Real-time measurement of the rate of sarcoplasmic reticulum calcium pumping in skinned muscle fibers

A method and a device had been developed to directly measure the accumulation of calcium in the sarcoplasmic reticulum and its release from the sarcoplasmic reticulum, depending on the free Ca²⁺ concentration in the solution. The sarcoplasmic reticulum occupies to 30% of the volume of the swim bladder muscles of the oyster toadfish Opsanus tau. To isolate and skin muscle fibers and to remove the accumulated calcium from the sarcoplasmic reticulum, a set of solutions containing EGTA as a pCa buffer was used. To measure the calcium exchange between a fiber ～10 nl in volume and the solution in a 5-μl cuvette, instead of EGTA, 50–100 μM FURA2 or bisFURA2 was used both as pCa buffer and as a fluorescent indicator of the calcium concentration in the cuvette. An increase in fluorescence intensity meant an increase in the free FURA concentration in the solution surrounding the fiber since the calcium entering the sarcoplasmic reticulum was taken from this solution. The slope of the fluorescence curve corresponded to a rate of calcium accumulation in the sarcoplasmic reticulum of 1.6 μmol per second per liter of the solution in the cuvette or 2.6 mmol per second per liter of the sarcoplasmic reticulum. A solution without oxalate and ruthenium red may exhibit oscillations of the free FURA concentration, which can be explained by calcium-activated calcium release from the sarcoplasmic reticulum.     

Isolation of highly active preparations of sarcoplasmic reticulum and Ca2-dependent ATPase from cardiac muscle]

A microsomal preparation with a high ability for Ca2+ uptake has been isolated from pigeon heart. A method of further purification of Ca2+-accumulating system of heart, based on the ability of sarcoplasmic reticulum for the energy-dependent Ca2+ accumulation in the presence of oxalate, has been developed. Upon centrifugation in the gradient of sucrose and KCl concentration the fragments of sarcoplasmic reticulum, rendered "heavy" by calcium oxalate, can be separated from foreign cell membranes. The main component of heart "calcium pump" is Ca2+-dependent ATPase (making up to about 50% of all proteins of the purified reticulum), having a molecular weight of 100.000--105.000. Specific activity of heart Ca2+-ATPase as well as the ability of purified heart sarcoplasmic reticulum for Ca2+ uptake are only slightly less than those of the skeletal muscle reticulum. The data obtained suggest that heart sarcoplasmic reticulum may be efficient for providing heart muscle relaxation.     

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.     

Electron cytochemistry of oxalate-stimulated calcium uptake in microsomes from the smooth muscle of the pig stomach

Summary A microsomal fraction was isolated from the smooth muscle of the antrum of the pig stomach by differential centrifugation. Electron microscopy of the negatively stained material showed that this fraction is heterogeneous in composition. The microsomes accumulated calcium in the presence of ATP, magnesium and oxalate. The amount of calcium taken up per mg protein was in the same range as observed for other smooth muscle microsomal preparations. Although this amount is much smaller than that in the microsomal fraction of skeletal muscle, calcium oxalate crystals were formed in some vesicles, as occurs in the skeletal muscle fragmented sarcoplasmic reticulum. Through the presence of the calcium oxalate crystals, many of these vesicles acquired sufficient mass and density to allow them to be isolated by centrifugation. A purification of about 40 fold in terms of calcium content was reached.     

Calcium in smooth muscle sarcoplasmic reticulum in situ. Conventional and X-ray analytical electron microscopy

Numerous electron-opaque deposits appear in the SR of the relaxed smooth muscle cells of the guinea pig Taenia coli that had been treated, before fixation, with a depolarizing medium containing oxalate to precipitate calcium ions in situ. X-ray spectra obtained by spot and line-scanning analyses of these deposits in situ show characteristic calcium signals, thus providing direct evidence for calcium accumulation inside the sarcoplasmic reticulum of smooth muscle.     

