1 alpha, 25-Dihydroxy-vitamin D-3 regulates ATP-dependent calcium transport in basolateral plasma membranes of rat enterocytes

Basolateral plasma membrane vesicles of rat small intestinal epithelium accumulate calcium through an ATP-dependent pumping system. The activity of this system is highest in duodenum and decreases towards the ileum. This distribution along the intestinal tract is similar as the active calcium absorption capacity of intact intestinal epithelial segments. ATP-dependent calcium uptake in basolateral membrane vesicles from duodenum and ileum increased significantly after repletion of young vitamin D-3-deficient rats with 1 alpha,25-dihydroxy-vitamin D-3. Ca2+ -ATPase activity in duodenal basolateral membranes increased to the same extend as ATP-dependent calcium transport, but (Na+ + K+)-ATPase activity remained unaltered.     

1α,25-dihydroxy-vitamin D-3 regulates ATP-dependent calcium transport in basolateral plasma membranes of rat enterocytes

Basolateral plasma membrane vesicles of rat small intestinal epithelium accumulate calcium through an ATP-dependent pumping system. The activity of this system is highest in duodenum and decreases towards the ileum. This distribution along the intestinal tract is similar as the active calcium absorption capacity of intact intestinal epithelial segments. ATP-dependent calcium uptake in basolateral membrane vesicles from duodenum and ileum increased significantly after repletion of young vitamin D-3-deficient rats with 1α,25-dihydroxy-vitamin D-3. Ca²⁺-ATPase activity in duodenal basolateral membranes increased to the same extend as ATP-dependent calcium transport, but (Na⁺ + K⁺)-ATPase activity remained unaltered.     

Characteristics of the Ca2+ pump and Ca2+-ATPase in the plasma membrane of rat myometrium.

A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 140 kDa Ca2+-pump protein of the myometrial plasma membrane. The Ca2+-ATPase activity of these membranes is not specific for ATP, and is not inhibited by mercurial agents, whereas Ca2+ uptake has the opposite properties. Ca2+-ATPase activity is also over 100 times that of calcium transport; it appears that the ATPase responsible for transport is largely masked by the presence of another Ca2+-ATPase of unknown function. Measurements of total Ca2+-ATPase activity are, therefore, probably not directly relevant to the question of intracellular Ca2+ control.     

cAMP-dependent phosphorylation inhibits potential-dependent passive transport of Ca2+ in myometrial sarcolemma vesicles

It is established that Ca2+ transport from the predominantly inverted vesicles of pig myometrium sarcolemma depends on the value of the membrane potential which is created on vesicles by the K+-valinomycin system. It is shown that variations in the membrane potential from -60 to +30 mV cause acceleration of the calcium transport from the vesicles, the maximal transport being observed at delta psi from 0 up to +30 mV. The endogenic and exogenic cAMP-dependent phosphorylation of plasma membrane proteins inhibits the passive transport of calcium at all the membrane potential values studied. A degree of potential-dependent Ca2+ transport inhibition correlates with the value of cAMP-dependent phosphorylation of sarcolemma proteins.     

Ca2+ uptake, Ca2+-ATPase activity, phosphoprotein formation and phosphate turnover in a microsomal fraction of smooth muscle

Vesicles capable of phosphate-stimulated calcium uptake were isolated from the microsomal fraction of the smooth muscle of the pig stomach according to a previously described procedure which consists in increasing the density of the vesicles by loading them with calcium phosphate and isolating them by centrifugation [Raeymaekers, L., Agostini, B., and Hasselbach, W. (1981) Histochemistry, 70, 139--150]. These vesicles, which contain calcium phosphate deposits, are able to accumulate an additional amount of calcium. This calcium uptake is accompanied by calcium-stimulated ATPase activity and by the formation of an acid-stable phosphoprotein. The acid-denatured phosphoprotein is dephosphorylated by hydroxylamine, which indicates that an acylphosphate is formed. This phosphoprotein probably represents a phosphorylated transport intermediate similar to that seen with the Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle. As with the Ca2+-ATPase of sarcoplasmic reticulum vesicles, this vesicular fraction catalyses an exchange between inorganic phosphate and the gamma-phosphate of ATP (ATP-Pi exchange) which is dependent on the presence of intravesicular calcium, and an exchange of phosphate between ATP and ADP (ATP-ADP exchange). The results further indicate that the turnover rate of the calcium pump, calculated from the ratio of calcium-stimulated ATPase activity to the steady-state level of phosphoprotein, is similar to that of Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle.     

