Regulation by Ca2+-calmodulin-dependent phosphorylation of passive transport of Ca2+ in the myometrial sarcolemma

Closed vesiculate preparations of pig myometrium sarcolemma (predominantly with inside-out orientation) are characterized by passive permeability for Ca2+. The kinetics of Ca2+ release from the vesicles is exponential. Using the grapho-analytical subtraction method, the kinetic parameters of this reaction were determined. Myometrium sarcolemma contains endogenous Ca2+-calmodulin-dependent protein kinase and phosphoprotein phosphatase which is inhibited by sodium o-vanadate. The Ca2+-calmodulin-dependent phosphorylation stimulates passive Ca2+ release from sarcolemmal vesicles. In the course of phosphorylation the capacity of the pool providing for rapid Ca2+ release increases by 61%, the initial rate of Ca2+ release showing a 28% increase. Trifluoroperazine, an inhibitor of Ca2+-calmodulin-dependent processes, eliminates the activating effect of phosphorylation on the rate of Ca2+ release from sarcolemmal vesicles.     

Role of cAMP-dependent phosphorylation in passive transport of Ca2+ by myocardial sarcolemma

The myocardial sarcolemma vesicles loaded with Na2+ can accumulate Ca2+ against the concentration gradient in exchange for Na2+; the rate of this process is about 10 nmole of Ca2+ per mg of protein per min. The cAMP-dependent phosphorylation of sarcolemmal preparations has no effect on the Na+/Ca2+ exchange. At the same time the cAMP-dependent phosphorylation of protein components of the sarcolemma causes inhibition of the passive Ca2+ efflux from the vesicles depending on the degree of membrane phosphorylation.     

Ca2+-calmodulin-dependent phosphorylation and passive transport of Ca2+ in the myocardial sarcolemma

Highly purified vesicles of rabbit myocardium sarcolemma with predominant inside-out orientation possess the Ca2+-calmodulin-dependent protein kinase activity. At optimal concentrations of calmodulin (0.5 microM) and Ca2+ (0.1 mM), the activity of protein kinase is 0.21 nmol 32P X min X mg of protein. The Km(app) value for ATP is 3.0 X 10(-6) M, V = 0.27 nmol 32P X mg of protein X min. Endogenous Ca2+-calmodulin-dependent protein kinase phosphorylates four protein substrates in sarcolemmal vesicles (Mr = 145, 22, 11.5, and 6-8 KD). Studies with passive efflux of Ca2+ from the SL vesicles showed that the Ca2+-calmodulin-dependent phosphorylation of protein components of sarcolemma inhibits this reaction.     

The effect of Ca2+-phospholipid dependent phosphorylation on passive calcium transport in the myocardial sarcolemma

Protein kinase C in vesicular preparations of the myocardium sarcolemma is shown to phosphorylate proteins with the molecular weight of 250, 140, 67, 58, 24 and 11 kD. The exogenic protein kinase C catalyzed phosphorylation of the sarcolemma preparations lowers the initial rate of the passive calcium transport from 0.56 down to 0.18 mmol per 1 mg second. Activation of endogenic protein kinase C by 4 beta-phorbol-12 beta-myristate-13 alpha-acetate is also accompanied by phosphorylation of vesicular preparations of sarcolemma and by inhibition of the passive calcium transport. Polymyxin B, being an inhibitor of protein kinase C, suppresses the phosphorylation and thus prevents the inhibitory action of phosphorylation on the passive calcium transport.     

Effect of the membrane potential on the passive transport of Ca2+ in fractions of vesicles of the myometrium sarcolemma

