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.     

3':5'-AMP-dependent phosphorylation of muscular membrane proteins and calcium transport

The data are presented concerning the role of cAMP-dependent phosphorylation of sarcoplasmic reticulum and sarcolemma proteins in the transport of Ca2+ through the cardiac skeletal and smooth muscle membranes. Phosphorylation of membrane proteins by soluble and membrane-bound cAMP-dependent protein kinases is shown to have a regulating effect on Mn2+-, Ca2+-ATPase activity and to change membrane permeability for Ca2+. The molecular mechanisms and the character of the interaction between the transport systems and the phosphorylating protein substrates have not been yet established.     

Ca2+-dependent K+ permeability of heart sarcolemmal vesicles. Modulation by cAMP-dependent protein kinase activity and by calmodulin

The Ca2+-dependent K+ permeability of heart sarcolemma vesicles was measured by following the transmembrane movement of the charge compensating tetraphenylborate anion. The increase in vesicles permeability induced by Ca2+ is lost when membrane proteins are dephosphorylated by an endogenous protein phosphatase and is restored by a phosphorylation process catalysed by a cAMP-dependent protein kinase. The calmodulin antagonist R 24571 lowers the Ca2+-dependent K+ permeability by decreasing the Ca2+ affinity of the K+ transporting system.     

Cyclic AMP-dependent protein phosphorylation in canine renal brush-border membrane vesicles is associated with decreased phosphate transport

It is known that the administration of parathyroid hormone to dogs results in phosphaturia and decreased phosphate transport in brush-border vesicles isolated from the kidneys of those dogs. Parathyroid hormone has been shown to activate adenylate cyclase at the basal-lateral membrane of the renal proximal tubular cell. It has been postulated that parathyroid hormone-induced phosphaturia is effected through phosphorylation of brush-border protein by membrane-bound cAMP-dependent protein kinase. An experimental system was designed such that phosphorylation of brush-border vesicles and Na+-stimulated solute transport could be studied in the same preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane vesicles revealed cAMP-dependent phosphorylation of 2 protein bands (Mr = 96,000 and 62,000), which was enhanced by exposure of the inside of the membrane vesicles to ATP and cAMP. Cyclic AMP-dependent phosphorylation of brush-border vesicles was accompanied by inhibition of Na+-stimulated Pi but not D-glucose transport or 22Na+ uptake. When renal brush-border vesicles from parathyroidectomized and normal dogs were phosphorylated in vitro in the presence and absence of cAMP, both the cAMP-dependent phosphorylation and inhibition of Na+-stimulated Pi transport were greater in vesicles isolated from kidneys of parathyroidectomized dogs relative to control animals. We conclude that the cAMP-dependent phosphorylation of brush-border membrane-vesicle proteins is associated with specific inhibition of Na+-stimulated Pi transport. The phosphaturic action of parathyroid hormone (PTH) could be mediated through the cAMP-dependent phosphorylation of specific brush-border membrane proteins.     

The mechanism of calcium control of smooth muscle tonic contraction

It has been shown in the experiments carried out on a fraction of inverted vesicles of myometrium sarcolemma that ATP-dependent Ca2+ transport system prevents dissipation of the calcium gradient directed from the intervesicular space outward with subsequent establishment of the stationary level of cation content inside the membrane vesicles (a blocker of electro-controlled calcium channels diltiasems was present in the incubation medium). Ortovanadatean inhibitor of the sarcolemma calcium pump suppressed Ca2+ stationary exchange in the vesicles fraction. The value of calcium stationary content in the vesicle membrane was regulated both by a change of the calcium pump activity (by varying Mg2+ concentration in the ATP-containing incubation medium), and by modification of calcium permeability of the vesicles (by varying concentration of ionophore A-23187 in this medium). In the presence of diltiasem and ortovanadate the Ca2+ basal current entering the myocytes from hyperpotassium washing solution activated the smooth muscle tonic contraction. In the absence of ortovanadate no contractile response was observed. On the basis of the evidence obtained a mechanism of calcium control of myometrium tonic contraction is proposed. According to this mechanism the Ca2+ current entering the unexcited myocytes under physiological conditions is efficiently compensated by the calcium pump of the sarcolemma. The inhibition of the latter (or an increase of the sarcolemma basal calcium permeability) provides further slow transition of the stationary value of Ca2+ concentration in the myoplasm to a new higher level and activation of the smooth muscle contraction accordingly.     

