Regulation of human platelet membrane Ca2+ transport by cAMP- and calmodulin-dependent phosphorylation

We have examined the effects of added cAMP-dependent protein kinase and endogenous calmodulin-dependent kinase on Ca2+ transport in purified internal membranes from human platelets. Both Ca2+ uptake and Ca2+-ATPase activity were maximally stimulated about 2-fold by addition of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase inhibitor reduced both Ca2+ uptake and Ca2+-ATPase activities at concentrations which also inhibited cAMP-dependent protein phosphorylation. In addition, concerted stimulation of Ca2+-ATPase by exogenous calmodulin and added catalytic subunit of cAMP-dependent protein kinase was observed. A 22-kDa protein was phosphorylated by both cAMP-dependent and calmodulin-dependent kinases at the same rate as stimulation of the Ca2+-ATPase. Cyclic AMP-dependent phosphorylation of the 22-kDa polypeptide was inhibited by the protein kinase inhibitor and calmodulin-dependent phosphorylation was inhibited by chlorpromazine and EGTA. These results are consistent with the hypothesis that one mode of control of Ca2+ homeostasis in platelets may be similar to the phospholamban system in cardiac muscle.     

Structure, function and subcellular localization of a human platelet Ca2+-ATPase

Human platelets contain a Ca2+-ATPase in internal membranes that is essential for Ca2+ homeostasis. This Ca2+ pump has enzymatic properties quite similar to the sarcoplasmic reticulum (SR) Ca2+ pumps. Antibodies against the SR Ca2+ pump crossreact with the human platelet protein. However, the platelet Ca2+-ATPase is approximately 10 kD larger than the SR pumps and exhibits a larger mRNA coding for the protein in a megakaryocyte tumor cell line. In addition, the platelet Ca2+-pump may be localized in specialized internal membrane structures that function in Ca2+ uptake and release. These results suggest that the platelet Ca2+-ATPase may represent a new class of internal membrane Ca2+-pumps.     

Protein kinase C stimulates dense tubular Ca2+ uptake in the intact human platelet by increasing the Vm of the Ca(2+)-ATPase pump: stimulation by phorbol ester, inhibition by calphostin C.

The effects of protein kinase C (PKC) on Ca2+ transport were investigated in human intact platelets. The indicator quin2 was used to measure the free cytoplasmic Ca2+ concentration ([Ca2+]cyt) and to search for possible PKC effects on the Ca(2+)-ATPase extrusion pump located in the plasma membrane. The Ca2+ indicator chlorotetracycline (CTC) was used to study PKC effects on the dense tubular Ca(2+)-ATPase uptake pump. The activity of PKC was stimulated by phorbol 12-myristate 13-acetate (PMA) and was inhibited with calphostin C. Neither PKC activation nor inhibition had any effect on [Ca2+]cyt or the Ca2+ extrusion pump. Substantial activation of the dense tubular pump was observed with PMA. In resting platelets bathed in 2 mM external Ca2+ giving [Ca2+]cyt = 102-106 nM, activation of PKC by PMA (100 nM) increases the rate and extent of dense tubular Ca2+ uptake to 1.62 +/- 0.35 and 1.25 +/- 0.3 times control value (respectively). The Vm of the dense tubular pump was measured by using ionomycin to manipulate [Ca2+]cyt. It is shown that PMA increases the Vm by a factor of 1.7 +/- 0.4 but has no effect on the Km value (= 180 nM). An unexpected finding was that PKC activity supports a portion of the basal activity of the dense tubular Ca2+ pump in resting platelets. Preincubation with the inhibitor calphostin C (100 nM) decreases the rate and extent of dense tubular Ca2+ uptake in resting platelets by 38 +/- 5% and 29 +/- 21% (respectively). This is due to a 28 +/- 9% decrease in the Vm of the dense tubular pump. This suggests that there is a low level of stimulation of dense tubular Ca2+ pump mediated by PKC in resting platelets.     

The influence of activating hormones on human platelet membrane Ca2+-ATPase activity

Intact platelets were pretreated with hormones and thereafter membranes were prepared and Ca2+-ATPase activity determined. Thrombin decreased the Vmax of Ca2+-ATPase after pretreatment of intact platelets. Platelet activating factor, vasopressin and ADP also decreased Ca2+-ATPase activity. 12-O-tetradecanoylphorbol-13-acetate (TPA) or A23187 or ionomycin alone had no effect, whilst the simultaneous pretreatment with TPA and Ca2+-ionophore decreased Ca2+-ATPase activity. cAMP elevating agents prostaglandin E1 (PGE1) and forskolin had no influence per se on Ca2+-ATPase, but antagonized the inhibitory effect of thrombin. The data suggest a close connection between phosphoinositide metabolism and the Ca2+-ATPase system.     