Using a potential-sensitive fluorescent probe diS-C3-(5), the formation of the membrane (K+-diffusion) potential, delta psi, in the myometrium sarcolemmal vesicular fraction was demonstrated. The magnitude of this potential corresponds to that calculated according to the Nernst equation, is time-stable (characteristic dissociation time--3-5 min) and temperature-dependent and is generated upon the substitution of the anion (Cl- for gluconate-) and the compensating cation (Na+ for Tris+, choline+). The change in delta psi from -61 to 0 mV leads to the activation of passive Ca2+ efflux from the vesicles (with choline+ as the compensating cation in the dilution medium). At the same value of the potential, i. e., -61 mV, the substitution of choline in the dilution medium for Na+ or Li+ stimulates the passive release of Ca2+. Co2+, Mn2+ and D-600 suppress this process by 15-20% in depolarized vesicles which points to the inhibition of Ca2+ release with an alteration of the membrane potential value from 0 to -61 mV (20%). The potential-dependent component of passive Ca2+ transport is characterized by saturation with the substrate (Km = 0.5 mM). The dependence of Ca2+ flux release from the sarcolemmal vesicles on the membrane potential value (-60-+27 mV) is bell-shaped and qualitatively relative to the volt-amper characteristics of the steady state Ca2+ flux in single smooth muscle cells. Analysis of experimental results revealed that the potential-dependent component of passive Ca2+ transport in myometrium sarcolemmal vesicles is determined by the non-activated Ca2+ conductivity of plasma membrane.     

Potential-dependent passive transport of calcium in myocardium sarcolemma vesicles

The effect of membrane potential on passive Ca2+ transport in isolated cardiac sarcolemmal vesicles was investigated. The membrane potentials were induced by creating potassium gradients across the vesicular membranes in the presence of valinomycin. The fluorescence changes in the voltage-sensitive dye, dis-C3(5), were consistent with the induction of potassium equilibrium potentials. The rate of 45Ca2+ efflux from inside-out vesicles was considerably greater at 0 than at -80 or +55 mV; prepolarization of the membrane to +90 mV did not enhance the 45Ca2+ efflux upon subsequent depolarization. The voltage-dependent 45Ca2+ efflux increased with a rise in internal Ca2+ concentration and exhibited a saturation effect. Furthermore, evaluation of the rate of 45Ca2+ efflux over a wide range of membrane potentials produced a profile similar to that of current-voltage relationships for single calcium channels in isolated cardiomyocytes. It is concluded that the voltage-dependent Ca2+ efflux from the vesicles occurs via Ca2+-channels.     

Active Na+-dependent transport of Ca 2+ into the fraction of smooth muscle sarcolemma vesicles

Studies on the vesicular fraction of myometrium sarcolemma showed that in the absence of initial Ca2+ gradient the vesicles activity accumulate Ca2+ by utilizing the energy of the antiport-directed Na+ gradient. Monensin (50 microM) suppresses practically completely the Ca2+ transport. The amount of Ca2+ entering the vesicles against the concentration gradient diminishes with a decrease in the oppositely directed Na+ gradient. Cd2+ (5 mM) causes a complete inhibition of active Ca2+ transport, whereas Mn2+ and Mg2+ inhibit this process by 85% and 35%, respectively; amiloride (500 microM) is fairly ineffective. In the absence of initial Ca2+ and Na+ gradients valinomycin (0.05-1 microM) does not affect the changes in Ca2+ concentration in the intravesicular volume both with and without K+ gradient. Under conditions of initial equilibrium for Ca2+ and Na+ the magnitude and sign of the membrane potential for the K(+)-valinomycin system have no effect on Ca2+ transport regardless of value of absolute Na+ concentration inside and outside the vesicles. Depolarization of membrane vesicles does not interfere with the Na(+)-driven active Ca2+ transport into the sarcolemma which is dependent on the energy of the Na+ gradient. Using calibration curves, it was shown that the physiologically significant (6-fold) Na+ gradient increases Ca2+ concentration in the intravesicular volume from 100 to 160-170 microM. Ac active potential-independent Ca2+ transport through the smooth muscle sarcolemma requires about one third (0.3 kcal/mol) of the Na+ gradient; energy the remainder is dissipated. It is concluded that in smooth muscles the Na+ gradient can provide the active transsarcolemmal transport of Ca2+.     

