentrations at low and saturating concentrations of Ca2+ as well as by data obtained in the study on moderate treatment of erythrocyte membranes with trypsin.     

Change in the molecular properties of sarcoplasmic reticulum Ca-ATPase during modification of the membranes with cholesterol

The thiol reagent NBD-chloride (4-chloro-7-nitro-benzo-2-oxo-1,3-diazole) was used to determine the amount and reactivity of SH-groups of Ca-ATPase of rat skeletal muscle sarcoplasmic reticulum during hypercholesterolemia. Modification of membranes with cholesterol brought about a decrease in the total amount and reactivity of SH-groups at the cost of reduction of rapid SH-groups and decrease of the modification constant of these SH-groups. The masking effect of high concentrations of ATP on the reactivity of SH-groups in hypercholesterolemia was noticed. It is inferred that the reduced efficacy of Ca-pump work found under the same experimental conditions before is a consequence of the modification of sarcoplasmic reticulum membranes with cholesterol and change in the molecular conformation of Ca-ATPase.     

Role of interprotein interactions in the regulation of the sarcoplasmic reticulum Ca-pump

The data on the structural state of sarcoplasmic reticulum membranes in skeletal muscles of rabbit were obtained by EPR-spectroscopy, fluorescent analysis and flash-photolysis and discussed in the paper. Comparison of the functional state of Ca-pump and variations in hydrophobic volume and membrane microviscosity permits concluding that thermoinduced anomalies of the enzymic activity are due to changes in the phase state of lipids. It is shown that changes in the physiochemical state of lipids affect the interprotein interactions in the oligomeric Ca-pump structure. In this case ATP weakens the interaction between Ca-pump globules, while a decrease in the hydrophobic membrane volume intensifies it. An assumption is advanced that the modifying ATP effect on Ca-pump is based on an increase in the functional independence of Ca2+-ATPase monomers, and therefore it is under the control of the membrane lipid phase.     

Variation in the lipid composition of rabbit muscle sarcoplasmic reticulum membrane with muscle type

The compositions of sarcoplasmic reticulum (SR) membranes from rabbit caudofemoralis, tibialus, and soleus muscles (fast, mixed, and slow twitch, respectively) were analyzed. Compared to caudofemoralis (fast twitch) SR, soleus (slow twitch) SR contained a significantly greater percentage of cholesterol, phosphatidylinositol, and sphingomyelin and a lesser percentage of phosphatidylcholine. Correlations between properties reported for the SR isolated from different muscle types and our analyses of the compositions are discussed. We suggest that the greater cholesterol content and the greater sphingomyelin to phosphatidylcholine ratio present in soleus SR contribute to decreased bilayer fluidity and, hence, decreased Ca2+-ATPase activity.     

Structural changes in membrane lipids of the sarcoplasmic reticulum of skeletal muscles during hypercholesterolemia

Membrane lipid phase has been studied in the sarcoplasmic reticulum (SR) of skeletal muscles, using spin probes. Hypercholesterolemia was found to increase rotation and decrease hydrophobicity of water-soluble probe medium located inside SR vesicles. This is probably indicative of the reduction in SR vesicle membranes. At the same time reduced rotation and increased hydrophobicity and regularity of fat-acid probe micro-environment are observed in hypercholesterolemia, with the differences disappearing nearer to the centre of the membrane. It is suggested that POL activation and cholesterol accumulation in SR membranes in hypercholesterolemia lead to greater density of phospholipid molecules in the membrane.     

Effect of cholesterol on the physical state and function of lymphocyte membranes

Effect of gradual increase of cholesterol content in T-lymphocyte membranes on the structure and physical state of plasmic membrane lipids and activities of the membrane-bound enzymes was investigated. The increase in cholesterol content was shown to result in a two-phase change of luminescence parameters of the fluorescent probes dimethylaminochalcone and pyrene, which indicates heterogeneity of cholesterol in the membranes. With the growth of steroid content in the cell membranes, at first, we observed a sharp decrease in the lipid bilayer fluidity and inhibition of Na+, K+-ATPase activity, which at the molar ratio cholesterol/phospholipids 0.6 in thymocyte membranes, remains at the same level. With higher cholesterol concentrations ATPase activity did not change. The effect of cholesterol on ATPase activity was in a good agreement with the effect of membrane lipids on fluidity. It is suggested that two pools of cholesterol molecules exist in the membranes, differing in their effects of bilayer fluidity and functional activity of the membranes.     

Peroxide modification of skeletal muscle sarcoplasmic reticulum in antioxidant deficiency and under the action of ionol. I. Calcium transport into sarcoplasmic reticulum membranes]

It is shown that in case of antioxidant insufficiency (AOI) activation of NADPH- and ascorbate-dependent lipid peroxidation (LPO) in sarcoplasmic reticulum (SR) of skeletal muscles proceeds 1.7 and 4.1 times faster, respectively. Activation of lipid peroxidation in AOI leads to damage of Ca2+ transport processes in SR of skeletal muscles. Under these conditions ATP-dependent accumulation of 45Ca (by 88%) and Ca(2+)-ATPase (by 14%) activity in SR of skeletal muscles falls. In case of AOI a significant disturbance of passive Ca2+ transport in SR of skeletal muscles takes place, being characterized by an increased passive 45Ca output from vesicles due to breakage of the biomembrane permeability as a result of lipid peroxidation of membranes. Treatment of animals with ionol, a synthetic antioxidant, causes a decrease of activated NADPH- and ascorbate-dependent LPO in SR of skeletal muscles and stabilization of Ca2+ transport processes.     

Ganglioside protection of the erythrocyte membranes in myocardial ischemia]

Myocardial ischemia was shown to lead to modification of structural and functional organization of rat erythrocyte membranes. Thus, it was found that the activity of Na+, K+-ATP-ase markedly decreased, while accumulation of LPO products and of lysophosphatidylcholine (lyso--PC) took place in erythrocyte membranes of rats subjected to myocardial ischemia. Using nonpenetrating modifier trinitrobenzosulfonic acid, an increase in the content of modified phosphatidylethanolamine in erythrocyte membranes of ischemic rats was revealed as compared to the membranes of control animals. The intravenous administration of gangliosides (30 mg/kg) resulted in partial normalization of Na+, K+(-)ATPase activity, of LPO product and lysoPC content and of transbilayer distribution of lipids.