A.M. Rubtsov

Histidine-containing dipeptides as endogenous regulators of the activity of sarcoplasmic reticulum Ca-release channels

It is shown that histidine-containing dipeptide carnosine (beta-alanyl-L-histidine), which is present in skeletal muscles in millimolar concentrations, decreases the rate of Ca2+ accumulation by the heavy fraction of sarcoplasmic reticulum from rabbit skeletal muscles. This effect results from the ability of carnosine to induce a rapid Ca2+ release from the heavy sarcoplasmic reticulum vesicles via activation of the ruthenium red-sensitive Ca-channels. The effect of carnosine is dose-dependent that indicates the presence of saturable site(s) for carnosine in the molecules of Ca-channels. The C0.5 value carnosine (the concentration that induces the half-maximal Ca2+ release) is 8.7 mM. The 1 N-methylated derivative of carnosine, i.e., anserine, also induces a rapid Ca2+ release with the half-maximal effect at 2.7 mM. Conversely, neither histidine nor beta-alanine (both separately and in the mixture) cause Ca2+ release. In addition, carnosine increases the sensitivity of Ca-channels to their well-known activators (caffeine, AMP, and Ca2+) and decreases inhibitory effect of low concentrations of Mg2+. It is concluded that carnosine as a component of skeletal muscles can be an endogenous regulator of the sarcoplasmic reticulum Ca-channel activity.

Molecular mechanisms of regulation of the activity of sarcoplasmic reticulum Ca-release channels (ryanodine receptors), muscle fatigue, and Severin's phenomenon.

In this short review of the literature and our own data the characteristics of structural organization of sarcoplasmic reticulum Ca-release channels (ryanodine receptors) in different types of muscles, the participation of other sarcoplasmic reticulum proteins in excitation–contraction coupling and Ca-release channel operation, and the regulation of the channel activity by endogenous low molecular weight compounds are analyzed. Special attention is given to changes that occur in muscle cells during exhausting work and to the role of sarcoplasmic reticulum Ca-release channels in the loss of muscle contractile activity during the development of fatigue. It is concluded that the protection of muscle fibers against fatigue in the presence of the histidine-containing dipeptide carnosine, called in the literature “Severins phenomenon”, is primarily connected with modulation of sarcoplasmic reticulum Ca-release channel activity by carnosine.

Caffeine interaction with the Ca-release channels of heavy sarcoplasmic reticulum. Evidence that 170 kD Ca-binding protein is a caffeine receptor of the Ca-channels

The study of Ca2+- and caffeine-induced Ca2+ release from heavy sarcoplasmic reticulum vesicles under the different conditions suggests that Ca2+ and caffeine can interact with the common receptor of the Ca-release channels. The reticulum membranes were solubilized using nonionic detergent polyoxyethylene 9-lauryl ether, and affinity chromatography on reactive red 120-agarose was carried out. The 170 kD Ca-binding protein which is eluted by caffeine is the most probable candidate for the caffeine receptor of the Ca-channels.

Characteristics of sarcoplasmic reticulum membrane preparations isolated from skeletal muscles of active and hibernating ground squirrel Spermophilus undulatus.

The total Ca-ATPase activity in the sarcoplasmic reticulum (SR) membrane fraction isolated from skeletal muscles of winter hibernating ground squirrel Spermophilus undulatus is ∼2.2-fold lower than in preparations obtained from summer active animals. This is connected in part with ∼10% decrease of the content of Ca-ATPase protein in SR membranes. However, the enzyme specific activity calculated with correction for its content in SR preparations is still ∼2-fold lower in hibernating animals. Analysis of the protein composition of SR membranes has shown that in addition to the decrease in Ca-ATPase content in hibernating animals, the amount of SR Ca-release channel (ryanodine receptor) is decreased ∼2-fold, content of Ca-binding proteins calsequestrin, sarcalumenin, and histidine-rich Ca-binding protein is decreased ∼3-4-fold, and the amount of proteins with molecular masses 55, 30, and 22 kD is significantly increased. Using the cross-linking agent cupric–phenanthroline, it was shown that in SR membranes of hibernating ground squirrels Ca-ATPase is present in a more aggregated state. The affinity of SR membranes to the hydrophilic fluorescent probe ANS is higher and the degree of excimerization of the hydrophobic probe pyrene is lower (especially for annular lipids) in preparations from hibernating than from summer active animals. The latter indicates an increase in the microviscosity of the lipid environment of Ca-ATPase during hibernation. We suggest that protein aggregation as well as the changes in protein composition and/or in properties of lipid bilayer SR membranes can result in the decrease of enzyme activity during hibernation.