Uptake of calcium by the endoplasmic reticulum of the frog photoreceptor

We studied retinal photoreceptors of Rana pipiens by using techniques designed to investigate calcium localization. Particularly useful were methods in which intracellular sites of calcium uptake were detected by incubation of saponin-treated isolated retinas in calcium-containing media, with oxalate present as a trapping agent. With these procedures, cell compartments accumulate deposits, which can be shown to contain calcium by x-ray microanalysis. Calcium accumulation was prominent in the rough endoplasmic reticulum in the myoid region. In addition, deposits were observed in agranular reticulum and in certain Golgi- associated compartments of the myoid region, in mitochondria, in axonal reticulum, and in agranular reticulum of presynaptic terminals. Calcium was also detected in the endoplasmic reticulum of retinas fixed directly upon isolation, by a freeze-substitution method. The factors influencing accumulation of calcium in the endoplasmic reticulum were evaluated by a semiquantitative approach based on determining the relative frequency of calcium oxalate crystals under varying conditions. Calcium accumulation was markedly enhanced by ATP. Studies with a nonhydrolyzable ATP analogue (adenylyl- imidodiphosphate ) and with inhibitors of the sarcoplasmic reticulum Ca2+-Mg2+ ATPase (mersalyl and tetracaine) indicated that this ATP-dependent calcium uptake reflects an energy-dependent process roughly comparable to that in the sarcoplasmic reticulum.     

The subcellular calcium distribution in the smooth muscle cells of the pig coronary artery

In order to complete preliminary investigations on the subcellular calcium localisation in smooth muscle cells, further experiments are presented using smooth muscle cells from the coronary artery of the pig. The methods used were a precipitation technique using potassium oxalate and autoradiography using ⁴⁵Ca. In all cases we were able to reproduce the results obtained in our preliminary study. The preparations clearly show calcium oxalate precipitates in the cell membrane, the sarcoplasmic reticulum, the microvesicles, mitochondria and the nucleus membrane. These findings were supported by silver grain distributions in autoradiograms obtained by means of ⁴⁵Ca. The qualitative results obtained histochemically are in good agreement with estimations of the calcium distribution in subcellular fractions obtained by atomic absorption spectrophotometry.     

The effect of calcium oxalate crystallization kinetics on the kinetics of calcium uptake and calcium ATPase activity of sarcoplasmic reticulum vesicles

The ionophore A23187 is a potent inhibitor of oxalate supported calcium uptake if added before uptake is initiated by ATP and is a much weaker inhibitor of uptake once uptake has been initiated. This observation is shown to be due to a failure of oxalate to capture the transported calcium at the beginning of uptake because the rate of calcium oxalate crystallization is initially slow, thereby allowing the ionophore to release the accumulated calcium. This hypothesis is supported by the observation that calcium oxalate crystallization shows a lag phase which is absent when calcium oxalate seeds are in the reaction system. Once calcium uptake has progressed, calcium oxalate seeds are present in the sarcoplasmic reticulum and calcium oxalate crystallization proceeds sufficiently rapidly that the ionophore cannot compete successfully for calcium. That A23187 and oxalate compete for intravesicular ionic calcium is shown by the stimulation which each produces in ATPase activity and by the dependence of ionophore activity on oxalate concentration.The failure of calcium oxalate crystallization to reach equilibrium during the early phase of calcium uptake caused us to examine whether at any time during calcium uptake, crystallization reaches equilibrium. Skeletal sarcoplasmic reticulum accumulated calcium at such a high rate that oxalate, in concentrations up to 20mM, was unable to clamp intravesicular calcium at equilibrium values. The lower rate of calcium accumulation by cardiac sarcoplasmic reticulum and/or perhaps its greater permeability to oxalate apparently allows intravesicular calcium to be clamped by oxalate.     

The Effect of Quinidine on Calcium Accumulation by Isolated Sarcoplasmic Reticulum of Skeletal and Cardiac Muscle

Quinidine potentiates twitch tension and (at higher concentrations) causes contracture of skeletal muscle whereas the same drug reduces tension development of cardiac muscle. To gain insight into the possible differences in the excitation-contraction coupling mechanism of the two types of muscle the effect of quinidine on calcium accumulation by isolated sarcoplasmic reticulum from skeletal and cardiac muscle was investigated. In a medium containing ATP, Mg⁺⁺, oxalate, and ⁴⁵Ca, pharmacologically active concentrations of the drug inhibited calcium accumulation by both skeletal and cardiac sarcoplasmic reticulum. The inhibition of the rates of calcium, uptake by the skeletal muscle preparation ranged from 11% with 10^-4 M quinidine to 90% with 10^-3 M quinidine. With the cardiac muscle preparation the inhibition ranged from 16% with 3 x 10^-6 M quinidine to 100% with 10^-3 M quinidine. With both preparations the inhibition of calcium transport was accompanied by an inhibition of the Ca⁺⁺-activated ATPase activity of the sarcoplasmic reticulum. The effect of quinidine on the skeletal sarcoplasmic reticulum supports the hypothesis that this compound produces twitch potentiation and contracture by interfering with intracellular calcium, sequestration. Its effect on cardiac sarcoplasmic reticulum. has been interpreted in terms of the hypothesis that cardiac contractility is a function of the amount of calcium released from the sarcoplasmic reticulum which is in turn dependent upon the absolute calcium content of the reticulum. Hence, following inhibition of calcium transport there would be less calcium available for coupling.     