Structural and functional properties of a Ca2+-ATPase from human platelets

An antibody prepared against highly purified rabbit muscle Ca2+-ATPase from sarcoplasmic reticulum has been observed to cross-react with proteins in human platelet membrane vesicles. The antibody specifically precipitated Ca2+-ATPase activity from solubilized human platelet membranes and recognized two platelet polypeptides denatured in sodium dodecyl sulfate with Mr = 107,000 and 101,000. Ca2+-ATPase activity from Brij 78-solubilized platelet membranes was purified up to 10-fold. The purified preparation consisted mainly of two polypeptides with Mr approximately 100,000, and 40,000. The lower molecular weight protein appeared unrelated to Ca2+-ATPase activity. The Ca2+-ATPase in human platelet membrane vesicles exhibited "negative cooperativity" with respect to the kinetics of ATP hydrolysis. The apparent Km for Ca2+ activation of ATPase activity was 0.1 microM. Ca2+-dependent phosphorylation of platelet vesicles by [gamma-32P]ATP at 0 degrees C yielded a maximum of 0.2-0.4 nmol of PO4/mg of protein that was labile at pH 7.0 and 20 degrees C. This result suggests that only about 2-4% of the total protein in platelet membrane vesicles is the Ca2+-ATPase, which agrees with an estimate based on the specific activity of the Ca2+-ATPase in platelet membranes (20-50 nmol of ATP hydrolyzed/min/mg of protein at 30 degrees C). Calmodulin resulted in only a 1.6-fold stimulation of Ca2+-ATPase activity even after extensive washing of membranes with a calcium chelator or chlorpromazine. It is concluded that human platelets contain a Ca2+-ATPase immunochemically related to the Ca2+ pump from rabbit sarcoplasmic reticulum and that the enzymatic characteristics and molecular weight of the platelet ATPase are quite similar to those of the muscle ATPase.     

Modulation of human erythrocyte Ca2+-ATPase activity by glycerol: The role of calmodulin

The effect of an intracellular cryoprotectant glycerol on human erythrocyte Ca2+-ATPase activity and possible involvement of calmodulin in the regulation of Ca2+-pump under these conditions were investigated. The experiments were carried out using saponin-permeabilized cells and isolated erythrocyte membrane fractions (white ghosts). Addition of rather low concentrations of glycerol to the medium increased Ca2+-ATPase activity in the saponin-permeabilized cells; the maximal effect was observed at 10% glycerol. Subsequent increase in glycerol concentrations above 20% was accompanied by inhibition of Ca2+-ATPase activity. Lack of stimulating effect of glycerol on white ghost Ca2+-ATPase may be attributed to removal of endogenous compounds regulating activity of this ion transport system. Inhibitory analysis using R24571 revealed that activation of Ca2+-ATPase by 10% glycerol was observed only in the case of inhibitor administration after modification of cells with glycerol; in the case of inhibitor addition before erythrocyte contact with glycerol, this phenomenon disappeared. These data suggest the possibility of regulation of human erythrocyte Ca2+-ATPase by glycerol; this regulatory effect may be attributed to both glycerol-induced structural changes in the membrane and also involvement of calmodulin in modulation of catalytic activity of the Ca2+-pump.     

Improved expression and characterization of Ca2+-ATPase and phospholamban in High-Five cells

The Ca2+-ATPase accounts for the majority of Ca2+ removed from the cytoplasm during cardiac muscle relaxation. The Ca2+-ATPase is regulated by phospholamban, a 52 amino acid phosphoprotein, which inhibits Ca2+-ATPase activity by decreasing the apparent affinity of the ATPase for Ca2+. To study the physical mechanism of Ca2+-ATPase regulation by phospholamban using spectroscopic and kinetic experiments, large amounts of both proteins are required. Therefore, we developed a Ca2+-ATPase and phospholamban preparation based on the baculovirus-insect cell expression system using High-Five insect cells to produce large amounts of microsomal vesicles that contain either Ca2+-ATPase expressed alone or Ca2+-ATPase co-expressed with phospholamban. The expressed proteins were characterized using immunofluorescence spectroscopy, Ca2+ -ATPase activity assays, Ca2+ uptake and efflux assays, and Western blotting. Our purification method yields 140 mg of microsomal protein per liter of infection (1.7 x 10(9)cells), and the Ca2+-ATPase and phospholamban account for 16 and 1.4%, respectively, of the total microsomal protein by weight, yielding a phospholamban:Ca2+-ATPase ratio of 1.6:1, similar to that observed in native cardiac SR vesicles. The enzymatic properties of the expressed Ca2+-ATPase are also similar to those observed in native cardiac SR vesicles, and when co-expressed with phospholamban, the Ca2+-ATPase is functionally coupled to phospholamban similar to that observed in cardiac SR vesicles.     