ATP-dependent transport of Ca2+ in myocardium sarcolemma vesicles and its activation by phorbol esters

Highly purified pig myocardium sarcolemma vesicles possess the Ca2+,Mg2+-ATPase activity (4.1 mumol Pi/mg protein/hour) and induce the ATP-dependent accumulation of 45Ca2+ (6.0 nmol/mg protein/min). This reaction is not stimulated by oxalate; Ca2+ are released from the vesicles by saponin and Na+ treatment, which suggests that Ca2+ transport against the concentration gradient is induced by myocardium sarcolemma vesicles and not by sarcoplasmic reticulum fragments. The phorbol ester possessing a biological activity of a growth-promoting factor and activating membrane-bound protein kinase C stimulates the Ca2+,Mg2+-ATPase activity and the ATP-dependent accumulation of Ca2+, whereas its counterpart devoid of biological activity does not influence Ca2+ transport. Polymixin B, a specific inhibitor of protein kinase C, prevents the activating effect of phorbol esters on Ca2+ accumulation inside the vesicles. It is suggested that the ATP-dependent transport of Ca2+ in myocardium sarcolemma is controlled by Ca2+-phospholipid-dependent phosphorylation catalyzed by protein kinase C.     

Ca2+-phospholipid-dependent phosphorylation of vesicular preparations of myocardial sarcolemma

A Ca2+-phospholipid-dependent protein kinase C was isolated from the soluble fraction of bovine brain, using hydrophobic chromatography on phenyl-Sepharose CL-4B and high performance liquid chromatography on a Mono Q column. The enzyme had a specific activity of 822 nmol 32P/mg protein/min with histone H1 as a substrate. Phosphorylation of pig myocardium sarcolemma protein substrates was stimulated by Ca2+ and phosphatidylserine; the optimal concentrations of these compounds were 10(-4) M and 200 micrograms/ml, respectively. The value of Km(app) for Ca2+ was 3.10(-6) M. An addition of exogenous dioleine increased the enzyme affinity for Ca2+ which led to a decrease of Ca2+ concentration necessary for the maximal activation to occur. The optimal concentration of ATP needed for sarcolemmal preparation phosphorylation was 0.3-0.4 mM, which seems to be due to the high activity of sarcolemmal ATPases. The proteins phosphorylated in sarcolemmal preparations were identified, using SDS polyacrylamide gel electrophoresis with subsequent autoradiography. The 250, 140, 67, 58, 25 and 11 kD proteins appeared to be phosphorylated in the greatest degree. Since in myocardial sarcolemma protein kinase C predominantly phosphorylates the same proteins as does the cAMP-dependent protein kinase, it was assumed that protein kinase C can also play a role in the regulation of Ca2+-transporting systems of sarcolemma.     

Kinetics of passive transport of Ca2+ in vesicular preparations of myocardial sarcolemma with inside-out oriented cytoplasm

Using affinity chromatography on a concanavalin A-Sepharose 4B column, two fractions of rabbit myocardium with oppositely oriented sarcolemmal vesicles have been obtained. Analysis of 45Ca2+ release from the vesicles with inside-out oriented cytoplasm demonstrates that this reaction is biphasic and obeys a pseudo-first-order kinetics. The initial rate of Ca2+ release is equal to 0.57 nmol/mg/s. The release of Ca2+ from the vesicles is inhibited via phosphorylation of sarcolemmal proteins by the catalytic subunit of cAMP-dependent protein kinase; the initial rate of this process drops to 0.08 nmol/mg of protein/s. The reaction is also inhibited by Cd2+ greater than Mn2+ greater than Co2+, when the latter are present inside the vesicles.     

Passive transport of Ca2+ in vesiculated preparations of myocardial sarcolemma

The highly purified vesicles of myocardial sarcolemma oriented outward mainly by the cytoplasmic side are used to show that Ca2+-calmodulin-dependent phosphorylation inhibits passive Ca2+-transport, while R24571, a blocking agent of calmodulin-dependent processes, removes this inhibitory effect. Passive Ca2+ transport is also inhibited by nicardipin with Ki (5 X 10(-8) M) and Mg2+. Tetrodotoxin and tetraethylammonium exert no effect on Ca2+-transport.     

