Ca2+-accumulating capacity of mitochondria, sarcolemma and sarcoplasmic reticulum of rat heart

Using the isotope exchange technique including 45Ca, the Ca2+-binding and Ca2+-accumulating capacity of mitochondria, sarcolemma and sarcoplasmic reticulum of rat heart was studied. The ATP-independent binding of Ca2+ to isolated membrane fractions is by 1--2 orders of magnitude less than the ATP-dependent Ca2+-accumulating capacity of the fractions. The Ca2+-accumulating capacity of mitochondria is increased 6--8 fold after addition of physiological concentrations of succinate and Pi to the incubation medium. Under these conditions the ratio of Ca2+-accumulating capacity of mitochondria, sarcolemma and sarcoplasmic reticulum of the heart is 100:3,12:2,9. The initial rate of Ca2+-uptake by the sarcoplasmic reticulum is much higher in comparison with sarcolemma and mitochondria. A high Ca2+-accumulating capacity of heart mitochondria probably determines a long-term regulation of the concentration of "troponin-accessible" Ca2+ in the sarcoplasm, whereas the high initial rate of Ca2+ accumulation by the sarcoplasmic reticulum provides for a rapid decrease of Ca2+ concentration during rhythmic contractions of the heart.     

Cardiomyocytes structure, function and associated pathologies

The heart is the first formed organ in the developing fetus. During fetal and postnatal development cardiomyocytes become terminally differentiated muscular cells that are connected end to end by gap junctions, allowing concerted contractile activity. The contraction-relaxation cycle of cardiomyocytes is orchestrated by cyclic increases and decreases in intracellular Ca(2+) initiated by depolarization of the sarcolemma and sustained by Ca(2+) release and re-uptake by the sarcoplasmic reticulum. When stressed, cardiomyocytes undergo hypertrophic growth and apoptotic responses in vivo as well as in cell culture models. Such changes predispose to heart failure in the longer term.     

Localization of Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in adult rat papillary muscle

Localization of the Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in rat papillary muscle was determined by indirect immunofluorescence and immunoferritin labeling of cryostat and ultracryotomy sections, respectively. The Ca2+ + Mg2+-ATPase was found to be rather uniformly distributed in the free sarcoplasmic reticulum membrane but to be absent from both peripheral and interior junctional sarcoplasmic reticulum membrane, transverse tubules, sarcolemma, and mitochondria. This suggests that the Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum is antigenically unrelated to the Ca2+ + Mg2+-ATPase of the sarcolemma. These results are in agreement with the idea that the sites of interior and peripheral coupling between sarcoplasmic reticulum membrane and transverse tubules and between sarcoplasmic reticulum and sarcolemmal membranes play the same functional role in the excitation-contraction coupling in cardiac muscle.     

Progress and problems in understanding the involvement of calcium in heart function

In this article we have briefly reviewed the role of Ca2+ in the excitation contraction coupling in the myocardium and have indicated that cardiac contraction and relaxation are initiated upon raising and lowering the intracellular concentration of free Ca2+, respectively. Different mechanisms for the entry of Ca2+ through sarcolemma as well as release of Ca2+ from sarcoplasmic reticulum and possibly mitochondria have been outlined for initiating cardiac contraction. Relaxation of the cardiac muscle appears to be intimately dependent upon efflux of Ca2+ through sarcolemma as well as sequestration of Ca2+ by the intracellular storage sites, particularly sarcoplasmic reticulum and possibly mitochondria. The actions of some pharmacological and pathophysiological interventions have been explained on the basis of changes in subcellular Ca2+ movements in myocardium. Quinidine, which produced an initial positive inotropic action on rat heart was also found to increase sarcolemmal Ca2+-ATPase activity without any changes in the Na+-K+ ATPase. Other antiarrhythmic agents, procainamide and lidocaine, also increased sarcolemmal Ca2+-ATPase activity without affecting the Na+-K+ ATPase. On the other hand, both Ca2+-ATPase and Na+-K+ ATPase activities were increased in heart sarcolemma obtained from cardiomyopathic hamsters. In this model the increased Ca2+-ATPase activity may promote the occurrence of intracellular Ca2+ overload in the cardiac cell whereas the increased Na+-K+ ATPase activity may increase Ca2+ efflux through Na+-Ca2+ exchange systems as an adaptive mechanism. It has been suggested that some caution should be exercised while interpreting the data from in vitro experiments in terms of functional changes in the myocardium. Furthermore, it has been proposed that the pathophysiology and pharmacology of Ca2+ movements at different membrane sites be understood fully in normal and diseased myocardium in order to improve the therapy of heart disease.     