Functional reconstitution of the cardiac sarcoplasmic reticulum Ca2(+)-ATPase with phospholamban in phospholipid vesicles

The Ca2(+)-ATPase in cardiac sarcoplasmic reticulum (SR) is under regulation by phospholamban, an oligomeric proteolipid. To determine the molecular mechanism by which phospholamban regulates the Ca2(+)-ATPase, a reconstitution system was developed, using a freeze-thaw sonication procedure. The best rates of Ca2+ uptake (700 nmol/min/mg reconstituted vesicles compared with 800 nmol/min/mg SR vesicles) were observed when cholate and phosphatidylcholine were used at a ratio of cholate/phosphatidylcholine/Ca2(+)-ATPase of 2:80:1. The EC50 values for Ca2+ were 0.05 microM for both Ca2+ uptake and Ca2(+)-ATPase activity in the reconstituted vesicles compared with 0.63 microM Ca2+ in native SR vesicles. Inclusion of phospholamban in the reconstituted vesicles was associated with a significant inhibition of the initial rates of Ca2+ uptake at pCa 6.0. However, phosphorylation of phospholamban by the catalytic subunit of the cAMP-dependent protein kinase reversed the inhibitory effect on the Ca2+ pump. Similar findings were observed when a peptide, corresponding to amino acids 1-25 of phospholamban, was used. These findings indicate that phospholamban is an inhibitor of the Ca2(+)-ATPase in cardiac SR and phosphorylation of phospholamban relieves this inhibition. The mechanism by which phospholamban inhibits the Ca2+ pump is unknown, but our findings with the synthetic peptide suggest that a direct interaction between the Ca2(+)-ATPase and the hydrophilic portion of phospholamban may be one of the mechanisms for regulation.     

Platelet free calcium concentrations measured with fura-2 are influenced by the transmembrane sodium gradient

The influence of the transmembrane Na+ gradient on the intracellular free calcium concentration, [Ca2+]i, was studied in Sepharose gel-filtered platelets from healthy human subjects, using the Ca-sensitive fluorescent dye, fura-2. Raising the internal Na+ concentration, [Na+]i, by Na+ pump inhibition with 0.05 mM ouabain, without changing external Na+ did not cause a significant increase in [Ca2+]i. Substitution of extracellular Na+ by iso-osmolar sucrose induced a rapid (half-time about 2 min) and significant rise in [Ca2+]i; this effect was amplified in Na-loaded platelets. Partial restitution of external Na+ in these cells with increased [Ca2+]i promoted a significant and rapid Na+ concentration-dependent fall in [Ca2+]i; little decline in [Ca2+]i was observed if K+ was used instead of Na+. These observations represent in vitro evidence for the existence of a Na/Ca exchange mechanism in human platelets that may, in vivo, participate in the control of [Ca2+]i.     

Is there a plasma membrane-located anion-sensitive ATPase? III. Identity of the erythrocyte enzyme with (Ca2+ + Mg2+)-ATPase.

The characteristics of the anion-sensitive Mg2+-ATPase activity of the rabbit erythrocyte have been studied in a lyophilized ghost preparation. The enzyme appears to be different from the anion-sensitive Mg2+-ATPase activity of other tissues in many parameters, such as optimal pH, effects of various anions, oligomycin sensitivity and effects of Triton X-100. The enzyme is insensitive towards inhibition by irreversibly bound 4,4'-diisothiocyano-dihydrostilbene-2,2'-disulfonic acid (H2DIDS). This excludes a relationship between the enzyme and the "band 3" protein, which is thought to be involved in the anion exchange over the erythrocyte membrane. From the effects of ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), CaCl2, chlorpromazine and ruthenium red it is concluded that the enzyme activity does not represent a separate entity but is part of the (Ca2+ + Mg2+)-ATPase system of the erythrocyte membrane. A reported stimulatory effect of carbonic anhydrase is attributed to a contamination of the carbonic anhydrase preparation by calcium and/or (Ca2+ + Mg2+)-ATPase activator protein.     

