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Biomedical subjects

R J Solaro

Publications and source records attributed to R J Solaro.

At least 127 records · Page 7Linked to original sources
PubMed1983

Ischaemia-induced changes in canine cardiac sarcoplasmic reticulum.

Effects of myocardial ischaemia on sarcoplasmic reticulum (SR) of dog hearts were investigated. Regional ischaemia was produced by occlusion of the left circumflex artery, and a microsomal fraction enriched in vesicles of SR was isolated from subendocardium (Endo) and subepicardium (Epi) of control and ischaemic areas of the heart. No significant changes occurred in ischaemic Epi. A loss of in vitro activities (ie calcium transport and ATPase) was found for SR from ischaemic Endo which paralleled the changes in the histology of the tissue. At 5 min of coronary occlusion, Ca2+ binding and Ca2+-ATPase activities of SR from ischaemic Endo were normal. A decrease in the activities of SR was first evident at 15 min after the occlusion, decreased further at about 30 min and remained at that level at 60 min of ischaemia. The maximal rate of Ca2+ uptake did not parallel the Ca2+-binding and Ca2+-ATPase activities. The degree of cAMP-dependent phosphorylation by endogenous and exogenous protein kinase was not different between SR from control and ischaemic areas. A participatory role of SR in the ischaemic impairment of left ventricular systolic and diastolic performance is discussed.

Animals↗
PubMed1983

Swimming exercise, thyroid state, and the distribution of myosin isoenzymes in rat heart.

We tested whether changes in cardiac myosin ATPase activity induced by swimming exercise in male rats are due to a redistribution of existing isoenzymic forms of ventricular myosin. The isoenzymic profiles were analyzed by nondissociating gel electrophoresis of ventricular samples and compared with ATPase activities of myofibrils prepared from the same ventricle. Myofibrils prepared from hearts of rats in the control sedentary group or from hearts of rats in the groups of 8- or 12-wk swimmers had the same actomyosin Mg2+-ATPase activities measured between pCa 8 and 5. However, the myosin Ca2+-ATPase activity of myofibrils prepared from hearts of 8- or 12-wk swimmers was 20% higher than the activity of control preparations. This increase in activity was in proportion to an increase in the relative amount of V1, the myosin isoenzyme with the highest Ca2+-ATPase activity. Thyroidectomized rats, whose hearts had no detectable V1, were also subjected to the swimming program. In the case of the hypothyroid rats, myofibrillar preparations from controls and 8- or 12-wk swimmers had the same actomyosin Mg2+-ATPase activity, myosin Ca2+-ATPase activity, and the same isoenzyme profiles. Co-electrophoresis of ventricular samples from the euthyroid and hypothyroid controls and swimmers showed no evidence for new variants of myosin. We conclude that the increase in myosin Ca2+-ATPase activity in the ventricles of euthyroid swimmers is due to a redistribution of existing isoforms of myosin and that the redistribution process may require thyroid hormone for its expression.

Actomyosin↗
PubMed1983

Coordination of cardiac sarcoplasmic reticulum and myofibrillar function by protein phosphorylation.

Adrenergic stimulation alters functional dynamics of the heart by mechanisms most likely involving cyclic AMP (cAMP)-dependent protein phosphorylation. In vitro studies indicate that the myofibrils and sarcoplasmic reticulum (SR) may act as effectors of the adrenergic stimulation. cAMP-dependent phosphorylation of troponin I (TnI), one of the regulatory proteins of cardiac myofibrils, results in a decreased steady-state affinity of troponin C (TnC) for calcium, an increase in the off-rate for Ca2+ exchange with TnC, and a rightward shift of the relation between free Ca2+ and myofibrillar force or ATPase. Phosphorylation of phospholamban, a regulatory protein of cardiac SR, results in an increased velocity of Ca2+ transport by SR vesicles, an increased affinity of the transport protein for Ca2+, and an increased turnover of elementary steps of the ATPase reaction. These in vitro findings support the hypothesis that the inotropic response of the heart to catecholamine stimulation involves phosphorylation of TnI and phospholamban. Our in vivo studies with perfused rabbit hearts show that during the peak of the inotropic response to isoproterenol there is a simultaneous phosphorylation of TnI and an 11,000-dalton protein in the SR, most likely the monomeric form of phospholamban.

Animals↗
PubMed1982

Phosphate and vanadate reduce the efficiency of the chemo-mechanical energy transformation in cardiac muscle.