Palmitylcarnitine inhibition of the calcium pump in cardiac sarcoplasmic reticulum: a possible role in myocardial ischemia.

Palmitylcarnitine is a time-dependent inhibitor of the Ca²⁺-ATPase activity of cardiac sarcoplasmic reticulum isolated from adult dogs. Half-maximal inhibition was obtained at approximately 20 μM (2 μmoles/mg). The extent of inhibition depended on the ratio of palmitylcarnitine to sarcoplasmic reticulum protein. Calcium uptake by cardiac sarcoplasmic reticulum (measured in the presence of sodium oxalate) was found to be even more sensitive to inhibition by palmitylcarnitine and complete inhibition was obtained at concentrations as low as 2.5 μM (0.25 μmole/mg) following preincubation. Calcium binding (measured in the absence of oxalate) was inhibited by palmitylcarnitine and calcium release was stimulated at similar ratios. The level of palmitylcarnitine has been reported to increase several fold in myocardial ischemia and inhibition of the sarcoplasmic reticulum calcium pump could conceivably contribute either to the initial loss of contractility or the subsequent inability to restore full contractile function after prolonged ischemia.     

Determination of calcium transport and phosphoprotein phosphatase activity in microsomes from respiratory and vascular smooth muscle.

1. Calcium transport into microsomal vesicles of respiratory (tracheal) smooth muscle was characterized. This calcium transport was ATP dependent and stimulated by the presence of the oxalate ion. The magnitude of transport was similar to that reported for microsomes from other types of smooth muscle. 2. Bovine and rabbit, heavy and light microsomes were isolated from respiratory (tracheal) and vascular (aortic) smooth muscle. Preincubation of these vesicles with cyclic AMP and protein kinase did not alter the transport of calcium into the vesicles. There uas no evidence of phosphate incorporation into microsomal membrane proteins. Similar results were obtained if phosphorylase b kinase replaced the combination of cyclic AMP and protein kinase during the preincubation. 3. The phosphoprotein phosphatase activity of cardiac sarcoplasmic reticulum and smooth muscle microsomes was determined. The activity of this enzyme was found to be several-fold less in the cardiac sarcoplasmic reticulum than in various smooth muscle microsome preparations.     

New methods for the isolation of skeletal muscle sarcolemma and sarcoplasmic reticulum allowing a comparison between the mammalian and amphibian beta(2)-adrenergic receptors and calcium pumps

New methods were established for the rapid and simultaneous isolation of multiple sarcolemmal and sarcoplasmic reticular fractions from very small amounts (0.25-2.0 g) of skeletal muscle. Thebeta(2)-adrenergic receptor and calcium transport systems were used as indices of purity and functional integrity as well as being the focal points of the study. These methods were found to be suitable for the special needs of small tissue samples, allowed rapid preparation and were appropriate for skeletal muscle from various species, frogs to mammals. The sarcolemmalbeta(2)-adrenergic receptor was expressed in frogs and mammals at similar levels of expression (336-454 fmol. x mg(-1)). The calcium pump was also present in sarcolemmal and sarcoplasmic reticular fractions in all species but notable species differences were found. In sarcolemmal fractions, while calcium binding was uniformly low (<1 nmol. x mg(-1)), oxalate stimulation was variable: low in frogs ( approximately 1.05-fold) high in mammals (120-450-fold). In sarcoplasmic reticular fractions, calcium binding was low in frogs (4-9 nmol. x mg(-1)) and much higher in mammals (322-383 nmol. x mg(-1)); oxalate stimulated calcium transport to a much greater extent in frogs (<70-fold) than in mammals (1.6-2-fold). It is concluded that thebeta(2)-adrenergic receptor appears to be strongly conserved in skeletal muscle while the use of calcium pumps evolves from reliance in Amphibia on the sarcoplasmic reticular calcium pump to the use in Mammalia of calcium pumps from both the sarcoplasmic reticulum and the plasma membrane.     