Characterization of Ca2+ uptake in plasma membrane vesicles isolated from guinea pig ileum smooth muscle

We have characterized ATP-dependent Ca2+ transport into highly purified plasma membrane fraction isolated from guinea pig ileum smooth muscle. The membrane fraction contained inside-out sealed vesicles and was enriched 30-40-fold in 5'-nucleotidase and phosphodiesterase I activity as compared to post nuclear supernatant. Plasma membrane vesicles showed high rate (76 nmol/mg/min) and high capacity for ATP dependent Ca2+ transport which was inhibited by addition of Ca2+ ionophore A23187. The inhibitors of mitochondrial Ca2+ transport, i.e., sodium azide, oligomycin and ruthenium red did not inhibit ATP-dependent Ca2+ uptake into plasma membrane vesicles. The energy dependent Ca2+ uptake into plasma membranes showed very high specificity for ATP as energy source and other nucleotide triphosphates were ineffective in supporting Ca2+ transport. Phosphate was significantly better as Ca2+ trapping anion to potentiate ATP-dependent Ca2+ uptake into plasma membrane fraction as compared to oxalate. Orthovanadate, an inhibitor of cell membrane (Ca2+-Mg2+)-ATPase activity, completely inhibited ATP-dependent Ca2+ transport and the Ki was approximately 0.6 microM. ATP-dependent Ca2+ transport and formation of alkali labile phosphorylated intermediate of (Ca2+-Mg2+)-ATPase increased with increasing concentrations of free Ca2+ in the incubation mixture and the Km value for Ca2+ was approximately 0.6-0.7 microM for both the reactions.     

The mechanism of calcium control of smooth muscle tonic contraction

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

ATP-dependent transport of Ca2+ in myocardium sarcolemma vesicles and its activation by phorbol esters

Highly purified pig myocardium sarcolemma vesicles possess the Ca2+,Mg2+-ATPase activity (4.1 mumol Pi/mg protein/hour) and induce the ATP-dependent accumulation of 45Ca2+ (6.0 nmol/mg protein/min). This reaction is not stimulated by oxalate; Ca2+ are released from the vesicles by saponin and Na+ treatment, which suggests that Ca2+ transport against the concentration gradient is induced by myocardium sarcolemma vesicles and not by sarcoplasmic reticulum fragments. The phorbol ester possessing a biological activity of a growth-promoting factor and activating membrane-bound protein kinase C stimulates the Ca2+,Mg2+-ATPase activity and the ATP-dependent accumulation of Ca2+, whereas its counterpart devoid of biological activity does not influence Ca2+ transport. Polymixin B, a specific inhibitor of protein kinase C, prevents the activating effect of phorbol esters on Ca2+ accumulation inside the vesicles. It is suggested that the ATP-dependent transport of Ca2+ in myocardium sarcolemma is controlled by Ca2+-phospholipid-dependent phosphorylation catalyzed by protein kinase C.     

Mechanisms of regulation of Ca2+ levels in myometrium cells

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

Isolation of plasma membrane vesicles from rabbit skeletal muscle and their use in ion transport studies