Stimulation of Ca2+ uptake by cyclic AMP and protein kinase in sarcoplasmic reticulum-rich and sarcolemma-rich microsomal fractions from rabbit heart.

The effect of cyclic AMP on Ca2+ uptake by rabbit heart microsomal vesicular fractions representing mainly fragments of either sarcoplasmic reticulum or sarcolemma was investigated in the presence and absence of soluble cardiac protein kinase and with microsomes prephosphorylated by cyclic AMP-dependent protein kinase. The acceleration of oxalate-promoted Ca2+ uptake by fragmented sarcoplasmic reticulum following cyclic AMP-dependent membrane protein phosphorylation, observed by other authors, was confirmed. In addition it was found that the acceleration was greatest at pH 7.2 and almost negligible at pH 6.0 and pH 7.8. A very marked increase in Ca2+ uptake by cyclic AMP-dependent membrane protein phosphorylation was observed in the presence of boric acid, a reversible inhibitor of Ca2+ uptake. In addition to the microsomal fraction thought to represent mainly fragments of the sarcoplasmic reticulum, the effect of protein kinase and cyclic AMP on Ca2+ uptake was investigated in a cardiac sarcolemma-enriched membrane fraction. Ca2+ uptake by sarcolemmal vesicles, unlike Ca2+ uptake by sarcoplasmic reticulum vesicles, was inhibited by low doses of digitoxin. The acceleration of oxalate-promoted Ca2+ uptake by cyclic AMP and soluble cardiac protein kinase, however, was quite similar to what was seen in preparations of fragmented sarcoplasmic reticulum, which suggests that it may reflect an acceleration of active Ca2+ transport across the myocardial cell surface membrane.     

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.     

Primary role of sarcoplasmic reticulum in phasic contractile activation of cardiac myocytes with shunted myolemma

Homogeneous populations of single myocytes showing good preservation of ultrastructure were obtained by enzymatic digestion of rabbit and rat hearts, and maintained in a relaxed state in the presence of free Ca2+ concentrations less than 10(-7) M. Ultrastructural details such as a cytoskeleton of 100-A filaments connected to the sarcolemma at the Z lines were demonstrated especially well in these preparations. In spite of seemingly normal structure, electron probe analysis of cryosections reveals similar concentrations of electrolytes in the medium and in the cytoplasm, indicating the presence of electrochemical shunting across the external membrane. The dissociated myocytes display Ca uptake and phasic contractions that are apparently dependent on mitochondrial respiration, but are not affected by mitochondrial uncouplers when ATP and phosphocreatine are added. The uptake is augmented by oxalate and, based on identification of calcium oxalate crystals by electron microscopy and electron probe analysis, is localized to the sarcoplasmic reticulum (SR). An advantageous feature of the dissociated myocytes is that they are suitable for experiments using large numbers of cells in suspension. Thereby, velocities of calcium transport were measured directly by isotopic tracer and filtration methods. It was then found that the lowest CA2+ concentrations (5 x 10(-7) M for the rabbit and 1 x 10(-7) M for the rat) sustaining Ca transport also induce phasic contractile activity in all myocytes, even though the external membrane is electrochemically shunted. A stepwise rise in the Ca2+ concentration of up to one order of magnitude, increases transport velocities in parallel with the rates of phasic contractions. Both these parameters are affected by Mg2+, temperature, cyclic-AMP, and methylxanthines, even though the Ca2+ concentration is maintained constant in the medium. Therefore, Ca transport by SR is a requirement and a rate limiting factor for the occurrence of phasic contractile activation in dissociated cardiac cells retaining an electrochemically shunted external membrane. It is suggested that transient Ca release required for phasic contractile activation is due to equilibrium oscillations across the SR membrane. The sequential pattern of sarcomere activation is consistent with a self propagating mechanism of calcium release. SR in dissociated skeletal muscle cells sustains a greater Ca transport activity than in dissociated heart cells. However, the heart cells display a much higher phasic contractile activity, indicating that cardiac SR has a greater tendency to release accumulated calcium. If free Ca2+ in the medium is raised above 10(-6) M, both cardiac and skeletal myocytes undergo contractures and degenerative phenomena, accompanied by Ca, Mg, and phosphate accumulation in cardiac mitochondria.     

