Polyamines decrease Ca(2+) sensitivity of tension and increase rates of activation in skinned cardiac myocytes

Owing in part to their interactions with membrane proteins, polyamines (e.g., spermine, spermidine, and putrescine) have been identified as potential modulators of membrane excitability and Ca(2+) homeostasis in cardiac myocytes. To investigate whether polyamines also affect cardiac myofilament proteins, we assessed the effects of polyamines on contractility using rat myocytes and trabeculae that had been permeabilized with Triton X-100. Spermine, spermidine, and putrescine reversibly increased the [Ca(2+)] required for half-maximal tension (i.e., right-shifted tension pCa curves), with the following order of efficacy: spermine (+4) > spermidine (+3) > putrescine (+2). However, synthetic analogs that differed from spermine in charge distribution were not as effective as spermine in altering isometric tension. None of the polyamines had a significant effect on maximal tension, except at high concentrations. After flash photolysis of DM-Nitrophen (a caged Ca(2+) chelator), spermine accelerated the rate of tension development at low and intermediate but not high [Ca(2+)]. These results indicate that polyamines, especially spermine, interact with myofilament proteins to reduce apparent Ca(2+) binding affinity and speed cross-bridge cycling kinetics at submaximal [Ca(2+)].     

Phospholipid and detergent effects on (Ca2+ + Mg2+)ATPase purified from human erythrocytes

(Ca2+ + Mg2+)ATPase (EC 3.6.1.3) was solubilized from human erythrocyte membranes by detergent extraction with Triton N-101 (0.5 mg/mg membrane protein) and purified by calmodulin affinity chromatography. ATPase activity was assayed in mixtures of Triton N-101 and phospholipid, without reconstitution into bilayer vesicles. At low levels of phospholipid (5 micrograms/ml), the ATPase activity was highly sensitive to the detergent concentration, with maximal activity occurring at or near the critical micelle concentration of the detergent. With increased amounts of phospholipid (50 micrograms/ml), detergent concentrations greater than the critical micelle concentration were required for maximal activity. Detergent alone did not support ATPase activity. Sonicated phospholipid in the form of vesicles was equally ineffective. Activity seemed to be dependent on the presence of detergent/phospholipid mixed micelles. The acidic phospholipids, phosphatidylserine and phosphatidylinositol, as well as the commercial phospholipid preparation, Asolectin, gave activities five to eight times greater than the same amount of phosphatidylcholine. Mixtures of phosphatidylserine and phosphatidylcholine produced intermediate ATPase activities, with the maximal value dependent on the phosphatidylserine concentration. Addition of phosphatidylcholine to fixed concentrations of phosphatidylserine caused a rise in activity that was independent of the ratio of the two phospholipids or the total phospholipid concentration. Phosphatidylcholine may therefore be irreplaceable for some aspect of ATPase function. The number of phospholipid molecules present in mixed micelles at maximal ATPase activity was calculated to be near 50. This value implied that the hydrophobic surface of the ATPase molecule must be completely coated by a single layer of phospholipid molecules for maximum activity to occur.     

Faster force transient kinetics at submaximal Ca2+ activation of skinned psoas fibers from rabbit.

The early, rapid phase of tension recovery (phase 2) after a step change in sarcomere length is thought to reflect the force-generating transition of myosin bound to actin. We have measured the relation between the rate of tension redevelopment during phase 2 (r), estimated from the half-time of tension recovery during phase 2 (r = t0.5(-1)), and steady-state force at varying [Ca2+] in single fibers from rabbit psoas. Sarcomere length was monitored continuously by laser diffraction of fiber segments (length approximately 1.6 mm), and sarcomere homogeneity was maintained using periodic length release/restretch cycles at 13-15 degrees C. At lower [Ca2+] and forces, r was elevated relative to that at pCa 4.0 for both releases and stretches (between +/- 8 nm). For releases of -3.4 +/- 0.7 nm.hs-1 at pCa 6.6 (where force was 10-20% of maximum force at pCa 4.0), r was 3.3 +/- 1.0 ms-1 (mean +/- SD; N = 5), whereas the corresponding value of r at pCa 4.0 was 1.0 +/- 0.2 ms-1 for releases of -3.5 +/- 0.5 nm.hs-1 (mean +/- SD; N = 5). For stretches of 1.9 +/- 0.7 nm.hs-1, r was 1.0 +/- 0.3 ms-1 (mean +/- SD; N = 9) at pCa 6.6, whereas r was 0.4 +/- 0.1 ms-1 at pCa 4.0 for stretches of 1.9 +/- 0.5 (mean +/- SD; N = 14). Faster phase 2 transients at submaximal Ca(2+)-activation were not caused by changes in myofilament lattice spacing because 4% Dextran T-500, which minimizes lattice spacing changes, was present in all solutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum.

ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold. Activation of K+-stimulated ATPase activity by Ca2+ was maximal at an ionized Ca2+ concentration of approx. 1 microM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.     

Localization of (Ca2+ + Mg2+)-ATPase, Ca2+ pump and other ATPase activities in cardiac sarcolemma

N-Ethylmaleimide was employed as a surface label for sarcolemmal proteins after demonstrating that it does not penetrate to the intracellular space at concentrations below 1.10(-4) M. The sarcolemmal markers, ouabain-sensitive (Na+ +K+)-ATPase and Na+/Ca2+-exchange activities, were inhibited in N-ethylmaleimide perfused hearts. Intracellular activities such as creatine phosphokinase, glutamate-oxaloacetate transaminase and the internal phosphatase site of the Na+ pump (K+-p-nitrophosphatase) were not affected. Almost 20% of the (Ca2+ +Mg2+)-ATPase and Ca2+ pump were inhibited indicating the localization of a portion of this activity in the sarcolemma. Sarcolemma purified by a recent method (Morcos, N.C. and Drummond, G.I. (1980) Biochim. Biophys. Acta 598, 27-39) from N-ethylmaleimide-perfused hearts showed loss of approx. 85% of its (Ca2+ +Mg2+-ATPase and Ca2+ pump compared to control hearts. (Ca2+ +Mg2+)-ATPase and Ca2+ pump activities showed two classes of sensitivity to vanadate ion inhibition. The high vanadate affinity class (K1/2 for inhibition approx. 1.5 microM) may be localized in the sarcolemma and represented approx. 20% of the total inhibitable activity in agreement with estimates from N-ethylmaleimide studies. Sucrose density fractionation indicated that only a small portion of Mg2+-ATPase and Ca2+-ATPase may be associated with the sarcolemma. The major portion of these activities seems to be associated with high density particles.     

Effect of lactate on depolarization-induced Ca(2+) release in mechanically skinned skeletal muscle fibers

Itis unclear whether accumulation of lactate in skeletal muscle fibersduring intense activity contributes to muscle fatigue. Usingmechanically skinned fibers from rat and toad muscle, we were able toexamine the effect of L(+)-lactate onexcitation-contraction coupling independently of other metabolicchanges. We investigated the effects of lactate on the contractileapparatus, caffeine-induced Ca²⁺ release from thesarcoplasmic reticulum, and depolarization-induced Ca²⁺release. Lactate (15 or 30 mM) had only a small inhibitory effect directly on the contractile apparatus and caused appreciable(20-35%) inhibition of caffeine-induced Ca²⁺ release,seemingly by a direct effect on the Ca²⁺ release channels.However, 15 mM lactate had no detectable effect on Ca²⁺release when it was triggered by the normal voltage sensor mechanism, and 30 mM lactate reduced such release by only <10%. These results indicate that lactate has only a relatively small inhibitory effect onnormal excitation-contraction coupling, indicating that lactate accumulation per se is not a major factor in muscle fatigue.

     

Neuronal-specific endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration in mixed primary hippocampal culture homogenates

Endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration is crucial for maintenance of neuronal Ca(2+) homeostasis. The use of cell culture in conjunction with modern Ca(2+) imaging techniques has been invaluable in elucidating these mechanisms. While imaging protocols evaluate endoplasmic reticulum Ca(2+) loads, measurement of Mg(2+)/Ca(2+) ATPase activity is indirect, comparing cytosolic Ca(2+) levels in the presence or absence of the Mg(2+)/Ca(2+) ATPase inhibitor thapsigargin. Direct measurement of Mg(2+)/Ca(2+) ATPase by isolation of microsomes is impossible due to the minuscule amounts of protein yielded from cultures used for imaging. In the current study, endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration was measured in mixed homogenates of neurons and glia from primary hippocampal cultures. It was demonstrated that Ca(2+) uptake was mediated by the endoplasmic reticulum Mg(2+)/Ca(2+) ATPase due to its dependence on ATP and Mg(2+), enhancement by oxalate, and inhibition by thapsigargin. It was also shown that neuronal Ca(2+) uptake, mediated by the type 2 sarco(endo)plasmic reticulum Ca(2+) ATPase isoform, could be distinguished from glial Ca(2+) uptake in homogenates composed of neurons and glia. Finally, it was revealed that Ca(2+) uptake was sensitive to incubation on ice, extremely labile in the absence of protease inhibitors, and significantly more stable under storage conditions at -80 degrees C.     