A method has been developed for the isolation of sealed plasma membrane vesicles from rabbit white skeletal muscle. The final preparation was highly purified as indicated by enrichment of plasma membrane marker enzymes (i.e. ouabain-sensitive (Na+,K+)-ATPase, adenylate cyclase, and acetylcholinesterase). The absence of sarcoplasmic reticulum and mitochondria as contaminants was indicated by the low specific activity of marker enzymes, i.e. Ca2+-ATPase, succinate-cytochrome c reductase, and monoamine oxidase. Thin section and negative staining electron microscopy confirmed the absence of sarcoplasmic reticulum and mitochondrial contamination. The plasma membrane preparation consisted largely of sealed vesicles as observed by electron microscopy and as also demonstrated by latency of enzymic activities, which were unmasked by preincubation with detergent (sodium dodecyl sulfate). Membrane sidedness was estimated from latency of ouabain-sensitive (Na+,K+)-ATPase activity and acetylcholinesterase activity. The latency studies suggest that most of the vesicles are oriented inside out with respect to the orientation of the sarcolemma membrane in the muscle fiber. The inside-out plasma membrane vesicles actively accumulated sodium ions upon addition of ATP. The sodium ions were concentrated greater than 8-fold inside the vesicles and were released upon addition of the ionophore monensin. The sodium ions were taken up in the presence of K+ or NH4+ but not of choline. Uptake was inhibited by low concentrations of vanadate or digitoxin. The Na+ uptake was concomitant with Rb+ efflux. Therefore, the sodium ion transport and the resulting gradients formed appear to have been generated by the ouabain-sensitive (Na+,K+)-ATPase. Batrachotoxin, which opens Na+ channels in excitable tissues, prevents most of the Na+ uptake, suggesting the presence of toxin-activated Na+ channels in these plasma membrane vesicles.     

Abnormal response to calmodulin in vitro of dystrophic chicken muscle membrane Ca2+-ATPase activity

A skeletal muscle membrane fraction enriched in sarcoplasmic reticulum (SR) contained Ca2+-ATPase activity which was stimulated in vitro in normal chickens (line 412) by 6 nM purified bovine calmodulin (33% increase over control, P less than 0.001). In contrast, striated muscle from chickens (line 413) affected with an inherited form of muscular dystrophy, but otherwise genetically similar to line 412, contained SR-enriched Ca2+-ATPase activity which was resistant to stimulation in vitro by calmodulin. Basal levels of Ca2+-ATPase activity (no added calmodulin) were comparable in muscles of unaffected and affected animals, and the Ca2+ optima of the enzymes in normal and dystrophic muscle were identical. Purified SR vesicles, obtained by calcium phosphate loading and sucrose density gradient centrifugation, showed the same resistance of dystrophic Ca2+-ATPase to exogenous calmodulin as the SR-enriched muscle membrane fraction. Dystrophic muscle had increased Ca2+ content compared to that of normal animals (P less than 0.04) and has been previously shown to contain increased levels of immuno- and bioactive calmodulin and of calmodulin mRNA. The calmodulin resistance of the Ca2+-ATPase in dystrophic muscle reflects a defect in regulation of cell Ca2+ metabolism associated with elevated cellular Ca2+ and calmodulin concentrations.     

The involvement of altered vesicle transport in redistribution of Ca2+, Mg2+-ATPase in cholestatic rat liver

Vectorial sorting of plasma membrane protein-containing vesicles is essential for the establishment and maintenance of cell polarity. In the present study, the involvement of altered vesicle transport in the redistribution of membrane-bound Ca2+, Mg2+-ATPase resulting from cholestasis was investigated in hepatocytes. Cholestasis was induced in rat liver by common bile duct ligation. Ca2+, Mg2+-ATPase activity was demonstrated histochemically at the light and electron microscopical levels. Microtubules, an important factor for transcellular transport of vesicles, were studied in situ by immunofluorescence microscopy and electron microscopy in detergent-extracted preparations. The results showed that microtubules underwent significant changes after common bile duct ligation. The most pronounced alteration was focal accumulation of -tubulin in the cytoplasm of hepato cytes after 7 days of common bile duct ligation. At the electron microscopical level, the number of microtubules was increased considerably. In control livers, the activity of Ca2+, Mg2+-ATPase was localized only at the apical plasma membrane of hepatocytes, but it was also present at the basolateral plasma membrane after common bile duct ligation. The number of intracellular vesicles containing Ca2+, Mg2+-ATPase activity was increased strikingly, and some of them were associated with lateral membrane domains in which Ca2+, Mg2+-ATPase activity was found. It is concluded that common bile duct ligation induces the rearrangement of microtubules, which may disturb vectorial transport of Ca2+, Mg2+-ATPase-containing vesicles in hepatocytes, leading to the redistribution of Ca2+, Mg2+-ATPase. © 1998 Chapman & Hall     