Effect of nitrite-anions on Ca2+ transport into the sarcolemma of myometrium

The influence of nitrite-anions physiological concentration on Ca2+ input into vesicles was investigated when using the "outside-out" vesicles of myometrial plasmalemma and 45Ca2+. It was established that nitrite-anions increased Ca(2+)-permeability of plasmalemma and increased the affinity of cation-transport system. The effects are probably connected with reversible modification of glutamate residues that bound and transported Ca2+ within the membrane. These findings showed that nitrite-anions are competitive activators of the passive calcium transport. On the other hand the decrease of Ca2+ affinity for the transport system under transmembrane proton scattering by the membrane, by rapid dissipation of transmembrane delta pH. It may be possible that the dissipation of transmembrane proton gradient changed the conformation of calcium transport system that calls the difference of kinetic mechanism of NO2- action in case of delta pH = 0 and delta pH = 1.5 on vesicle membranes.     

Regulation of the Ca2+ pump ATPase by cAMP-dependent phosphorylation of phospholamban

Ca2+ transients in myocardial cells are modulated by cyclic AMP-dependent phosphorylation of a protein in the sarcoplasmic reticulum. This protein, termed phospholamban, serves to regulate the Ca2+ pump ATPase of this membrane, thus altering the mode of Ca2+ transients and the myocardial contractile response. Elucidating the structure of phospholamban and its intimate interaction with the Ca2+ pump ATPase should provide the basis for understanding, at the molecular level, how the cAMP system contributes to excitation-contraction coupling in muscle cells.     

Ca2+ transport in the sarcolemma of the myometrium under its chemical modification

The effect of some chemical modifying agents of the sarcolemma surface--dicyclohexicarbodiimide, trinitrobenzolsulphuric acid, dithiotreitol and nitrit-anions on passive transsarcolemmic accumulation of Ca2+ by the pig myometrium sarcolemma property oriented vesicules was studied. The comparative analysis of these substances action under proton transmembrane gradient dispersion is presented. The significance of surface amino-, carboxy groups and disulphide membrane bounds in Ca2+ transport mechanisms as a corresponding aspect to some contemporary ideas about Ca(2+)-channel has been defined. The hypothesis is made that the proton transmembrane gradient dissipation leads to dislocation of carboxyl groups and disulphid bounds in relation to the membrane surface resulting in increasing Ca(2+)-permeability of the sarcolemma. The increase of sarcolemma permeability for Ca2+ under the action of 10 nM NO-2 was demonstrated. On the base of experimental data the supposition was made that NO-2 modified amino- and carboxylate groups of the sarcolemma surface. Under the chemical modification of these groups the nitrit-anions inhibit transsarcolemmic Ca2+ movement. NO-2 is not a result of sarcolemma surface SH-groups oxidation, while possibly NO2- protects some functionally important disulphide bridges of Ca(2+)-channel from their chemical restoration.     

A fast passive Ca2+ efflux mediated by the (Ca2+ + Mg2+)-ATPase in reconstituted vesicles

The (Ca2+ + Mg2+)-ATPase from skeletal muscle sarcoplasmic reticulum was reconstituted into phospholipid bilayers. The permeability of lipid bilayers to Co2+ and glucose was increased slightly by incorporation of the ATPase, and the permeability of mixed bilayers of phosphatidylethanolamine and phosphatidylcholine increased with increasing content of phosphatidylethanolamine both in the presence and absence of the ATPase. The presence of the ATPase, however, resulted in a marked increase in permeability to Ca2+, the permeability decreasing with increasing phosphatidylethanolamine content. Permeability to Ca2+ was found to be dependent on pH and the external concentrations of Mg2+ and Ca2+, was stimulated by adenine nucleotides but was unaffected by inositol trisphosphate. A kinetic model is presented for Ca2+ efflux mediated by the ATPase. It is shown that the kinetic parameters that describe Ca2+ efflux from vesicles of sarcoplasmic reticulum also describe efflux from the vesicles reconstituted from the purified ATPase and phosphatidylcholine. It is shown that the effects of phosphatidylethanolamine on efflux can be simulated in terms of changes in the rates of the transitions linking conformations of the ATPase with inward- and outward-facing Ca2+-binding sites, and that effects of phosphatidylethanolamine on the ATPase activity of the ATPase can also be simulated in terms of effects on the corresponding conformational transitions. We conclude that the ATPase can act as a specific pathway for Ca2+ efflux from sarcoplasmic reticulum.     