Skeletal muscle sarcolemma in malignant hyperthermia: evidence for a defect in calcium regulation

Sarcolemmal properties implicated in the skeletal muscle disorder, malignant hyperthermia (MH), were examined using sarcolemma-membrane vesicles isolated from normal and MH-susceptible (MHS) porcine skeletal muscle. MHS and normal sarcolemma did not differ in the distribution of the major proteins, cholesterol or phospholipid content, vesicle size and sidedness, (Na+ + K+)-ATPase activity, ouabain binding, or adenylate cyclase activity (total and isoproterenol sensitivity). The regulation of the initial rates of MHS and normal sarcolemmal ATP-dependent calcium transport (calcium uptake after 1 min) by Ca2+ (K1/2 = 0.64-0.81 microM), calmodulin, and cAMP-dependent protein kinase were similar. However, when sarcolemmal calcium content was measured at either 2 or 20 min after the initiation of active calcium transport, a significant difference between MHS and normal sarcolemmal calcium uptake became apparent, with MHS sarcolemma accumulating approximately 25% less calcium than normal sarcolemma. Calcium transport by MHS and normal sarcolemma, at 2 or 20 min, had a similar calmodulin dependence (C1/2 = 150 nM), and was stimulated to a similar extent by cAMP-dependent protein kinase or calmodulin. Halothane inhibited MHS and normal sarcolemmal active calcium uptake in a similar fashion (half-maximal inhibition at 10 mM halothane), while dantrolene (30 microM) and nitrendipine (1 microM) had little effect on either MHS or normal sarcolemmal calcium transport. After 20 min of ATP-supported calcium uptake, 2 mM EGTA plus 10 microM sodium orthovanadate were added to initiate sarcolemmal calcium efflux. Following an initial rapid phase of calcium release, an extended slow phase of calcium efflux (k = 0.012 min-1) was similar for both MHS and normal sarcolemma vesicles. We conclude that although a number of sarcolemmal properties, including passive calcium permeability, are normal in MH, a small but significant defect in MHS sarcolemmal ATP-dependent calcium transport may contribute to the abnormal calcium homeostasis and altered contractile properties of MHS skeletal muscle.     

Effect of the membrane potential on the Mg2+,ATP-dependent transport of Ca2+ across smooth muscle sarcolemma

The effect of the membrane potential (K(+)-valinomycin system) on the Mg2+, ATP-dependent transport of Ca2+ in inside-out vesicles of myometrium sarcolemma has been studied. The membrane potential was identified by using a cyanine potential-sensitive probe, diS-C3-(5). In the presence of valinomycin (5.10(-8) M) the inside-out directed K+ gradient (delta psi = -86 mV, with a negative charge inside) stimulated the initial rate of the energy-dependent accumulation of Ca2+ transfer whereas the oppositely directed K+ gradient (delta psi = +72 mV, with a positive charge inside) had no effect on this process. The K+ gradient was formed by isotonic substitution of K+ in intra- or extravesicular space for choline +. At the same time, in the absence of K+ gradient the Mg2+, ATP-dependent accumulation of Ca2+ in membrane vesicles did not depend on the chemical nature of the cations (K+ or choline+) used for isotonicity. The decrease of delta psi from 0 to -86 mV affects the initial rate of Ca2+ accumulation but not the maximal content of the accumulated cation. Preliminary dissipation of the membrane potential (delta psi = -86 mV) in Mg2(+)-free isotonic (with respect of K+ and choline+) media containing ATP and Ca2+ resulted in the inhibition of Mg2+, ATP-dependent Ca2+ transport induced by subsequent addition of Mg2+. These results indicate that the negative (intravesicular) electrical potential activates the Ca-pump of smooth muscle sarcolemma. This activation is based on the increase in the turnover number of the Ca2+ transporting system but not on its affinity for the transfer substrate. The use of the absolute reaction rates theory made it possible to establish that the Ca-pump effectuates the transport of a single positive charge in inside-out vesicles of smooth muscle plasma membranes, i.e., the energy-dependent transport of Ca2+ occurs either as a symport (with an anion (Cl-) or an antiport with a monovalent cation (K+) or a proton. It is assumed that the potential dependence of the Ca-pump in the smooth muscle plasma membrane plays a role in the realization of effects of mediators and physiologically active substances that are manifested as stimulation of the contractile response and depolarization of the sarcolemma. In is quite probable that the delta psi-dependent Ca-pump is also responsible for the maintenance of intracellular homeostasis of monovalent cations (K+, H+, Cl-) in smooth muscle tissues.     