Characterization of the effects of Mg2+ on Ca2+- and Sr2+-activated tension generation of skinned rat cardiac fibers

Submaximum and maximum forces of the cardiac muscle contractile apparatus, activated by Ca2+ or Sr2+, were determined as a function of Mg2+ concentration. Apical left ventricular tissue from Sprague-Dawley rats was broken by homogenization into small bundles of fibers with disrupted sarcolemmas (skinned). Tension generation was activated by and graded according to the concentration of Ca2+ or Sr2+ in solutions bathing the skinned fibers and measured with a photodiode force transducer. Steady-state tensions for various levels of activation at each of four concentrations of Mg2+ (5 x 10(-5), 1 x 10(-3), 5 x 10(-3), and 10 x 10(-3) M) in the bathing solutions were analyzed. Other bathing solution constituents and parameters mimicked significant normal intracellular conditions while providing adequate buffering of [H+], [Ca2+], and [MgATP2-] (magnesium adenosine triphosphate). To assess changes in sensitivity of the mechanical system to activation by Ca2+ (or Sr2+), each submaximum tension was expressed as a percentage of the given fiber bundle's maximum force generated at saturating [Ca2+] (or [Sr2+]) at the same [Mg2+]. When plotted as saturation curves these data demonstrate that increasing [Mg2+] depresses Ca2+ sensitivity of the force-generating mechanism. The Ca2+ and Sr2+ sensitivity of the cardiac force-generating apparatus is similar at every [Mg2+], indicating that the magnitude of Mg2+ effect is similar for both types of activation. However, absolute maximum tensions at saturating activating cation concentration increased as [Mg2+] increased; the effect of Mg2+ on maximum force was proportionately the same for Ca2+ and Sr2+ activation. But because saturating [Ca2+] always resulted in a lower maximum force than saturating [Sr2+], this site of Ca2+-Mg2+ interaction appears distinct from the one influencing Ca2+ sensitivity.     

Ca2+ and cross-bridge-induced changes in troponin C in skinned skeletal muscle fibers: effects of force inhibition

Changes in skeletal troponin C (sTnC) structure during thin filament activation by Ca2+ and strongly bound cross-bridge states were monitored by measuring the linear dichroism of the 5' isomer of iodoacetamidotetramethylrhodamine (5'IATR), attached to Cys98 (sTnC-5'ATR), in sTnC-5'ATR reconstituted single skinned fibers from rabbit psoas muscle. To isolate the effects of Ca2+ and cross-bridge binding on sTnC structure, maximum Ca2+-activated force was inhibited with 0.5 mM AlF4- or with 30 mM 2,3 butanedione-monoxime (BDM) during measurements of the Ca2+ dependence of force and dichroism. Dichroism was 0.08 +/- 0.01 (+/- SEM, n = 9) in relaxing solution (pCa 9.2) and decreased to 0.004 +/- 0.002 (+/- SEM, n = 9) at pCa 4.0. Force and dichroism had similar Ca2+ sensitivities. Force inhibition with BDM caused no change in the amplitude and Ca2+ sensitivity of dichroism. Similarly, inhibition of force at pCa 4.0 with 0.5 mM AlF4- decreased force to 0.04 +/- 0.01 of maximum (+/- SEM, n = 3), and dichroism was 0.04 +/- 0.03 (+/- SEM, n = 3) of the value at pCa 9.2 and unchanged relative to the corresponding normalized value at pCa 4.0 (0.11 +/- 0.05, +/- SEM; n = 3). Inhibition of force with AlF4- also had no effect when sTnC structure was monitored by labeling with either 5-dimethylamino-1-napthalenylsulfonylaziridine (DANZ) or 4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD). Increasing sarcomere length from 2.5 to 3.6 microm caused force (pCa 4.0) to decrease, but had no effect on dichroism. In contrast, rigor cross-bridge attachment caused dichroism at pCa 9.2 to decrease to 0.56 +/- 0.03 (+/- SEM, n = 5) of the value at pCa 9. 2, and force was 0.51 +/- 0.04 (+/- SEM, n = 6) of pCa 4.0 control. At pCa 4.0 in rigor, dichroism decreased further to 0.19 +/- 0.03 (+/- SEM, n = 6), slightly above the pCa 4.0 control level; force was 0.66 +/- 0.04 of pCa 4.0 control. These results indicate that cross-bridge binding in the rigor state alters sTnC structure, whereas cycling cross-bridges have little influence at either submaximum or maximum activating [Ca2+].     