Divalent cations and nifedipine action on Ca(2+) transport into myometrial plasmalemma vesicles

While using 45Ca2+ on the model of "outside-out configuration" vesicules of the myometrium cells sarcolemma an investigation of Cd2+, Zn2+, Co2+ and niphedipin on Ca2+ transport into the vesicules in the conditions of protons gradient transmembrane dissipation has been conducted. The above listed substances blocking effect corresponds to their physicochemical properties. Cadmium and zinc ions are considerably more effective in suppressing Ca2+ transport into the vesicules under the dissipation of delta pH on the membrane if compare with the case of delta pH = 0. In the case of niphedipin inhibiting action an opposite result is observed. The hypothesis has been made, that dissipation of delta pH on the sarcolemma is capable to strengthen the transmembrane Ca2+ transport by means of changing the channel structures conformation.     

Mechanisms of Ca2+ transport in plasma membrane vesicles prepared from cultured pituitary cells. II. (Ca2+ + Mg2+)-ATPase-dependent Ca2+ transport activity

Purified plasma membrane vesicles from GH3 rat anterior pituitary cells exhibit a Mg2+-ATP-dependent Ca2+ transport activity. Concentrative uptake of Ca2+ is abolished by exclusion of either Mg2+ or ATP or by inclusion of the Ca2+ ionophore A23187. Furthermore, addition of A23187 to vesicles which have reached a steady state of ATP-supported Ca2+ accumulation rapidly and completely discharges accumulated cation. Ca2+ uptake is unaffected by treatment of vesicles with oligomycin, the uncoupler CCCP, or valinomycin and is greatly reduced in non-plasma membrane fractions. Likewise, Ca2+ accumulation is not stimulated by oxalate, consistent with the plasma membrane origin of this transport system. (Na+, K+)-ATPase participation in the Ca2+ transport process (i.e. via coupled Na+/Ca2+ exchange) was eliminated by omitting Na+ and including ouabain in the reaction medium. Ca2+ transport activity in GH3 vesicles has a similar pH dependence as that seen in a number of other plasma membrane systems and is inhibited by orthovanadate in the micromolar range. Inhibition is enhanced if the membranes are preincubated with vanadate for a short time. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ and ATP are 0.7 and 125 microM, respectively. The average Vmax is 3.6 nmol of Ca2+/min/mg of protein at 37 degrees C. Addition of exogenous calmodulin or calmodulin antagonists had no significant effect on these kinetic properties. GH3 plasma membranes also contain a Na+/Ca2+ exchange system. The apparent Km for Ca2+ is almost 10-fold higher in this system than that for ATP-driven Ca2+ uptake. When both processes are compared under similar conditions, the Vmax of the exchanger is approximately 2-3 times that of ATP-dependent Ca2+ accumulation. Similar results are obtained when purified plasma membranes from bovine anterior pituitary glands were investigated. It is suggested that both Na+/Ca2+ exchange and the (Ca2+ + Mg2+)-ATPase are important in controlling intracellular levels of Ca2+ in anterior pituitary cells.     

Change in Ca2+ transport in myometrial plasma cell membrane in proton gradient dissipation

By means of delta pH 14C-methylamine indicator the myometrium vesicle sarcolemma fraction was shown to be capable, while applying a "delta pH-leap", for developing in it a proton transmembrane gradient, dissipating in time. The proton gradient dissipation under Ca ions transmembrane equilibrium concentration is a driving force of these ions transposition against the concentration gradient. The blocking agents of H+ transport--Cd ions and DCCD decrease the proton-dependent 45Ca2+ accumulation in the vesicle sarcolemma fraction. The conclusion has been made about the possibility of Ca2+(H(+)-exchange on the uterus smooth cells sarcolemma. The possible physiological value of this exchange is under discussion.