THERMAL UNCOUPLING OF THE CA2+-TRANSPORTING ATPASE IN SARCOPLASMIC RETICULUM : CHANGES IN SURFACE PROPERTIES OF LIGHT VESICLES

It is known that the light fraction of rabbit skeletal muscle sarcoplasmic reticulum vesicles can release Ca2+ from the intravesicular space, although the Ca(2+)-conductive channels are present only in the heavy fraction of sarcoplasmic reticulum vesicles. To study the possible pathways of the Ca2+ leakage from light vesicles we have used a short-term treatment for 4.5 min at 45 degrees C which quickly decreases the efficiency of Ca(2+)-transporting ATPase operation without any visible effects on the hydrolytic activity of the Ca(2+)-ATPase in the membranes. The treatment of the vesicles decreased the negative membrane surface potential created by the Ca(2+)-ATPase. Comparative titration of control and heat-treated preparations of light sarcoplasmic reticulum vesicles by K+, Na+, Mg2+, and Ca2+ revealed clear differences in their surface properties. The short-term heating resulted in release of Ca2+ from the vesicles previously loaded with 45Ca2+, which indicates an increase in passive membrane permeability to Ca2+. Study of Ca(2+)-ATPase protein arrangement in the membrane indicated that the heat treatment induced protein oligomerization and some of the Ca(2+)-ATPase molecules acquired intermolecular and intramolecular covalent bonds. From these data, we have concluded that the changes in the surface and structure properties of the vesicle membranes after the short-term heat treatment were the result of clustering of the Ca(2+)-ATPase molecules. This protein rearrangement may create channels for calcium leakage from light sarcoplasmic reticulum vesicles.

Ca-ATPase Activity and Protein Composition of Sarcoplasmic Reticulum Membranes Isolated from Skeletal Muscles of Typical Hibernator, the Ground Squirrel Spermophilus undulatus

Ca-ATPase activity in sarcoplasmic reticulum (SR) membranes isolated from skeletal muscles of the typical hibernator, the ground squirrel Spermophilus undulatus, is about 2-fold lower than that in SR membranes of rats and rabbits and is further decreased 2-fold during hibernation. The use of carbocyanine anionic dye Stains-All has revealed that Ca-binding proteins of SR membranes, histidine-rich Ca-binding protein and sarcalumenin, in ground squirrel, rat, and rabbit SR have different electrophoretic mobility corresponding to apparent molecular masses 165, 155, and 170 kDa and 130, 145, and 160 kDa, respectively; the electrophoretic mobility of calsequestrin (63 kDa) is the same in all preparations. The content of these Ca-binding proteins in SR membranes of the ground squirrels is decreased 3–4 fold and the content of 55, 30, and 22 kDa proteins is significantly increased during hibernation.

Pathways of calcium release from heavy sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle

The active uptake and efflux of Ca2+ from suspensions of vesicles from heavy rabbit muscle sarcoplasmic reticulum have been examined using the antipyrylazo III dye method in the presence of various nueleotide triphosphate substrates to support active Ca2+ accumulation. On addition of ATP, Ca2+ is rapidly accumulated and maintained at high internal concentrations until the substrate for pump protein is exhausted. Ca2+ ‐induced Ca2+ release which is inhibited by ruthenium red can be demonstrated. The kinetics of Ca2+ release via these channels is different from the Ca2+ efflux observed after substrate exhaustion. This rate was found to be dependent on the type of nucleotide triphosphate, decreasing in the order ATP > GTP > CTP > ITP UTP. It is suggested that different conformations of the Ca2+ pump protein induced by the different substrates may result in the creation of pathways for the facilitated diffusion of Ca2+.