Evidence for the presence of calsequestrin in two structurally different regions of myocardial sarcoplasmic reticulum

Localization of calsequestrin in chicken ventricular muscle cells was determined by indirect immunofluorescence and immuno-Protein A-colloidal gold labeling of cryostat and ultracryotomy sections, respectively. Calsequestrin was localized in the lumen of peripheral junctional sarcoplasmic reticulum, as well as in the lumen of membrane-bound structures present in the central region of the I-band, while being absent from the lumen of the sarcoplasmic reticulum in the A-band region of the cardiac muscle cells. Since chicken ventricular muscle cells lack transverse tubules, the presence of calsequestrin in membrane bound structures in the central region of the I-band suggests that these cells contain nonjunctional regions of sarcoplasmic reticulum that are involved in Ca2+ storage and possibly Ca2+ release. It is likely that the calsequestrin containing structures present throughout the I-band region of the muscle cells correspond to specialized regions of the free sarcoplasmic reticulum in the I-band called corbular sarcoplasmic reticulum. It will be of interest to determine whether Ca2+ storage and possibly Ca2+ release from junctional and nonjunctional regions of the sarcoplasmic reticulum in chicken ventricular muscle cells are regulated by the same or different physiological signals.     

The effect of trifluoroperazine on the sarcoplasmic reticulum membrane

The inhibitory effect of trifluoroperazine (25-200 microM) on the sarcoplasmic reticulum calcium pump was studied in sarcoplasmic reticulum vesicles isolated from skeletal muscle. It was found that the lowest effective concentrations of trifluoroperazine (10 microM) displaces the Ca2+ dependence of sarcoplasmic reticulum ATPase to higher Ca2+ concentrations. Higher trifluoroperazine concentrations (100 microM) inhibit the enzyme even at saturating Ca2+. If trifluoroperazine is added to vesicles filled with calcium in the presence of ATP, inhibition of the catalytic cycle is accompanied by rapid release of accumulated calcium. ATPase inhibition and calcium release are produced by identical concentrations of trifluoroperazine and, most likely, by the same enzyme perturbation. These effects are related to partition of trifluoroperazine ino the sarcoplasmic reticulum membrane, and consequent alteration of the enzyme assembly within the membrane structure, and of the bilayer surface properties. The effect of trifluoroperazine was also studied on dissociated ('chemically skinned') cardiac cells undergoing phasic contractile activity which is totally dependent on calcium uptake and release by sarcoplasmic reticulum, and is not influenced by inhibitors of slow calcium channels. It was found that trifluoroperazine interferes with calcium transport by sarcoplasmic reticulum in situ, as well as with the role of sarcoplasmic reticulum in contractile activation.     

Regulation of intracellular calcium in chick embryo fibroblast: calcium uptake by the microsomal fraction.

The total membrane fraction of a chick embryo fibroblast (CEF) homogenate accumulates calcium in an energy-dependent manner. This activity can be dissociated into azide-sensitive and azide-insensitive components. The azide-sensitive component of calcium uptake is believed to represent mitochondrial calcium uptake. The azide-insensitive component of calcium uptake is enhanced by the presence of a calcium trapping agent such as oxalate, and cannot utilize, ADP, inorganic phosphate and a Krebs cycle substrate to support uptake. The distribution of the azide-insensitive calcium uptake in subcellular fractions suggests that this uptake occurs in other than mitochondrial membranes. The membranes most likely to contribute to the azide-insensitive component of calcium uptake are the endoplasmic reticulum and plasma membrane. A microsomal preparation from CEF cells is essentially devoid of the azide-sensitive calcium uptake activity. This microsomal activity is similar in characteristics to the sarcoplasmic reticulum of skeletal muscle. However the specific activity of CEF microsomal calcium uptake system is much less than that found in the skeletal muscle system. The transport of calcium by these membranes provide a mechanism for the regulation of cytosol calcium levels and may play a role in the control of movement and growth of cultured cells.     