Trabecular preparations from the hog heart right ventricle were "skinned" by treatment with Lubrol WX and glycerol. Ca++ activated isometric contractions were gradedly relaxed by inorganic phosphate (Pi) in the millimolar range or vanadate (Vi) in the micromolar range while tension cost (ATP split/force generated) was increased by a factor of 1.75. From measurements of force, ATPase activity, immediate stiffness and stretch activation, evidence is provided that the mechanical deactivation and the increase in tension cost may result from an acceleration of the myosin cross-bridge cycle, due to a direct interference of Pi and Vi with the chemomechanical energy transformation at the contractile proteins. The possible significance of such a mechanism in cardiac failure or muscle fatigue is discussed.

Animals↗
PubMed1982

Activation of thin-filament-regulated muscle by calcium ion: considerations based on nearest-neighbor lattice statistics.

We discuss the activation of thin-filament-regulated muscles by calcium ion in terms of a qualitative model based on nearest-neighbor lattice statistics. For the most part, the model takes into account only the essential features of the phenomenon--that there must be an interaction between calcium adsorption to troponin and crossbridge reaction with actin for calcium ion to activate contraction and that the relevant stationary states are nonequilibrium ones. Even so, the model predicts the following features which are seen experimentally but have generally not been considered in previous models: (i) the relative activations of stationary-state isometric force and ATPase are not equal; (ii) in general, neither activation of force nor that of ATPase is proportional to calcium adsorption to the activating sites; and (iii) the slopes of the relations between the activations and the logarithm of the calcium ion concentration generally depend on the necessary interaction between calcium ion adsorption and crossbridge reaction with actin. Thus, these relations show cooperative effects even if these is no interaction between calcium adsorption sites.

Actins↗
PubMed1982

Stimulation of Ca++ binding and ATPase activity of dog cardiac myofibrils by AR-L 115BS, a novel cardiotonic agent.

AR-L 115BS, a benzimidazole derivative, is a positive inotropic agent that has been shown to increase active tension development and unloaded shortening velocity of chemically skinned heart muscle preparations at submaximal activating levels of free Ca++. We measured the effect of AR-L on relations between free Ca++, bound Ca++ and ATPase activity of dog cardiac myofibrils. At pCa 6, 100-300 micrometers AR-L increased myofibrillar ATPase activity maximally by about 30%. The concentration of AR-L giving half-maximal activation of myofibrillar ATPase activity was about 10 micrometers, and is similar to plasma concentrations associated with elevated contractility in intact animals. There was no effect of AR-L on myofibrillar ATPase activity at pCa 5 or 8, and the relation between pCa and percent activation of myofibrillar ATPase activity was shifted to the left by 0.4-0.5 pCa units in the presence of 100 micrometers AR-L. Calcium binding by cardiac myofibrils was increased by AR-L in the presence and absence of MgATP by 0.2-0.3 nmol/mg myofibrillar protein over a broad range of free Ca++ concentrations, a result suggesting that AR-L increases the affinity of myofibrillar troponin C for Ca++. The shift in the pCa giving half maximal and myofibrillar ATPase activity induced by raising the free Mg++ from 1.0 to 10 mm was unaffected by AR-L. These results indicate that the positive actions of AR-L 115BS on cardiac contractility may involve direct activation of myofibrils by virtue of an increased affinity of thin filament receptors for Ca++.

Adenosine Triphosphatases↗
PubMed1981

Fluorescence studies of the interaction of calmodulin with myosin light chain kinase.

The interaction of calmodulin with myosin light chain kinase produces an approximately 30% increase in myosin light chain kinase tryptophan fluorescence. This represents the first report of calmodulin-induced structural changes in a protein which it activates. We fund that the calmodulin-myosin light chain kinase interaction is: 1) dependent on [Ca2+] (half-maximal binding at pCa 6.2) and essentially independent of [Mg2+], 2) occurs before saturation of all four reported Ca2+-specific sites on calmodulin. 3) saturates with 1 mol of calmodulin bound per mol of kinase with an apparent affinity of approximately 2.0 X 10(7) M-1, 4) is specific for calmodulin over troponin-C, 5) is directly related to the activation of myosin light chain kinase for phosphorylation of myosin light chain. Fluorescence stopped flow studies of these calmodulin-induced fluorescence changes in myosin light chain kinase indicate that Ca2+ binding to calmodulin occurs very rapidly and is not rate-limiting while the calmodulin-induced fluorescence increase in myosin light chain kinase occurs as a biphasic process with rates of approximately 65 s-1 and 6 s-1. The fluorescence increase produced by calmodulin binding to myosin light chain kinase is completely reversed by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at a rate of approximately 2 s-1.

