Differentiation of Ca pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle

ATP promotes ⁴⁵Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca²⁺-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the ⁴⁵Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5′-nucleotidase. In contrast, the additional Ca²⁺-transport activity elicited by oxalate behaved like NADH-cytochrome c reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca²⁺-storage capacity was not detectable in the absence of Ca²⁺-trapping agent. Low digitonin concentrations selectively increased the Ca²⁺ permeability of the plasmalemmal vesicles. The two Ca²⁺-transport activities were further differentiated by their distinct sensitivities to K⁺, vanadate and calmodulin. In this respect, the oxalte-insensitive and oxalate-stimulated Ca²⁺-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca²⁺ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.     

Vesicularization of the endoplasmic reticulum is a fast response to plasma membrane injury

The endoplasmic reticulum of most cell types mainly consists of an extensive network of narrow sheets and tubules. It is well known that an excessive increase of the cytosolic Ca²⁺ concentration induces a slow but extensive swelling of the endoplasmic reticulum into a vesicular morphology. We observed that a similar extensive transition to a vesicular morphology may also occur independently of a change of cytosolic Ca²⁺ and that the change may occur at a time scale of seconds. Exposure of various types of cultured cells to saponin selectively permeabilized the plasma membrane and resulted in a rapid swelling of the endoplasmic reticulum even before a loss of permeability barrier was detectable with a low-molecular mass dye. The structural alteration was reversible provided the exposure to saponin was not too long. Mechanical damage of the plasma membrane resulted in a large-scale transition of the endoplasmic reticulum from a tubular to a vesicular morphology within seconds, also in Ca²⁺-depleted cells. The rapid onset of the phenomenon suggests that it could perform a physiological function. Various mechanisms are discussed whereby endoplasmic reticulum vesicularization could assist in protection against cytosolic Ca²⁺ overload in cellular stress situations like plasma membrane injury.     

Gibberellic-acid-stimulated Ca2+ accumulation in endoplasmic reticulum of barley aleurone: Ca2+ transport and steady-state levels

The steady-state levels of Ca²⁺ within the endoplasmic reticulum (ER) and the transport of ⁴⁵Ca²⁺ into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the ER was measured using the Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 M. Transport of ⁴⁵Ca²⁺ into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA₃) and Ca²⁺. Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of ⁴⁵Ca²⁺ occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that ⁴⁵Ca²⁺ transport was into the ER. The sensitivity of ⁴⁵Ca²⁺ transport to inhibitors and the K_m of ⁴⁵Ca²⁺ uptake for ATP and Ca²⁺ transport in the microsomal fraction of barley aleurone cells. The rate of ⁴⁵Ca²⁺ transport is stimulated several-fold by treatment with GA₃. This effect of GA₃ is mediated principally by an effect on the activity of the Ca²⁺ transporter rather than on the amount of ER.Abbreviations CCR cytochrome-c reductase - DCCD dicyclohexylcarbodiimide - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - FCCP carbonylcyanide p-trifluoromethoxyphenyl hydrazone - GA₃ gibberellic acid - IDPase inosine diphosphatase - Mon monensin     

Effect of Na3VO4 and membrane potential on the structure of sarcoplasmic reticulum membrane

Summary Two-dimensional crystalline arrays of Ca²⁺-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na₃VO₄ in a Ca²⁺-free medium. The influence of membrane potential upon the rate of crystallization was studied by ion substitution using oxonol VI and 3,3-diethyl-2,2-thiadicarbocyanine (Di–S–C₂(5)) to monitor inside positive or inside negative membrane, potentials, respectively. Positive transmembrane potential accelerates the rate of crystallization of Ca²⁺-ATPase, while negative potential disrupts preformed Ca²⁺-ATPase crystals, suggesting an influence of transmembrane potential upon the conformation of Ca²⁺-ATPase.     

The characterization of plasma membrane Ca2+-ATPase in rich sphingomyelin-cholesterol domains

According to the raft hypothesis, sphingolipid-cholesterol (CHOL) microdomains are involved in numerous cellular functions. Here, we have prepared liposomes to simulate the lipid composition of rafts/caveolae using phosphatidylchone, sphingomyelin (SPM)-CHOL in vitro. Experiments of both 1,6-diphenyl-1,3,5-hexatriene and merocyanine-540 fluorescence showed that a phase transition from l(d) to l(o) can be observed clearly. In particular, we investigated the behavior of a membrane protein, plasma membrane Ca(2+)-ATPase (PMCA), in lipid rafts (l(o) phase). Three complementary approaches to characterize the physical appearance of PMCA were employed in the present study. Tryptophan intrinsic fluorescence increase, fluorescence quenching by both acrylamid and hypocrellin B decrease, and MIANS fluorescence decrease, indicate that the conformation of PMCA embedded in lipid l(o) phase is more compact than in lipid l(d) phase. Also, our results showed that PMCA activity decreased with the increase of SPM-CHOL content, in other words, with the increase of l(o) phase. This suggests that the specific domains containing high SPM-CHOL concentration are not a favorable place for PMCA activity. Finally, a possible explanation about PMCA molecules concentrated in caveolae/rafts was discussed.     