Stimulatory effect of regucalcin on ATP-dependent calcium transport in rat liver plasma membranes

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytoplasm, on ATP-dependent calcium transport in the plasma membrane vesicles of rat liver was investigated. (Ca2+-Mg2+)-ATPase activity in the liver plasma membranes was significantly increased by the presence of regucalcin (0.1-0.5 \sgmaelig;M) in the enzyme reaction mixture. This increase was completely inhibited by the presence of sulfhydryl group modifying reagent Nethylmaleimide (5.0 mM NEM) or digitonin (0.04%), which can solubilize the membranous lipids. When ATP-dependent calcium uptake by liver plasma membrane vesicles was measured by using 45CaCl2, the presence of regucalcin (0.1-0.5 \sgmaelig;M) in the reaction mixture caused a significant increase in the 45Ca2+ uptake. This increase was about 2-fold with 0.5 \sgmaelig;M regucalcin addition. An appreciable increase was seen by 5 min incubation with regucalcin addition. The regucalcin-enhanced ATP-dependent 45Ca2+ uptake by the plasma membrane vesicles was completely inhibited by the presence of NEM (5.0 mM) or digitonin (0.04%). These results demonstrate that regucalcin activates (Ca2+-Mg2+)-ATPase in the liver plasma membranes and that it can stimulate ATP-dependent calcium transport across the plasma membranes.     

Characterization of the Ca2+- or Mg2+-ATPase of transverse tubule membranes isolated from rabbit skeletal muscle

Transverse tubule membranes isolated from rabbit skeletal muscle have high levels of a Ca2+- or Mg2+-ATPase with Km values for Ca-ATP or Mg-ATP in the 0.2 mM range, but do not display detectable levels of ATPase activity activated by micromolar [Ca2+]. The transverse tubule enzyme is less temperature or pH dependent than the Ca2+-ATPase of sarcoplasmic reticulum and hydrolyzes equally well ATP, ITP, UTP, CTP, and GTP. Of several ionic, non-ionic, and zwitterionic detergents tested, only lysolecithin solubilizes the transverse tubule membrane while preserving ATPase activity. After extraction of about 50% of the transverse tubule proteins by solubilization with lysolecithin most of the ATPase activity remains membrane bound, indicating that the Ca2+- or Mg2+-ATPase is an intrinsic membrane enzyme. A second extraction of the remaining transverse tubule proteins with lysolecithin results in solubilization and partial purification of the enzyme. Sedimentation of the Ca2+- or Mg2+-ATPase, partially purified by lysolecithin solubilization, through a continuous sucrose gradient devoid of detergent leads to additional purification, with an overall 3- to 5-fold purification factor. The purified enzyme preparation contains two main protein components of molecular weights 107,000 and 30,000. Cholesterol, which is highly enriched in the transverse tubule membrane, copurifies with the enzyme. Transverse tubule membrane vesicles also display ATP-dependent calcium transport which is not affected by phosphate or oxalate. The possibility that the Ca2+- or Mg2+-ATPase is the enzyme responsible for the Ca2+ transport displayed by isolated transverse tubules is discussed.     

Effects of lanthanum on the heart sarcolemmal ATPase and calcium binding activities.

Effects of lanthanum on Ca2+-ATPase, Mg2+-ATPase, Na+-K+-ATPase, and calcium binding activities were studied in rat heart sarcolemma. Ten to 100 micrometers lanthanum depressed significantly the Ca2+-ATPase activity and 50--200 micrometers lanthanum inhibited the calcium binding activity. Lineweaver-Burk plots of the Ca2+-ATPase activity showed that the inhibition by lanthanum was competitive with calcium concentration. Neither Mg2+-ATPase nor Na+-K+-ATPase activities were affected by lanthanum when the assay medium contained 1 mM EDTA; however, in the absence of EDTA, these enzyme activities were significantly decreased by 10--100 micrometers lanthanum. Rat hearts perfused with HEPES buffer containing 0.5 mM lanthanum showed electron-dense deposits restricted to the outer cell surface and the sarcolemma obtained from these hearts also had the deposits, indicating that the membrane fraction isolated by the hypotonic shock--LiBr treatment method is of sarcolemmal origin. The Ca2+-ATPase activity of the sarcolemma isolated from lanthanum-perfused hearts, unlike the Mg2+-ATPase, Na+-K+-ATPase, and calcium binding activities, was significantly less than the control value. From these observations it is suggested that lanthanum may influence calcium movement across the sarcolemma by affecting sarcolemmal ATPase and calcium binding activities.     