Diabetic alterations in cardiac sarcoplasmic reticulum Ca2+-ATPase and phospholamban protein expression.

Diabetic cardiomyopathy has been suggested to be caused by abnormal intracellular Ca2+ homeostasis in the myocardium, which is partly due to a defect in calcium transport by the cardiac sarcoplasmic reticulum (SR). In the present study, the underlying mechanism for this functional derangement was investigated with respect to SR Ca2+-ATPase and phospholamban (the inhibitor of SR Ca2+-ATPase). The maximal Ca2+ uptake and the affinity of Ca2+-ATPase for Ca2+ were decreased, and exogenous phosphorylation level of phospholamban was higher in streptozotocin-induced diabetic rat SR. Levels of both mRNA and protein of phospholamban were significantly increased in the diabetic hearts, whereas those of SR Ca2+-ATPase were significantly decreased. Consequently, the relative phospholamban/Ca2+-ATPase ratio was 1.88 in the diabetic hearts, and these changes were correlated with changes in the rates of SR Ca2+ uptake. However, phosphatase pretreatment of phospholamban for dephosphorylation of the sites phosphorylated in vivo did not change the levels of subsequent phospholamban phosphorylation in either control or diabetic rat hearts. The above data indicated that the increased phospholamban phosphorylation was not due to autonomic dysfunction but possibly due to increased phospholamban expression. These findings suggest that reduction of the SR Ca2+-ATPase level would contribute to decreased rates of SR Ca2+ uptake and that this function is further impaired by the enhanced inhibition by phospholamban due to its increased expression in the diabetic heart.     

Immunoelectron microscopical localization of phospholamban in adult canine ventricular muscle

The subcellular distribution of phospholamban in adult canine ventricular myocardial cells was determined by the indirect immunogold-labeling technique. The results presented suggest that phospholamban, like the Ca2+-ATPase, is uniformly distributed in the network sarcoplasmic reticulum but absent from the junctional portion of the junctional sarcoplasmic reticulum. Unlike the Ca2+-ATPase, but like cardiac calsequestrin, phospholamban also appears to be present in the corbular sarcoplasmic reticulum. Comparison of the relative distribution of phospholamban immunolabeling in the sarcoplasmic reticulum with that of the sarcolemma showed that the density of phospholamban in the network sarcoplasmic reticulum was approximately 35-fold higher than that of the cytoplasmic side of the sarcolemma, which in turn was found to be three- to fourfold higher than the density of the background labeling. However, a majority of the specific phospholamban labeling within 30 nm of the cytoplasmic side of the sarcolemma was clustered and present over the sarcoplasmic reticulum in the subsarcolemmal region of the myocardial cells, suggesting that phospholamban is confined to the junctional regions between the sarcolemma and the sarcoplasmic reticulum, but absent from the nonjunctional portion of the sarcolemma. Although the resolution of the immunogold-labeling technique used (60 nm) does not permit one to determine whether the specific labeling within 30 nm of the cytoplasmic side of the sarcolemma is associated with the sarcolemma and/or the junctional sarcoplasmic reticulum, it is likely that the low amount of labeling in this region represents phospholamban associated with sarcoplasmic reticulum. These results suggest that phospholamban is absent from the sarcolemma and confined to the sarcoplasmic reticulum in cardiac muscle.     

Membrane crystals of Ca2+-ATPase in sarcoplasmic reticulum of fast and slow skeletal and cardiac muscles

Crystalline arrays of Ca2+ transport ATPase develop in sarcoplasmic reticulum membranes after treatment with Na3VO4 in a calcium-free medium [ Dux , L. and Martonosi , A. (1983) J. Biol. Chem. 258, 2599-2603]. The proportion of vesicles containing Ca2+-ATPase crystals in microsome preparations isolated from rat muscle of different fiber types (semimembranosus, levator ani, extensor digitorum longus, diaphragm, soleus, and heart) correlates well with the Ca2+-ATPase content and Ca2+-modulated ATPase activity. This implies that the concentration of Ca2+-ATPase in sarcoplasmic reticulum membranes of fast and slow skeletal or cardiac muscles differs only slightly, and the low Ca2+ transport activity of 'sarcoplasmic reticulum' preparations isolated from slow-twitch skeletal and cardiac muscles is due to the presence of large amount of non-sarcoplasmic-reticulum membrane elements. This is in accord with the relatively small differences in the density of 8.5-nm intramembranous particles seen by freeze-etch electron microscopy in sarcoplasmic reticulum of red and white muscles. The dimensions of the Ca2+-ATPase crystal lattice are similar in sarcoplasmic reticulum membranes of different fiber types; therefore if structural differences exist between 'isoenzymes' of Ca2+-ATPase, these are not reflected in the crystal-lattice.     