Calmodulin-mediated regulation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity in isolated cardiac sarcoplasmic reticulum

A severalfold activation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity by micromolar concentrations of calmodulin was observed in sarcoplasmic reticulum vesicles obtained from canine ventricles. This activation was seen in the presence of 120 mM KCl. The ratio of moles of calcium transported per mol of ATP hydrolyzed remained at about 0.75 when calcium transport and (Ca2+ + Mg2+)-activated ATPase activity were measured in the presence and absence of calmodulin. Thus, the efficiency of the calcium transport process did not change. Stimulation of calcium transport by calmodulin involves the phosphorylation of one or more proteins. The major 32P-labeled protein, as determined by sodium dodecyl sulfate slab gel electrophoresis, was the 22,000-dalton protein called phospholamban. The Ca2+ concentration dependency of calmodulin-stimulated microsomal phosphorylation corresponded to that of calmodulin-stimulated (Ca2+ + Mg2+)-activated ATPase activity. Proteins of 11,000 and 6,000 daltons and other proteins were labeled to a lesser extent. A similar phosphorylation pattern was obtained when microsomes were incubated with cAMP-dependent protein kinase and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Phosphorylation produced by added cAMP-dependent protein kinase and calmodulin was additive. These studies provided further evidence for Ca2+-dependent regulation of calcium transport by calmodulin in sarcoplasmic reticulum that could play a role in the beat-to-beat regulation of cardiac relaxation in the intact heart.     

Role of phospholamban in regulating cardiac sarcoplasmic reticulum calcium pump

Cardiac sarcoplasmic reticulum plays a critical role in the excitation-contraction cycle and hormonal regulation of heart cells. Catecholamines exert their ionotropic action through the regulation of calcium transport into the sarcoplasmic reticulum. Cyclic 3'-5'-adenosine monophosphate (cAMP) causes the cAMP-dependent protein kinase to phosphorylate the regulatory protein phospholamban, which results in the stimulation of calcium transport. Calmodulin also phosphorylates phospholamban by a calcium-dependent mechanism. We have reported the isolation and purification of phospholamban with low deoxycholate (DOC) concentrations (5 X 10(-6) M). We have also reported the isolation and purification of Ca2+ + Mg2+-ATPase with a similar procedure. Both phospholamban and Ca2+ + Mg2+-ATPase retained their native properties associated with sarcoplasmic reticulum vesicles. Further, we have shown that the removal of phospholamban from membranes of sarcoplasmic reticulum vesicles uncouples Ca2+-uptake from ATPase without any effect on Ca2+ + Mg2+-ATPase activity or Ca2+ efflux. Phospholamban appears to be the substrate for both the Ca2+-calmodulin system and the cAMP-dependent protein kinase system. It is found that the phosphorylation of phospholamban by the Ca2+-calmodulin system is required for the normal basal level of Ca2+ transport, and that the phosphorylation of phospholamban at another site by the cAMP-dependent protein kinase system causes the stimulation of Ca2+-transport above the basal level. The functional effects of the phosphorylation of phospholamban by cAMP-dependent protein kinase system are expressed only after the phosphorylation of phospholamban with Ca2+-calmodulin system. We propose a model for the cardiac Ca2+ + Mg2+-ATPase, whereby the enzyme is normally uncoupled from Ca2+ uptake. The enzyme becomes coupled to Ca2+ transport after the first site of phospholamban is phosphorylated with the Ca2+-calmodulin system. When the second site of phospholamban is phosphorylated with cAMP-dependent protein kinase both Ca2+ transport and ATPase are stimulated and phospholamban becomes inaccessible to DOC solubilization and trypsin.     

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.     

Effects of cAMP- and cGMP-dependent protein kinases, and calmodulin on Ca2+ uptake by highly purified sarcolemmal vesicles of vascular smooth muscle

Sarcolemmal fractions of vascular smooth muscles were prepared from porcine thoracic aortae by differential and sucrose density gradient centrifugation. In these fractions, there was a high activity of 5'-nucleotidase, a putative marker enzyme of plasma membrane, and a low activity of rotenone insensitive NADH-cytochrome c reductase a marker of sarcoplasmic reticulum. In these fractions, the Ca2+ uptake was ATP-dependent. A low concentration of saponin which inhibited Ca2+ uptake by the plasma membrane but not by the sarcoplasmic reticulum, inhibited 65% of the Ca2+ uptake of this fraction. The Ca2+ uptake of this fraction was enhanced by cAMP- and cGMP-dependent protein kinases, and by calmodulin. The cAMP-dependent protein kinase enhanced the phosphorylation of 28 and 22 kDa proteins, while the cGMP-dependent protein kinase phosphorylated the 35 kDa protein. The phosphorylation of 100, 75, 65, 41 and 22 kDa proteins was enhanced by Ca2+ and calmodulin. These results indicate that cAMP- and cGMP-dependent protein kinases as well as calmodulin play important roles in Ca2+ transport in the sarcolemma, and that the phosphorylated proteins may be associated with an enhancement of Ca2+ transport in the sarcolemma.     