Animals↗
PubMed1981

Interaction of calmodulin with skeletal muscle myosin light chain kinase.

Studies on myosin light chain kinase isolated from rabbit skeletal muscle show that the enzyme has a molecular weight of 80,000--84,000 with a sedimentation coefficient of 3.2 S and an apparent Stokes radius of 53 A. Gel filtration chromatography with a 3H-labeled calmodulin using a Hummel--Dryer technique shows that the enzyme will bind 1 mol of calmodulin per mol of enzyme, with an affinity of (1.9 +/- 0.5) x 10(7) M-1 in the absence of substrate. The calmodulin dependence of enzyme activation at limiting Mg2+ and light chain concentrations confirms this observation. The calcium dependence of activation of the enzyme--calmodulin complex is characterized by a Hill coefficient of 2.5, with half-activation occurring at 6.6 x 10(-7) M Ca2+. The amino acid composition shows a high percentage (9.1%) of proline, which may account for the large apparent Stokes radius and no clear resemblance to other skeletal muscle proteins. A comparison of the amino acid composition with that from turkey gizzard shows some resemblance.

Animals↗
PubMed1981

Ca2+, calmodulin and cyclic AMP-dependent modulation of actin-myosin interactions in aorta.

Incubation of bovine aortic native actomyosin with cyclic AMP and bovine aortic cyclic AMP-dependent protein kinase produced a rightward shift in the relation between free Ca2+ and both superprecipitation and actomyosin ATPase activity. The relation between free Ca2+ and phosphorylation of myosin light chains was also shifted to the right. The concentration of free Ca2+ required for half-maximal activation of both ATPase activity and myosin light chain phosphorylation was approximately 1.0 microM for control actomyosin and 2.5 microM for actomyosin incubated with cyclic AMP-protein kinase. Neither basal nor maximal activities were significantly affected by incubation with cyclic AMP-protein kinase. Addition of e microM calmodulin to cyclic AMP-protein kinase-treated actomyosin relieved inhibition of both superprecipitation and myosin light chain phosphorylation. These findings suggest that cyclic AMP-protein kinase-mediated inhibition of actin-myosin interactions in vascular smooth muscle involve a shift in the Ca2+ sensitivity of the system. This shift probably involves Ca2+-calmodulin interactions and the control of phosphorylation of the myosin light chains.

Actins↗
PubMed1981

Vanadate and phosphate ions reduce tension and increase cross-bridge kinetics in chemically skinned heart muscle.

Tension development, immediate stiffness and ATPase of chemically skinned myocardial strips were measured in solutions with varying concentrations of phosphate (Pi) or vanadate (predominantly H2VO4 at pH 7) ion. Vanadate and Pi decreased stiffness in proportion to tension. The results show that, like Pi, vanadate accelerates the turnover rate of cross-bridges, but is effective at about 1/500 the concentration required for the Pi effect. Both Pi and vanadate increased the energy cost of isometric tension maintenance (that is, the ratio of ATPase to tension) and increased the velocity of delayed tension development following quick stretch of the chemically skinned myocardial strips. The results also show that changes in the rate of rise of delayed tension during stretch activation probably reflect changes in the kinetics of the biochemical cycle of the cross-bridges.

Animals↗
PubMed1981

The effects of modifiers on enzyme catalysis: a non-classical nearest neighbor approach.

We present a nearest neighbor lattice model of the effects of modifiers on two-state enzyme catalysis of the reaction S in equilibrium with p. We do not in general make the assumptions of the classical approach to cooperative catalysis that yield (1) adsorption isotherms of the same form as those for the corresponding equilibrium system and (2) a rate of the catalyzed reaction proportional to the number of occupied catalytic sites. Closed form results are obtained for two approximations, the Bragg-Williams and the quasi-chemical. The latter requires (1), but is exact for several simple cases, including the concerted model, under this condition. Under (1) it is found that an interaction between modifier and catalytic sites, whether attractive or repulsive, increases the magnitudes of the slopes of the adsorption isotherms but that interactions between identical sites (catalytic or modifier) increase these magnitudes if attractive and decrease them if repulsive. Thus, the former interaction allows for phase transitions if sufficiently attractive or repulsive, but the latter only if sufficiently attractive. Herein also lies the explanation for why the concerted model displays only "positive cooperativity". It is further seen that it is not possible to classify a modifier as an activator or inhibitor of the catalyzed reaction solely on the basis of the sign of the interaction energy between catalytic and modifier sites. For a given energy, the rate of the reaction may increase or decrease in response to the modifier, or it may respond biphasically. Similarly, the rate may respond biphasically to the activities of s or p, leading to instabilities. Thus, possibilities of multiple nonequilibrium stationary states or spatio-temporal patterns are raised.