Membrane localization of myocardial type II cyclic AMP-dependent protein kinase activity

Crude cardiac membrane vesicles were separated into subfractions of sarcolemma and sarcoplasmic reticulum. The subfractions were used to determine the origin and type of cyclic AMP-dependent protein kinase activity present in myocardial membranes. A cyclic AMP-binding protein of molecular weight 55,000 was covalently labeled with the photoaffinity probe 8-azido adenosine 3',5'-mono[32P]phosphate, and found to copurify with the (Na+ + K+)-ATPase activity of sarcolemma, and away from the (Ca2+ + K+)-ATPase activity of sarcoplasmic reticulum. Endogenous cyclic AMP-dependent protein kinase activity also copurified with sarcolemma. Protein substrates phosphorylated by cyclic AMP-dependent protein kinase activity had apparent molecular weights of 21,000 and 8000 and were present in both sarcolemma and sarcoplasmic reticulum. However, while addition of cyclic AMP alone resulted in phosphorylation of sarcolemma proteins, both cyclic AMP and exogenous, soluble cyclic AMP-dependent kinase were required for phosphorylation of sarcoplasmic reticulum proteins. Addition of the calcium-binding protein, calmodulin, to either sarcolemma or sarcoplasmic reticulum resulted in phosphorylation of the 21,000 and 8000-dalton proteins, as well. The results suggest that cardiac sarcolemma contains an intrinsic type II cyclic AMP-dependent protein kinase activity that is not present in sarcoplasmic reticulum. On the other hand, Ca2+- and calmodulin-dependent protein kinase activity is present in both sarcolemma and sarcoplasmic reticulum.     

Excitation-contraction coupling in myocardium: implications of calcium release and Na+-Ca2+ exchange

In this paper, we present evidence in support of the hypothesis that electrogenic Na+-Ca2+ exchange is responsible for three phenomena in rat cardiac muscle: the slow repolarization phase of the action potential, the time course of the mechanical recovery process, and the development of triggered arrhythmias. It was shown that the duration of the slow phase of repolarization of the action potential varies in proportion to the Na+ concentration gradient and inversely with the Ca2+ concentration gradient over the cell membrane. This suggested that Na+-Ca2+ exchange can generate a current of sufficient magnitude to maintain the membrane depolarized at a level of -60 mV. The mechanical restitution process of rat cardiac trabeculae was shown to exhibit three phase. The first phase, alpha, probably reflects rapid transport of calcium in the sarcoplasmic reticulum from the uptake sites to the release sites. After the initial increase of force during alpha, force rises further during phase beta and then declines during phase gamma. During all phases, force increases with the extracellular calcium concentration. beta is accelerated by preceding extrasystoles, while an increase of the heart rate causes force to increase at approximately the same rate but to a higher level during phase beta. These observations are compatible with a model in which the sarcoplasmic reticulum sequesters calcium from the cytosol, while the membrane of the sarcoplasmic reticulum is assumed to exhibit also a small leak of calcium into the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional characterization of junctional terminal cisternae from mammalian fast skeletal muscle sarcoplasmic reticulum