Phosphorylation of the Ca2+-pumping ATPase of heart sarcolemma and erythrocyte plasma membrane by the cAMP-dependent protein kinase

The Ca2+ ATPase of heart sarcolemma was stimulated by the exposure of sarcolemma vesicles to ATP and the catalytic subunit of the cAMP-dependent protein kinase. The effect of the phosphorylation system was primarily on the Km(Ca2+) of the pumping ATPase. The ATPase purified from heart sarcolemma or erythrocytes became phosphorylated under the conditions mentioned above. Hydroxylamine treatment of the labeled ATPase has shown that the phosphorylation was additive to be acylphosphate formed on the ATPase during the reaction cycle. The stoichiometry of the kinase-promoted phosphorylation (i.e. the fraction of the ATPase molecules that became labeled) approached 30% with both the heart and the erythrocyte enzyme.     

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.     

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+.     

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.     

Effects of vitamin D-3 on phosphate and calcium transport across and composition of skeletal muscle plasma cell membranes

The effects of vitamin D-3 on calcium and phosphate transport in skeletal muscle plasma membranes were studied. Sarcolemma vesicles were isolated from vitamin D-deficient and vitamin D-treated (one week) chicks by sucrose density gradient centrifugation of a crude muscle plasma membrane fraction. Measurement of (Na+ + K+)-ATPase activity, cholesterol to phospholipid molar ratios and levels of intracellular marker enzymes showed a high degree of purification of the preparations. Administration of vitamin D-3 significantly increased active Ca2+ and phosphate uptake into the vesicles. The efflux of both ions from preloaded vesicles was only slightly altered by the sterol. Ca2+-ATPase activity was higher in sarcolemma from treated animals. This confirms that the effects of vitamin D-3 on calcium transport are related to the Ca2+ pump and not to the passive permeability properties of the membrane. No changes in the protein composition of vesicles from both experimental groups were observed. However, treatment with vitamin D-3 increased sphingomyelin and phosphatidylcholine concentrations. These changes in lipid structure may play a role in the effects of vitamin D-3 on transport characteristics of sarcolemma.     

Phosphorylation of purified bovine cardiac sarcolemma and potassium-stimulated calcium uptake

Sarcolemmal vesicles were prepared from bovine cardiac muscle by differential and discontinuous sucrose density gradient centrifugation. Na+/K+-ATPase was purified 33-fold to a specific activity of 53 +/- 0.5 (12) mumol Pi X mg-1 X h-1, binding sites for strophantin 20-fold to a density of 56.3 +/- 5.3 (14) pmol/mg and that for the calcium antagonist nitrendipine 5.5-fold to a density of 0.72 +/- 0.07 (6) pmol/mg. The specific activity of the Na+/Ca2+ exchanger was 61.1 +/- 3.7 (6) nmol/mg. The vesicles had an intravesicular volume of 20 +/- 4 (4) microliter/mg and 56.9 +/- 6 (4)% of the vesicles were right-side-out oriented. Several peptides of the purified membranes were phosphorylated in the presence of Mg . ATP and EGTA. Most of the radioactive phosphate was incorporated into a peptide with an apparent molecular mass of 22 kDa. Denaturation of the membranes at 100 degrees C changed the mobility of this peptide to 15 kDa and 11 kDa. This peptide could not be distinguished from a sarcoplasmic reticulum peptide of similar molecular mass. The phosphorylation of the sarcolemmal peptide was stimulated by Ca2+/calmodulin, cAMP and the catalytic subunit of cAMP-dependent protein kinase. A comparison of the phosphorylation of sarcolemmal membranes with that of sarcoplasmic reticulum showed that Ca2+/calmodulin stimulated in each membrane, the phosphorylation of the 22-kDa peptide and a 44-kDa peptide, and in the sarcoplasmic reticulum the phosphorylation of an additional peptide of 55-kDa. Ca2+/calmodulin-dependent phosphorylation of a 55-kDa peptide could not be demonstrated in sarcolemma, regardless if sarcolemmal membranes were incubated together with sarcoplasmic reticulum or if the phosphorylation was carried out in the presence of purified cardiac myosin light chain kinase or phosphorylase kinase. 'Depolarization' induced Ca2+ uptake which was measured according to Bartschat, D.K., Cyr, D.L. and Lindenmayer, G.E. [(1980) J. Biol. Chem. 255, 10044-10047] was 5 nmol/mg protein. This uptake was not enhanced after preincubation of the vesicles with Mg . ATP or Mg . ATP and cAMP-dependent protein kinase. The value of 5 nmol/mg protein is in agreement with the theoretical amount of Ca2+ which can be accumulated by the bovine cardiac sarcolemma in the absence of a driving force other than the Ca2+ gradient. The potassium-stimulated Ca2+ uptake was not blocked by the organic Ca2+ channel blockers. Prolonged incubation of Mg . ATP with sarcolemmal vesicles in the presence of various ATPase inhibitors led to the hydrolysis of ATP. The liberated phosphate precipitated with Ca2+ in the presence of LaCl3. These precipitates amounted to an apparent Ca2+ uptake ranging from 50 to over 1000 nmol/mg. The results suggest that potassium-stimulated Ca2+ uptake of bovine cardiac sarcolemmal vesicles is not enhanced in the presence of ATP or by phosphorylation of a 22-kDa peptide.     