Ruthenium red affects the intrinsic fluorescence of the calcium-ATPase of skeletal sarcoplasmic reticulum.

We have studied the effect of Ruthenium red on the sarcoplasmic reticulum Ca(2+)-ATPase. Ruthenium red does not modify the Ca2+ pumping activity of the enzyme, despite its interaction with cationic binding sites on sarcoplasmic reticulum vesicles. Two pools of binding sites were distinguished. One pool (10 nmol/mg) is dependent upon the presence of micromolar Ca2+ and may therefore represent the high-affinity Ca2+ transport sites of the Ca(2+)-ATPase. However, Ruthenium red only slightly competes with Ca2+ on these sites. The other pool (15-17 nmol/mg) is characterized as low-affinity cation binding sites of sarcoplasmic reticulum, distinct from the Mg2+ site involved in the ATP binding to the Ca(2+)-ATPase. The interaction of Ruthenium red with these low-affinity cation binding sites, which may be located either on the Ca(2+)-ATPase or on surrounding lipids, decreases tryptophan fluorescence level of the protein. As much as 25% of the tryptophan fluorescence of the Ca(2+)-ATPase is quenched by Ruthenium red (with a dissociation constant of 100 nM), tryptophan residues located near the bilayer being preferentially affected.     

Activation of the Ca2+-ATPase of human erythrocyte membrane by an endogenous Ca2+-dependent neutral protease

Limited proteolysis of the plasma membrane calcium transport ATPase (Ca2+-ATPase) from human erythrocytes by trypsin produces a calmodulin-like activation of its ATP hydrolytic activity and abolishes its calmodulin sensitivity. We now demonstrate a similar kind of activation of the human erythrocyte membrane Ca2+-ATPase by calpain (calcium-dependent neutral protease) isolated from the human red cell cytosol. Upon incubation of red blood cell membranes with purified calpain in the presence of Ca2+ the membrane-bound Ca2+-ATPase activity was increased and its sensitivity to calmodulin was lost. In contrast to the action of other proteases tested, proteolysis by calpain favors activation over inactivation of the Ca2+-ATPase activity, except at calpain concentrations more than 2 orders of magnitude higher. Exogenous calmodulin protects the Ca2+-ATPase against calpain-mediated activation at concentrations which also activate the Ca2+-ATPase activity. Calcium-dependent proteolytic modification of the Ca2+-ATPase could provide a mechanism for the irreversible activation of the membrane-bound enzyme.     

Calcium influx evoked by Ca2+ store depletion in human platelets is more susceptible to cytochrome P-450 inhibitors than receptor-mediated calcium entry.

We have previously reported that a component of ADP-evoked Ca2+ entry in human platelets appears to be promoted following the release of Ca2+ from intracellular stores. Other agonists may employ a similar mechanism. Here we have further investigated the relationship between the state of filling of the Ca2+ stores and plasma membrane Ca2+ permeability in Fura-2-loaded human platelets. Ca2+ influx was promoted following store depletion by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase, thapsigargin (TG) and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ). Divalent cation entry was confirmed by quenching of Fura-2 fluorescence with externally added Mn2+. It has been suggested that cytochrome P-450 may couple Ca2+ store depletion to an increased plasma membrane Ca2+ permeability. In apparent agreement with this, Mn2+ influx promoted by TG and tBuBHQ, or by preincubation of cells in Ca(2+)-free medium, was inhibited by the imidazole antimycotics, econazole and miconazole, which inhibit cytochrome P-450 activity. Agonist-evoked Mn2+ influx was only partially inhibited by these compounds at the same concentration (3 microM). Econazole (3 microM) reduced the Mn2+ quench evoked by ADP by 38% of the control value and that evoked by vasopressin, platelet activating factor (PAF) and thrombin no more than 15% of control, 20 s after agonist addition. Stopped-flow fluorimetry indicated that econazole had no detectable effect on the early time course of agonist-evoked Mn2+ entry or rises in [Ca2+]i. These data confirm the existence of a Ca2+ entry pathway in human platelets which is activated by depletion of the intracellular Ca2+ stores. Further, the results support the suggestion that cytochrome P-450 may participate in such a pathway. However, any physiological role for the cytochrome or its products in agonist-evoked events appears to be in the long-term maintenance or restoration of store Ca2+ content, rather than in promoting Ca2+ influx in the initial stages of platelet Ca2+ signal generation.     