The effect of lipid intermediates on Ca2+ and Na+ permeability and (Na+ + K+)-ATPase of cardiac sarcolemma. A possible role in myocardial ischemia

The effect of fatty acid and acylcarnitine on Ca2+ and Na+ transporting enzymes and carriers was studied in sealed cardiac sarcolemma vesicles of mixed polarity. Palmitoylcarnitine markedly reduced the Na+ gradient-induced Ca2+ uptake. Half-maximal reduction was obtained at 15 microM of the carnitine derivative. In a same concentration range palmitoylcarnitine caused a rapid release of accumulated Ca2+ when added to Ca2+-filled vesicles, which suggests that palmitoylcarnitine increases the permeability of the sarcolemma vesicles to Ca2+. A rapid release of Ca2+ was also observed if Ca2+ was taken up by action of the Ca2+ pump. The (Ca2+ + Mg2+)-ATPase, which most likely drives this active Ca2+ uptake, was 90% increased by 50 microM palmitoylcarnitine and evidence was presented that the acylcarnitine effect again was linked to an alteration of Ca2+ permeability of the vesicles. At the same concentration acylcarnitine was not able to unmask the latent protein kinase, so that probably the sarcolemma ATP permeability was not affected. Palmitoylcarnitine at 25 microM did not affect the ouabain-sensitive (Na+ + K+) -ATPase in native sarcolemma vesicles, however, it inhibited markedly if the enzyme was measured in SDS-treated vesicles. The effect of increased free fatty acid concentration on some of the sarcolemma transporting properties was tested by adding oleate-albumin complexes with different molar ratios to the sarcolemma vesicles. In contrast to molar ratios 1 and 5, the ratio of 7 was able to induce a rapid Ca2+ release and to inhibit (Na+ + K+)-ATPase in either native or SDS-treated vesicles markedly. 22Na release from 22Na-preloaded sarcolemma vesicles was shown to be stimulated by either palmitoylcarnitine (50 microM) or oleate-albumin complex (with a molar ratio of 7). The possible significance of the observed effects of lipid intermediates on ion permeability and (Na+ + K+)-ATPase activity in isolated sarcolemma vesicles for the derangement of cardiac cell function in ischemia is discussed.     

Ca2+ transport by the synaptosomal plasma membrane Ca2+-ATPase and the effect of thioridazine

Thioridazine inhibits the activity of the synaptic plasma membrane Ca(2+)-ATPase from pig brain and slightly decreases the rate of Ca(2+) accumulation by synaptic plasma membrane vesicles in the absence of phosphate. However, in the presence of phosphate, thioridazine increases the rate of Ca(2+) accumulation into synaptic plasma membrane vesicles. Phosphate anions diffuse through the membrane and form calcium phosphate crystals, reducing the free Ca(2+) concentration inside the vesicles and the rate of Ca(2+) leak. The higher levels of Ca(2+) accumulation obtained in the presence of thioridazine could be explained by a reduction of the rate of slippage on the plasma membrane ATPase.     