Voltage sensitivity of H+/Ca2+ antiport in higher plant tonoplast suggests a role in vacuolar calcium accumulation

The electrogenicity of H+/Ca2+ exchange in vacuolar membrane (tonoplast) vesicles from Beta was studied to elucidate the role of this transport system in vacuolar Ca2+ accumulation. To overcome the inherently high proton permeability of tonoplast vesicles, the pH difference established by the primary H(+)-ATPase was titrated to a uniform value by variation of the concentration either of ATP or of a permanent anion (Cl-). This enabled manipulation of membrane potential independently of the transmembrane pH difference, with a higher inside-positive membrane potential produced at lower Cl- concentrations. The rate and the extent of uncoupler-sensitive Ca2+ uptake are both stimulated about 2-fold in conditions of more positive membrane potential, suggesting that the transport system translocates positive charge outward during Ca2+ uptake. A minimum integral H+:Ca2+ stoichiometry of 3 results in a driving force for Ca2+ accumulation in the vacuole amounting to -140 mV in typical physiological conditions. It is concluded that the antiporter is thermodynamically competent to account for Ca2+ accumulation in plant vacuoles and that its reversal in vivo is unlikely.     

Mechanisms of regulation of Ca2+ levels in myometrium cells

The ways and mechanisms of the Ca2+ concentration regulation in myometrium cells are analyzed. The plasma membrane is thoroughly studied for its role in the calcium control provision for the contractile activity of the uterus. The systems of Mg2+-ATP-dependent transport of Ca2+, sodium-calcium metabolism as well as regularities of the Ca2+ passive transfer in the sarcolemma vesicles are considered. The systems of the Mg2+-ATP- and N+-dependent transport of calcium are discussed for their contribution into regulation of calcium concentration in the myoplasm. Oxytocin and ions of bivalent metals (stimulators of the contractile activity of the uterus) are studied for their effect on the activity of the sarcolemma calcium pump.     

The effect of thyroxine on the calmodulin-dependent (Ca2+-Mg2+)ATPase activity and protein phosphorylation in rabbit fast skeletal muscle sarcolemma

Enzymatic properties and the protein pattern of sarcolemma fractions isolated from three groups of rabbits: euthyroid, hyperthyroid and hypothyroid, were studied. The amount of phosphorylated intermediate formed by the calmodulin-dependent (Ca2+-Mg2+)ATPase and the activity of this enzyme as well as that of (Na+-K+)ATPase were the highest in membranes isolated at the hyperthyroid state. On the other hand, sarcolemma obtained from the hypothyroid animals exhibited a decreased activity of (Na+-K+)ATPase, while the activity of calmodulin-dependent (Ca2+-Mg2+)ATPase was the same as in the preparations obtained from euthyroid animals. Thyroid hormones also changed the protein pattern of muscle sarcolemma. Membranes isolated from hyperthyroid animals lacked peptides of apparent molecular masses of 41 kDa and 53 kDa, while a peptide of the apparent molecular mass of 63 kDa was enriched in the preparation from hypothyroid animals. Thyroid hormones affected endogenous cAMP-dependent protein phosphorylation. The sarcolemma fraction obtained from hyperthyroid animals exhibited a decreased phosphorylation of peptides of apparent molecular masses of 30 kDa and 47 kDa, while the cAMP-independent phosphorylation of several other peptides was augmented. Moreover, sarcolemma preparations isolated from hyperthyroid animals showed higher activity of cAMP-independent protein kinase(s) and lower activity of cAMP-dependent protein kinase when compared to the euthyroid preparations. It is proposed that thyroxine increases the content of calmodulin-dependent (Ca2+-Mg2+)ATPase protein and affects the activity of cAMP-independent and cAMP-dependent protein kinases bound to sarcolemma.