Influence of certain fish meals on (Na+ + K+)-ATPase and Ca2+-ATPase activity in rat small intestine

The effect of high-protein content fish meal on (Na+ + K+)-ATPase and Ca2+-ATPase activity in rat small intestine was studied. 5 groups of Wistar rats, weighing between 40-60 g, were fed diets with 12% protein content of dry matter for 10 days. The protein source was casein for the control group and fish meal derived from Coryphaenoides rupestris, Chimaera monstruosa and Merluccius merluccius for the test group. The results show a decrease in (Na+ + K+)-ATPase and a rise in Ca2+-ATPase activity in animals fed with fish meal protein compared to those fed on casein. No significant variations were observed between the groups fed on fish meal derived from C. rupestris and Ch. monstruosa. The calcium ion, which is abundant in fish, may be a factor responsible for these variations which produce inhibition of the (Na+ + K+)-ATPase and stimulation of the Ca2+-ATPase.     

Cyclic AMP stimulates Ca(2+)-ATPase-mediated Ca2+ extrusion from human platelets.

The effect of cAMP on active Ca2+ extrusion across the plasma membrane of intact human platelets was studied using quin2, a fluorimetric indicator of free Ca2+ in the cytoplasmic compartment ([Ca2+]cyt). Elevations of cAMP were achieved by incubation with dibutyryl-cAMP or by forskolin, which was found to selectively elevate cAMP without affecting cGMP levels. Progress curves of Ca2+ extrusion from quin2-overloaded platelets were measured. The rate vs. [Ca2+]cyt characteristic was calculated as previously described (Johansson, J.S. and Haynes, D.H. (1988) J. Membr. Biol. 104, 147-163). Forskolin, at a maximally effective concentration of 10 microM, was shown to stimulate Ca2+ extrusion by increasing by a factor of 1.6 +/- 0.5 the Vm of a saturable component, previously identified with a Ca(2+)-Mg(2+)-ATPase located in the plasma membrane. Neither the Km (80 nM) or Hill coefficient (1.7 +/- 0.3) of the Ca(2+)-ATPase was affected. Forskolin had no effect on the linear, non-saturable component of extrusion (previously identified with a Na+/Ca2+ exchanger) over the [Ca2+]cyt range examined (50-1500 nM). Dibutyryl-cAMP (Bt2-cAMP, 1 mM) stimulated the Ca(2+)-Mg(2+)-ATPase component of Ca2+ extrusion by a factor of 2.0 +/- 0.6. Separate experiments showed that 10 microM forskolin reduces the resting [Ca2+]cyt from 112 nM to 96 nM. Mathematical analysis showed that this can be accounted for by the above-mentioned increase in Vm of the pump, countered by a 37-74% increase in the rate constant for passive Ca2+ leakage across the plasma membrane. The results suggest two mechanisms by which prostacyclin-induced elevation of cAMP inhibits platelet aggregation: (a) lowering of resting [Ca2+]cyt and (b) increasing the rate of Ca2+ extrusion after the initial influx or triggered release event.     

A monoclonal antibody (PL/IM 430) to human platelet intracellular membranes which inhibits the uptake of Ca2+ without affecting the Ca2+ +Mg2+-ATPase.

To probe the structure-function relationships of proteins present in the endoplasmic reticulum-like intracellular membranes of human blood platelets a panel of monoclonal antibodies have been raised, using as immunogen highly purified platelet intracellular membrane vesicles isolated by continuous flow electrophoresis [Menashi, Weintroub & Crawford (1981) J. Biol. Chem. 256, 4095-4101]. Four of these antibodies recognize a single 100 kDa polypeptide in the platelet membrane by immunoblotting. One antibody PL/IM 430 (of IgG1 subclass) inhibited (approximately 70%) the energy-dependent uptake of Ca2+ into the vesicles without affecting the Ca2+ +Mg2+-ATPase activity or the protein phosphorylation previously shown to proceed concomitantly with Ca2+ sequestration [Hack, Croset & Crawford (1986) Biochem. J. 233, 661-668]. The inhibition is independent of ATP concentration over a range 0-2 mM-ATP but shows dose-dependency for external [Ca2+] with maximum inhibition of Ca2+ translocation at concentrations of Ca2+ greater than 500 nM. This capacity of the antibody PL/IM 430 functionally to dislocate components of the intracellular membrane Ca2+ pump complex may have value in structural studies.     