Investigation of mechanism of p38 MAPK activation in renal epithelial cell from distal tubules triggered by cardiotonic steroids

Ouabain and other cardiotonic steroids (CTS) kill renal epithelial cells from distal tubules (C7-MDCK) via interaction with Na,K-ATPase but independently of inhibition of Na,K-ATPase-mediated ion fluxes. Recently, we demonstrated that modest intracellular acidification and inhibition of p38 MAPK suppress death of C7-MDCK cells triggered by ouabain. In the present study we investigate the mechanism of p38 MAPK activation in renal epithelial cell from distal tubules evoked by cardiotonic steroids. Using Na+/K+ ionophores (monensin, nigericin) and media with different content of monovalent cations, we revealed that p38 MAPK phosphorylation in ouabain-treated renal epithelial cells is not caused by Na,K-ATPase inhibition and inversion of the [Na+]i/[K+]i ratio. We also demonstrated that attenuation of pH from 7.45 to 6.75 did not alter the level of p38 MAPK phosphorylation observed in ouabain-treated cells. Inhibitors of PKA, PKC, and PKG as well as protein phosphatases were unable to abolish p38 MAPK activation triggered by ouabain. Using phosphotyrosine antibodies we did not detect any effect of ouabain on activation of tyrosine kinases. Thus, our results show that activation of p38 MAPK and cytotoxic action of CTS are independent of intracellular Na+, K+, and H+ concentrations. The molecular origin of intermediates of death signaling induced by CTS via conformation changes of Na,K-ATPase with following activation of p38 MAPK should be examined further.

Effect of colchicine on sensitivity of duck salt gland Na,K-ATPase to Na+

Low molecular mass proteins of the FXYD family that affect the sensitivity of Na,K-ATPase to Na+ and K+ are known to be present in Na,K-ATPases in various tissues. In particular, in Na,K-ATPase from kidney a γ-subunit (with electrophoretic mobility corresponding to molecular mass of about 10 kD) is present, and Na,K-ATPase preparations from heart contain phospholemman (electrophoretic mobility of this protein corresponds to molecular mass of 13–14 kD), which provides for the interaction of heart Na,K-ATPase with cytoskeletal microtubules. Disruption of microtubules by colchicine removes phospholemman from heart Na,K-ATPase preparations. The goal of the present study was to reveal a low molecular mass protein (probably a member of FXYD family) in preparation of Na,K-ATPase from duck salt glands. Immunoprecipitation of solubilized duck salt gland Na,K-ATPase using antibodies against α1-subunit results in the coprecipitation of a 13 kD protein with the Na,K-ATPase complex. Treatment of homogenate from duck salt glands with colchicine removes this protein from the purified preparation of Na,K-ATPase. Simultaneously, we observed a decrease in the sensitivity of Na,K-ATPase to Na+ at pH 6.5. However, colchicine treatment of homogenate from rabbit kidney does not affect either the sensitivity of Na,K-ATPase obtained from this homogenate to Na+ or the content of 10 kD protein (presumably γ-subunit). The data suggest that phospholemman (or a similar member of the FXYD family) tightly interacts with Na,K-ATPase from duck salt glands and binds it to microtubules, simultaneously participating in the regulation of the sensitivity of Na,K-ATPase to Na+.

A protein whose binding to Na,K-ATPase is regulated by ouabain

Immunoprecipitation of Na,K-ATPase from kidney homogenate by antibodies against α1-subunit results in the precipitation of several proteins together with the Na,K-ATPase. A protein with molecular mass of about 67 kD interacting with antibodies against melittin (melittin-like protein, MLP) was found in the precipitate when immunoprecipitation was done in the presence of ouabain. If immunoprecipitation was done using antibodies against melittin, MLP and Na,K-ATPase α1-subunit were detected in the precipitate, and the amount of α1-subunit in the precipitate was increased after the addition of ouabain to the immunoprecipitation medium. MLP was purified from mouse kidney homogenate using immunoaffinity chromatography with antibodies against melittin. The addition of MLP to purified FITC-labeled Na,K-ATPase decreases fluorescence in medium with K+ and increases it in medium with Na+. The enhancement of fluorescence depends upon the MLP concentration. The N-terminal sequence of MLP determined by the Edman method is the following: HPPKRVRSRLNG. No proteins with such N-terminal sequence were found in the protein sequence databases. However, we revealed five amino acid sequences that contain this peptide in the middle part of the chain at distance 553 amino acids from the C-terminus (that corresponds to protein with molecular mass of about 67 kD). Analysis of amino acid sequence located between C-terminus and HPPKRVRSRLNG in all found sequences has shown that they were highly conservative and include WD40 repeats. It is suggested that the 67-kD MLP either belongs to the found protein family or was a product of proteolysis of one of them.