Junctional terminal cisternae are a recently isolated sarcoplasmic reticulum fraction containing two types of membranes, the junctional face membrane with morphologically intact "feet" structures and the calcium pump membrane [Saito, A., Seiler, S., Chu, A., & Fleischer, S. (1984) J. Cell Biol. 99, 875-885]. In this study, the Ca2+ fluxes of junctional terminal cisternae are characterized and compared with three other well-defined fractions derived from the sarcotubular system of fast-twitch skeletal muscle, including light and heavy sarcoplasmic reticulum, corresponding to longitudinal and terminal cisternae regions of the sarcoplasmic reticulum, and isolated triads. Functionally, junctional terminal cisternae have low net energized Ca2+ transport measured in the presence or absence of a Ca2+-trapping anion, as compared to light and heavy sarcoplasmic reticulum and triads. Ca2+ transport and Ca2+ pumping efficiency can be restored to values similar to those of light sarcoplasmic reticulum with ruthenium red or high [Mg2+]. In contrast to junctional terminal cisternae, heavy sarcoplasmic reticulum and triads have higher Ca2+ transport and are stimulated less by ruthenium red. Heavy sarcoplasmic reticulum appears to be derived from the nonjunctional portion of the terminal cisternae. Our studies indicate that the decreased Ca2+ transport is referable to the enhanced permeability to Ca2+, reflecting the predominant localization of Ca2+ release channels in junctional terminal cisternae. This conclusion is based on the following observations: The Ca2+, -Mg2+ -dependent ATPase activity of junctional terminal cisternae in the presence of a Ca2+ ionophore is comparable to that of light sarcoplasmic reticulum when normalized for the calcium pump protein content; i.e., the enhanced Ca2+ transport cannot be explained by a faster turnover of the pump. Ruthenium red or elevated [Mg2+] enhances energized Ca2+ transport and Ca2+ pumping efficiency in junctional terminal cisternae so that values approaching those of light sarcoplasmic reticulum are obtained. Rapid Ca2+ efflux in junctional terminal cisternae can be directly measured and is blocked by ruthenium red or high [Mg2+]. Ryanodine at pharmacologically significant concentrations blocks the ruthenium red stimulation of Ca2+ loading. Ryanodine binding in junctional terminal cisternae, which appears to titrate Ca2+ release channels, is 2 orders of magnitude lower than the concentration of the calcium pump protein. By contrast, light sarcoplasmic reticulum has a high Ca2+ loading rate and slow Ca2+ efflux that are not modulated by ruthenium red, ryanodine, or Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of creatine phosphokinase in supplying energy for the calcium pump system of heart sarcoplasmic reticulum.

An investigation of isolated and purified heart sarcoplasmic reticulum performed in the current study indicates the presence of significant creatine phosphokinase (CPK) activity in this preparation. The localization of CPK on the membrane of sarcoplasmic reticulum has been revealed also by an electron microscopic histochemical method. Under the conditions of the Ca(2+)-ATPase reaction in the presence of creatine phosphate, the release of creatine into the reaction medium is observed, the rate of the latter process being dependent on the MgATP concentration in accordance with the kinetic parameters of the Ca2+-ATPase reaction. CPK localized on the reticular membrane is able to maintain the high rate of calcium consumption by the sarcoplasmic reticulum vesicles. The results obtained demonstrate the close functional coupling between CPK and Ca2+-ATPase in the membrane of sarcoplasmic reticulum and indicate the important functional role of CPK in supplying energy for the Ca(2+)-ATPase reaction and ion transport across the membrane of heart sarcoplasmic reticulum.     

Regulation of the ATP dependent calcium uptake activity of heart sarcolemmal vesicles by endogenous cytosolic proteins

In a previous study we described the inhibitory action of a cytosolic protein fraction from heart muscle on ATP-dependent Ca2+ uptake by sarcoplasmic reticulum; further, this inhibition was shown to be blocked by an inhibitor antagonist, also derived from the cytosol (Narayanan et al. Biochim Biophys Acta 735: 53-66, 1983). The present study examined the effects of the endogenous cytosolic Ca2+ transport inhibitor and its antagonist on ATP-dependent Ca2+ uptake by sarcolemmal vesicles isolated from rat and canine heart. The cytosolic inhibitor caused strong inhibition (up to 97%) of Ca2+ uptake by sarcolemma (SL); this inhibition could be reversed by the cytosolic inhibitor antagonist. Studies on the characteristics of inhibition revealed the following: a) Inhibition was dependent on the concentration of the inhibitor (50% inhibition with approximately 80 micrograms inhibitor protein). b) The inhibitor reduced the velocity of Ca2+ uptake without appreciably influencing the apparent affinity of the transport system for Ca2+ but caused greater than 2-fold decrease in its apparent affinity for ATP. c) The rates of unidirectional passive Ca2+ release from actively Ca2+ loaded SL vesicles were not altered by low concentrations of the inhibitor (less than 100 micrograms/ml) which were effective in producing marked inhibition of Ca2+ uptake; at higher concentrations (greater than 100 micrograms/ml), the inhibitor caused increase in the rates of passive Ca2+ release. These findings demonstrate that the activity of the ATP-driven Ca2+ pump of cardiac SL can be regulated in vitro by endogenous cytosolic proteins.     