The Ca2+-pumping ATPase and the major substrates of the cGMP-dependent protein kinase in smooth muscle sarcolemma are distinct entities

It has been proposed that the plasma membrane Ca2+ pump of smooth muscle tissues may be regulated by cGMP-dependent phosphorylation [Popescu, L. M., Panoiu, C., Hinescu, M. & Nutu, O. (1985) Eur. J. Pharmacol. 107, 393-394; Furukawa, K. & Nakamura, H. (1987) J. Biochem. (Tokyo) 101, 287-290]. This hypothesis has been tested on a smooth muscle sarcolemma preparation from pig thoracic aorta. The actomyosin-extracted membranes showed ATP-dependent Ca2+ uptake as well as cGMP-dependent protein kinase (G-kinase) activity. The molecular masses of the major protein substrates of the G-kinase (G1) and that of the Ca2+ pump were compared. Electrophoretic analysis of the phosphorylated intermediate of the sarcolemmal Ca2+-ATPase and the G1 phosphoprotein showed that these two proteins are not identical. The results were confirmed by using a 125I-calmodulin overlay technique and an antibody against human erythrocyte Ca2+-ATPase. Ca2+-uptake experiments with prephosphorylated membrane vesicles were carried out to elucidate possible effects of cGMP-dependent phosphorylation of membrane proteins on the activity of the Ca2+ pump. The cGMP-dependent phosphorylation was found to be extremely sensitive to temperature leading to very low steady-state phosphorylation levels at 37 degrees C. The difficulty was overcome by ATP[gamma S], which produced full and stable thiophosphorylation of G1 during the Ca2+-uptake experiments at 37 degrees C. However, the cGMP-dependent thiophosphorylation failed to influence the Ca2+-uptake properties of sarcolemmal vesicles. The results show that the Ca2+ pump of smooth muscle plasma membrane is not a direct target of the cGMP-dependent protein kinase and is not regulated by the cGMP-dependent phosphorylation of membrane proteins.     

Defective Ca2+-pumping ATPase of heart sarcolemma from cardiomyopathic hamster

The Syrian cardiomyopathic hamster has a hereditary disease characterized by a progressive myocyte necrosis and intracellular calcium overload. Several systems in the heart sarcolemma that regulate the rate of Ca2+ entry or efflux were examined. There is a selective decrease of Ca2+-pumping ATPase activity in the heart sarcolemma of 40-day-old myopathic hamsters, while the Na+-Ca2+ exchange system and the ouabain-sensitive (Na+ + K+)-ATPase activity remain intact. This age-dependent decrease in Ca2+-ATPase activity closely parallels the time course of lesion development. Both the affinity for Ca2+ (Km) and the maximal velocity (Vmax) of the Ca2+-dependent ATP hydrolysis are altered. In addition, there is also an increased number of calcium channel receptor binding sites. Thus the data suggest that the imbalance in Ca2+ fluxes across the cardiac plasma membrane may be involved in the pathogenesis of this cardiomyopathy.     

(Ca2+ + Mg2+)-ATPase activity in plasma membrane of circulating mononuclear cells. Lack of a direct effect of vitamin D

The in vivo effect of vitamin D on (Ca2+ + Mg2+)-ATPase activity was examined in a plasma membrane fraction of rat circulating mononuclear cells (MPM). Although there was no significant difference in the ATPase activities in red blood cell ghosts, (Ca2+ + Mg2+)-ATPase activity in MPM was significantly higher (p less than 0.05) in long-term vitamin D3-replete rats (100 IU/day for 6 months) than that in vitamin D-deplete rats (for 6 months). In rats maintained on vitamin D-deficient diets for 5-7 weeks, in vivo administration of either vitamin D3, 2,000 IU orally, 5 days prior to killing or 1,25-dihydroxyvitamin D3, 2.4 nmol, intraperitoneally, 24 h prior to killing failed to show any significant effect on (Ca2+ + Mg2+)-ATPase activity in MPM. (Ca2+ + Mg2+)-ATPase activity in MPM from rats maintained on vitamin D-deficient diet with high calcium content (1.8%) was significantly higher (p less than 0.05) than that from rats maintained on vitamin D-deficient diet with low calcium content (0.3%). Moreover, in vitro addition of vitamin D3 metabolites did not show any effect on (Ca2+ + Mg2+)-ATPase activity in MPM. These data suggest that decreased (Ca2+ + Mg2+)-ATPase activity in MPM from long-term vitamin D-deplete rats resulted from an adaptation to low extracellular calcium rather than vitamin D depletion.