Association of (Ca+Mg)-ATPase activity with ATP-dependent Ca uptake in vesicles prepared from human erythrocytes

Ghost membranes prepared from human erythrocytes exhibit 2 distinct (Ca+Mg)-ATPase¹ activities (Quist and Roufogalis, Arch Biochem Biophys 168:240, 1975). (Ca+Mg)-ATPase activity dependent on a water soluble protein fraction is selectively lost from ghost membranes during preparation of vesicles under low ionic strength, slightly alkaline conditions. In this study, the Ca²⁺ dependence of the remaining membrane bound (Ca+Mg)-ATPase activity and ATP-dependent Ca uptake in vesicles were compared. The C²⁺ activation curves for (Ca+Mg)-ATPase activity and Ca uptake into vesicles were parallel over a Ca²⁺ range of 0.3–330 μM, and both curves have 2 apparent K_A values for Ca²⁺ of 0.45 and 100 μM. Addition of a concentrated soluble protein fraction containing predomintly spectrin to the vesicles increased (Ca+Mg)-ATPase activity over twofold but did not affect the rate of Ca uptake. These findings suggest that the (Ca+Mg)-ATPase activity remaining in vesicles after extraction of the water soluble proteins is associated with the Ca pump whereas (Ca+Mg)-ATPase activity dependent on the soluble protein fraction is associated with some other function.     

Ca2+-activated ATPase in microsomes from human liver

Human liver microsomal fractions exhibit ATP-supported Ca2+ uptake which is half-maximal at 7 X 10(-7) M free Ca2+ in the presence of oxalate. Ca2+ uptake is coupled to a Ca2+-stimulated ATPase activity, which is half-maximal at 4 X 10(-7) M free Ca2+. Catalysis involves formation of an Mr = 116,000 phosphoprotein with stability characteristics of an acylphosphate compound suggested to represent a phosphoryl protein intermediate of the Ca2+-ATPase. Phosphorylation is half-maximal at about 10(-6) M free Ca2+. The Mr = 116,000 protein is highly susceptible to proteolysis with trypsin. The phosphorylated active site was localized in an Mr = 58,000 primary tryptic fragment and in an Mr = 34,000 subfragment. Analyses on the mechanism of the Ca2+-ATPase suggest the following reaction sequence: formation of an ADP-reactive phosphoenzyme (Mr = 116,000) with bound Ca2+, which can transphosphorylate its Pi to ADP, giving rise to synthesis of ATP; reversible transformation of the ADP-reactive phosphoenzyme into an isomer without bound Ca2+, which cannot further react with ADP; hydrolytical cleavage, probably catalyzed by Mg2+, of the ADP-unreactive phosphoenzyme with liberation of Pi. Comparison with the Ca2+-transport ATPase in sarcoplasmic reticulum of skeletal muscle led us to suggest that the Mr = 116,000 Ca2+-ATPase belongs to the class of E1P . E2P-ATPases and might be operative as a Ca2+-transport ATPase at the level of the endoplasmic reticulum in human liver.     

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.     

Changes in microsomal Na+, K+-, Mg2+- and Ca2+-ATPase activities during proliferation of Chinese hamster V-79 and human HeLaS-3 cells

Changes in microsomal Na+, K+-, Mg2+- and Ca2+-ATPase activities during cell proliferation were examined in Chinese hamster V-79 (V-79) cells (normal cells) and human HeLaS-3 (HeLaS-3) cells (malignant cells). For V-79 cells, the Mg2+-ATPase activity per cell (pmol Pi/h/cell) in the confluent phase was higher than that in the logarithmically growing (log) phase. The amount of microsomal protein per cell was also high in the confluent phase. Specific activities (mumol Pi/h/mg protein) of Na+, K+-, Mg2+- and Ca2+-ATPase were significantly lower in the confluent phase than in the log phase. For HeLaS-3 cells, an increase in Ca2+-ATPase activity per cell was observed. The amount of microsomal protein per cell did not change between the log and confluent phase. The specific activity of Ca2+-ATPase in the confluent phase was also markedly higher than in the log phase. The relation between changes in ATPase activities and cell proliferation is discussed.