Evidence that coated vesicles isolated from brain are calcium- sequestering organelles resembling sarcoplasmic reticulum

Coated vesicles from the brain have been purified to near morphological homogeneity by a modification of the method of Pearse. These vesicles resemble sarcoplasmic reticulum fragments isolated from skeletal muscle. They contain proteins with 100,000- and 55,000-dalton mol wt which co-migrate on polyacrylamide gels, in the presence of sodium dodecyl sulfate, with the two major proteins of the sarcoplasmic reticulum fragment. These vesicles contain adenosine triphosphatase (ATPase) activity which is stimulated by calcium ions in the presence of Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.), displaying maximal activity at 8 x 10(-7) M Ca ++. They take up calcium ions from the medium, and this uptake is stimulated by ATP and by potassium oxalate, a calcium-trapping agent. The 100,000-dalton protein of the coated vesicles displays immunological reactivity with an antiserum directed against the 100,000-dalton, calcium-stimulated ATPase of the sarcoplasmic reticulum. As with the sarcoplasmic reticulum fragment, this protein becomes radiolabeled when coated vesicles are briefly incubated with gamma-labeled [32P]ATP. The possible functions of coated vesicles as calcium-sequestering organelles are discussed.     

The localization of calcium by X-ray microanalysis in myopathic muscle fibers

An electron histochemical study was undertaken to localize calcium with ammonium oxalate precipitation technique in soleus muscle of rat in normal cases and in myopathy induced experimentally by a prolonged treatment of 2,4-dichlorophenoxyacetate (2,4-D). The calcium content of precipitates was detected by energy-dispersive X-ray microanalysis. In normal cases, the electron dense precipitates containing calcium were mainly found in the vesicles of sarcoplasmic reticulum, whereas in 2,4-D induced myopathy the deposits were shifted near the Z line into the myofibrils. Calcium, because the uptake into sarcoplasmic vesicles was inhibited by 2,4-D, could attach to other binding sites, such as to the troponin-C.A long-lasting binding of calcium might lead to a prolonged activation of the actin-myosin system.     

Ca-pumps in smooth muscle: one in plasma membrane and another in endoplasmic reticulum

For several years it has been debated whether the Ca-pump in smooth muscle is located in the plasma membrane or in the endoplasmic reticulum (alias sarcoplasmic reticulum). Experimental evidence using skinned smooth muscle cells and subcellular membrane fractions isolated from a number of smooth muscles is reviewed here to hopefully resolve this issue. The inescapable conclusion is that there are two modes of nonmitochondrial ATP-dependent Ca-transport. The first one, unaffected by oxalate, is localized in the plasma membranes and the second, potentiated by oxalate, is localized in the endoplasmic reticulum. Clear experiments to delineate the roles of the two pumps in the excitation-contraction cycle of the smooth muscle remain to be conducted.     

A 45Ca autoradiographic and stereological study of freeze-dried smooth muscle of the guinea pig vas deferens

In an effort to more clearly elucidate the role of cellular structures as calcium sinks and sources in smooth muscle cells, the intracellular distribution of radioactive calcium was evaluated by a new method based on freeze-drying. The guinea pig vas deferens was exposed to a physiological salt solution that contained 45Ca. The muscle was then freeze-dried and prepared for electron microscope autoradiography. The grain density over the plasma membrane, mitochondria, and sarcoplasmic reticulum (SR) was significantly greater than that of the matrix. These results suggest that the plasma membrane, mitochondria and SR have the capacity to accumulate calcium. Which of these structures serve as a source of calcium for contraction remains to be determined. A stereological comparison between freeze-dried and conventionally prepared smooth muscles revealed several differences. The cross- sectional area of freeze-dried cells was about twice that of conventionally prepared cells. Moreover, mitochondria and sub-surface vesicles occupied a significantly smaller percentage of the cell in the freeze-dried tissue than they did in the conventionally prepared tissue.     

Calcium accumulation by isolated sarcoplasmic reticulum of skeletal muscle during development in tissue culture

The calcium uptake and ATPase activities of isolated sarcoplasmic reticulum were studied during the first six days of chick skeletal muscle maturation in tissue culture. Statistically significant increases in these activities were observed between the second and the sixth day of maturation. Increases in oxalate-dependent calcium uptake were demonstrated at concentrations of 2.5 × 10^?5 M calcium and 10^?4 M calcium. Calcium-binding determinations conducted in the absence of oxalate displayed changes manifested by an increase at day 5 followed by a significant decrease at day 6. Increases in total ATPase activity during maturation paralleled the sequential increases in calcium uptake. Calcium-stimulated ATPase activity, however, did not change significantly during periods of marked increase in calcium uptake, suggesting that these activities are dissociated during development of the sarcoplasmic reticulum. These data demonstrate that calcium uptake and total ATPase activity increase during muscle maturation in tissue culture and that these activities are present prior to spontaneous contractions.