The base-exchange enzyme activities of sarcolemma and sarcoplasmic reticulum from rat heart

The Ca2+ dependent incorporation of [14C]ethanolamine, L-[14C]serine and [14C]choline into phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine, respectively, were investigated in membrane preparations from rat heart. The ethanolamine and serine base-exchange enzyme-catalyzed reactions were associated with the sarcolemma and sarcoplasmic reticulum. There was a 17.2-fold and 6.8-fold enrichment, respectively, of the serine and the ethanolamine base-exchange enzyme activities in the sarcolemma compared to the starting whole homogenate. The sarcoplasmic reticulum was enriched in the ethanolamine and serine base-exchange enzyme activities. The choline base-exchange enzyme activity of all membranes fractions was negligible compared to the ethanolamine or serine base-exchange enzyme activities. The apparent Km for the ethanolamine and serine base-exchange enzyme in sarcolemma was 14 microM and 25 microM, respectively. The pH optimum for these base-exchange activities was 7.5-8.0. There was a dependence upon Ca2+ for these reactions with a 1 or 4 mM concentration required for maximal activity. The properties of the sarcoplasmic reticulum base-exchange enzymes were similar to the sarcolemmal base-exchange enzymes.     

Effect of pH on calcium accumulation and release by isolated fragments of cardiac and skeletal muscle sarcoplasmic reticulum.

The ability of a sudden increase in pH to initiate a release of calcium from isolated skeletal and cardiac muscle sarcoplasmic reticulum following calcium accumulation in the absence of a precipitating anion (calcium binding) is described. In skeletal sarcoplasmic reticulum a sudden increase in pH caused a rapid release of accumulated calcium. In cardiac sarcoplasmic reticulum a sudden increase in pH before the calcium binding process was complete caused the release of a small amount of calcium at a relatively slow rate. A sudden change in pH after the completion of calcium binding failed to trigger a release of calcium. The effect of pH on oxalate supported calcium uptake and on unidirectional calcium efflux rate by cardiac sarcoplasmic reticulum was also studied. Both the rate of calcium uptake and of unidirectional calcium efflux increased as the pH was raised from 6.4 to 7.2, reflecting an increased permeability of the sarcoplasmic reticulum membrane to calcium. These results indicate that in cardiac muscle a sudden increase in pH is unlikely to be the in vivo signal for calcium release from the sarcoplasmic reticulum. However, the effect of pH on calcium uptake and efflux by cardiac sarcoplasmic reticulum may contribute to the negative inotropic effect of an acidosis on the heart.     

(Ca2+ + Mg2+)-dependent ATPase mRNA from smooth muscle sarcoplasmic reticulum differs from that in cardiac and fast skeletal muscles

We have investigated some characteristics of the sarcoplasmic reticulum (Ca2+ + Mg2+)-dependent ATPase (Ca2+-ATPase) mRNA from smooth muscle using specific cDNA probes isolated from a rat heart cDNA library. RNA blot analysis has shown that the Ca2+-ATPase mRNA expressed in smooth muscle is identical in size to the cardiac mRNA but differs from that of fast skeletal muscle. S1 nuclease mapping has moreover shown that the cardiac and smooth muscle isoforms possess different 3'-end sequences. These results indicate that a distinct sarcoplasmic reticulum Ca2+-ATPase mRNA is present in smooth muscle.     

Thyroid hormone markedly increases the mRNA coding for sarcoplasmic reticulum Ca2+-ATPase in the rat heart

Previous findings have shown that thyroid hormone markedly increases the speed of diastolic relaxation in the heart. This thyroid hormone-dependent change is also accompanied by an increased Ca2+ pumping ability in the sarcoplasmic reticulum. In an effort to determine the underlying cause of improved Ca2+ transport, mRNA levels of the slow Ca2+-ATPase of the sarcoplasmic reticulum were quantified on Northern blots. In hypothyroid rat hearts, the steady state level of Ca2+-ATPase mRNA was only 36% of control levels, whereas hyperthyroid rat heart mRNA levels were 136% of control. Ca2+-ATPase mRNA responded rapidly to T3, as the mRNA level was significantly increased by 2 h and normalized by 5 h after T3 injection into hypothyroid rats. The well established effect of thyroid hormone on improved myocardial contractility and increased speed of diastolic relaxation may in part relate to specific alterations in the level of the mRNA coding for Ca2+-ATPase, resulting in increased pump units.     

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.