Enzymes↗
PubMed1981

Phosphorylation and adenosine triphosphatase activity of myofibrils from thyrotoxic rabbit hearts.

Cardiac hypertrophy induced by thyrotoxic stress leads to an increase in the rate of force development, velocity of shortening, tension-dependent heat generation, and myosin ATPase activity. We did studies to see whether alterations in covalent phosphorylation of myofibrillar proteins correlate with these changes. The protein preparations were isolated from control and thyrotoxic hearts of male albino rabbits freeze-clamped in situ. We measured myofibrillar ATPase, and the covalent phosphate content of ventricular myosin 19,000 (mol wt) light chain (P-light chain) and troponin I (TnI). The myofibrillar ATPase activity was increased 2-fold in the thyrotoxic preparations with no change in the level of myofibrillar phosphorylation. The covalent phosphate content of TnI was 1.21 +/- 0.09 mol P/mol TnI in control hearts and 1.14 +/- 0.04 mol P/mol TnI in thyrotoxic hearts. The covalent phosphate content of the light chain fraction was 0.41 +/- 0.06 mol P/mol P-light chain in control hearts and 0.37 +/- 0.04 mol P/mol P-light chain in thyrotoxic hearts. The dependence of the normalized myofibrillar ATPase on free calcium concentration was the same in control and thyrotoxic preparations. Thus the mechanical, thermal, and biochemical changes found in hearts from thyrotoxic animals probably occur with no change in phosphorylation of TnI or myosin light chains.

Adenosine Triphosphatases↗
PubMed1980

The calcium and magnesium binding sites on cardiac troponin and their role in the regulation of myofibrillar adenosine triphosphatase.

The cardiac troponin (Tn) complex, consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT), has been reconstituted from purified troponin subunits isolated from bovine heart muscle. The Ca2+-binding properties of cardiac Tn were determined by equilibrium dialysis using either EGTA or EDTA to regulate the free Ca2+ concentration. Cardiac Tn binds 3 mol Ca2+/mol and contains two Ca2+-binding sites with a binding constant of 3 X 10(8) M-1 and one binding site with a binding constant of 2 X 10(6) M-1. In the presence of 4 mM MgC12, the binding constant of the sites of higher affinity is reduced to 3 X 10(7) M-1, while Ca2+ binding to the site at the lower affinity is unaffected. The two high affinity Ca2+-binding sites of cardiac Tn are analogous to the two Ca2+-Mg2+ sites of skeletal Tn, while the single low affinity site is similar to the two Ca2+-specific sites of skeletal Tn (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4625-5633). The Ca2+-binding properties of the complex of TnC and TnI (1:1 molar ratio) were similar to those of Tn. Cardiac TnC also binds 3 mol of Ca2+/mol and contains two sites with a binding constant of 1 X 10(7) M-1 and a single site with a binding constant of 2 X 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the high affinity sites of TnC and Tn, the binding constants for Mg2+ were 0.7 and 3.0 X 10(3) M-1, respectively. The Ca2+ dependence of cardiac myofibrillar ATPase activity was similar to that of an actomyosin preparation regulated by the reconstituted troponin complex. Comparison by the Ca2+-binding properties of cardiac Tn and the cardiac myofibrillar ATPase activity as a function of [Ca2+] and at millimolar [Mg2+] suggests that activation of the ATPase occurs over the same range of [Ca2+] where the Ca2+-specific site of cardiac Tn binds Ca2+.

Adenosine Triphosphatases↗
PubMed1980

The site of phosphorylation of troponin I in the perfused rabbit heart. The effect of adrenaline.

1. On treatment of the perfused rabbit heart with adrenaline, the total covalently bound phosphate of troponin I increased from 1.14 mol of phosphate/mol to 1.86 mol of phosphate/mol. 2. Covalently bound phosphate could be identified only in the region of the molecule of cardiac troponin I consisting of residues 1--48. 3. When 32P-labelled orthophosphate was present in the perfusion medium the phosphate at serine-20 became radioactively labelled. This residue was the only significant site of phosphorylation that could be identified. 4. The addition of adrenaline caused a 4--5-fold increase in covalently bound [32P]phosphate. Virtually all of the 32P was located at serine-20. 5. It was concluded from these studies that the extent of phosphorylation of serine-20 of cardiac troponin I increased from 30--40% in the control perfused heart to about 100% in the presence of adrenaline.

Amino Acids↗