p-Nitrophenylphosphatase activity catalyzed by plasma membrane (Ca(2+) + Mg(2+)ATPase: correlation with structural changes modulated by glycerol and Ca(2+)

The plasma membrane (Ca²⁺+Mg²⁺)ATPase hydrolyzes pseudo-substrates such as p-nitrophenylphosphate. Except when calmodulin is present, Ca²⁺ ions inhibit the p-nitrophenylphosphatase activity. In this report it is shown that, in the presence of glycerol, Ca²⁺ strongly stimulates phosphatase activity in a dose-dependent manner. The glycerol- and Ca²⁺-induced increase in activity is correlated with modifications in the spectral center of mass (average emission wavenumber) of the intrinsic fluorescence of the enzyme. It is concluded that the synergistic effect of glycerol and Ca²⁺ is related to opposite long-term hydration effects on the substrate binding domain and the Ca²⁺ binding domain.     

A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity.

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.     

Calmodulin. An activator of human erythrocyte (Ca2+ + Mg2+)ATPase phosphorylation

The effect of purified calmodulin on the calcium-dependent phosphorylation of human erythrocyte membranes was studied. Under the conditions employed, only one major peak of phosphorylation was observed when solubilized membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of this phosphorylated protein band was estimated to be 130000 and in the presence of purified red blood cell calmodulin, the rate of phosphorylation of this band was increased. These data suggest that calmodulin activation of (Ca2+ + Mg2+)-ATPase could be a partial reflection of an increased rate of phosphorylation of the (Ca2+ + Mg2+)-ATPase of human erythrocyte membranes.     

Influence of gramicidin S on cardiac membrane Ca2+/Mg2+ ATPase activities and contractile force development

The effects of gramicidin S (GS), an antibiotic, on the rat heart membrane ATPases and contractile activity of the right ventricle strips were investigated. GS inhibited sarcolemmal Ca2+-stimulated ATPase (IC50 = 3 microM), Ca2+/Mg2+ ATPase which is activated by millimolar Ca2+ or Mg2+ (IC50 = 3.4 microM), and sarcoplasmic reticulum Ca2+-stimulated ATPase (IC50 = 6 microM). The type of inhibition for the sarcolemmal Ca2+/Mg2+ ATPase by GS was apparently uncompetitive, while that for Ca2+-stimulated ATPases in sarcolemma or sarcoplasmic reticulum was of mixed type. Other ATPases, including mitochondrial ATPase, sarcolemmal Na+-K+ ATPase, and myofibrillar ATPase, were not inhibited by this agent. GS also decreased the rat right ventricle maximum force development (half-maximal inhibitory concentration was 2-4 microM), maximum velocity of contraction, and maximum velocity of relaxation. The resting tension was increased by GS to over 200%. The contractile actions of GS were mostly irreversible upon washing the muscle 3 times over a 10-min period. Decreased Ca2+, Mg2+, Na+, K+ concentrations in the perfusate increased the effects of GS. These findings showed that GS was a potent inhibitor of divalent cation ATPases of heart sarcolemma and sarcoplasmic reticulum and it is suggested that these membrane effects may explain the cardiodepressant action of this agent.     

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.     

Ca2+ activation of membrane-bound (Ca2++Mg2+)-dependent ATPase from human erythrocytes prepared in the presence or absence of Ca2+.

The kinetics of Ca2+ activation of membrane-bound (Ca2+ + Mg2+)-dependent ATPase (ATP phosphohydrolase EC 3.6.1.3) from human erythrocytes was studied. The ATPase from membrane prepared in the presence of 0.7-500muM Ca2+ showed positively cooperative behaviour and a Km for Ca2+ of between 1 and 4 muM. If the membranes were prepared in the absence of Ca2+ the Km increased, and an enzyme model with at least four calcium-binding sites accounted for the kinetic change assuming that one calcium-binding site decreased its affinity. Mg2+ or Mg-ATP could not replace Ca2+. Continuous-flow centrifugation involving a shear stress on membranes was necessary to obtain the high affinity ATPase activity. Using ordinary centrifugation the Ca2+-prepared membranes behaved as membranes prepared in the absence of Ca2+. The Ca2+-stimulated ATPase from membranes prepared without Ca2+ showed reduced maximum activity, but dialyzed, membrane-free hemolysates, whether prepared with Ca2+ present or not, recovered the activity when the hemolysate was present during the ATPase assay. It is suggested that the different Ca2+-affinities of the Ca2+-stimulated ATPase correspond to two different states of the calcium-pump.