The mechanism of inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase by paxilline

Although cis-diamminedichloroplatinum (II) (cisplatin) is a potent anticancer drug, clinical use of this agent is highly limited predominantly because of its strong side effects on the kidney and gastrointestinal tracts. We found that cisplatin impaired respiratory function and DNA of mitochondria in renal proximal tubules and small intestinal mucosal cells, thereby inducing apoptosis of epithelial cells. Cisplatin-induced mitochondrial dysfunction and DNA (mtDNA) injury, lipid peroxidation, and apoptosis of epithelial cells in the kidney and small intestine were strongly inhibited by L-carnitine. However, carnitine had no appreciable effect on the tumoricidal action of cisplatin against cancer cells inoculated in the peritoneal cavity. These results indicate that L-carnitine may have therapeutic potential for inhibiting the side effects of cisplatin and other anticancer agents in the kidney and small intestine.     

Expression of the sarco/endoplasmic Ca(2+)-ATPase, SERCA1a, in fibroblasts induces the formation of organelle membrane arrays

Members of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) family are transmembrane proteins that are essential for the function of intracellular Ca(2+) storage organelles. We found that overexpression of avian muscle SERCA1a in transfected mouse fibroblasts led to the appearance of tubular membrane bundles that we termed plaques. These structures were generated in transfected cells when SERCA1a protein expression approached the endogenous level measured in chicken skeletal muscle. Plaque membranes had associated ribosomes and contained endoplasmic reticulum (ER) proteins. Endogenous ER protein levels were not elevated in SERCA1a-expressing cells, indicating that plaques were not generalized proliferations of ER but rather a reorganization of existing organelle membrane. Plaque formation also was observed in cells expressing a green fluorescent protein-SERCA1a fusion protein (GFP-SERCA1a). GFP-SERCA1a molecules displayed extensive lateral mobility between plaques, suggesting the presence of membrane continuities between these structures. Plaques were induced in cells expressing cDNA encoding a catalytically silent SERCA1a mutant indicating that ER redistribution was driven by a structural feature of the enzyme. SERCA1a-induced plaque formation shares some characteristics of sarcoplasmic reticulum (SR) biogenesis during muscle differentiation, and high-level SERCA1a expression in vivo may contribute to the formation of SR from ER during embryonic myogenesis.     

The plasma membrane Ca pump catalyzes the hydrolysis of ATP at low rate in the absence of Ca

The plasma membrane Ca²⁺ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca²⁺ at a rate near 1.5% of that attained at saturating concentrations of Ca²⁺. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca²⁺-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca²⁺-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca²⁺ and is catalyzed by E₂-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.     

Rearrangement of the endoplasmic reticulum and calcium transient formation: the computational approach

Experiments affecting calcium signaling often lead to changes in the calcium transient height. The present work is designed to approach this effect theoretically. Use of computational model let us to follow results of precisely designed changes in the endoplasmic reticulum distribution as a possible cause of cytoplasmic free calcium ion level. Obtained results suggest that indeed, rearrangement of the endoplasmic reticulum elements may be responsible for modulation of calcium signal's strength. We have also noticed that even if the endoplasmic reticulum concentration levels are local, the resulting changes in free calcium concentration are global and evenly distributed throughout the cell. The used mathematical method proved to be a powerful tool which made us understand the chemical dynamics of nonequilibrium processes of calcium transient formation. Presented data show how Ca2+ signal resulting from IP3 provoked release of calcium from the endoplasmic reticulum may depend on the cytoskeleton structure.     

Modulation of the kinetic characteristics of the sarcoplasmic reticulum ATPase by membrane fluidity

(Ca²⁺ + Mg²⁺)-ATPase from sarcoplasmic reticulum has been reconstituted with dipalmitoylphosphatidylcholine, and the activating effect of ATP and Ca²⁺ on this enzyme has been studied at different temperatures. It has been found that two kinetic forms of the enzyme are interconverted at about 31°C, and this is possibly related to a phase change in the phospholipid which is more directly associated with the protein. Above 31°C the enzyme is less dependent on ATP activation at high ATP concentrations but shows positive cooperativity for Ca²⁺ activation. On the other hand, below 31°C, the reconstituted enzyme is more dependent on ATP for activation at high ATP concentrations than the purified ATPase and does not show cooperativity for Ca²⁺ activation.     

Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

Bax and Bak play a redundant but essential role in apoptosis initiated by the mitochondrial release of apoptogenic factors. In addition to their presence at the mitochondrial outer membrane, Bax and Bak can also localize to the ER. Agents that initiate ER stress responses can induce conformational changes and oligomerization of Bax on the ER as well as on mitochondria. In wild-type cells, this is associated with caspase 12 cleavage that is abolished in bax-/-bak-/- cells. In bax-/-bak-/- cells, introduction of Bak mutants selectively targeted to either mitochondria or the ER can induce apoptosis. However, ER-targeted, but not mitochondria-targeted, Bak leads to progressive depletion of ER Ca2+ and induces caspase 12 cleavage. In contrast, mitochondria-targeted Bak leads to enhanced caspase 7 and PARP cleavage in comparison with the ER-targeted Bak. These findings demonstrate that in addition to their functions at mitochondria, Bax and Bak also localize to the ER and function to initiate a parallel pathway of caspase activation and apoptosis.     

Markers of squamous cell carcinoma in sarco/endoplasmic reticulum Ca ATPase 2 heterozygote mice keratinocytes

A mutation of Atp2a2 gene encoding the sarco/endoplasmic reticulum Ca²⁺-ATPase 2 (SERCA2) causes Darier's disease in human and null mutation in one copy of Atp2a2 leads to a high incidence of squamous cell tumor in a mouse model. In SERCA2 heterozygote (SERCA2^+/−) mice keratinocytes, mechanisms involved in partial depletion of SERCA2 gene and its related tumor induction have not been studied. In this study, we investigated Ca²⁺ signaling and differential gene expression in primary cultured keratinocytes from SERCA2^+/− mice. SERCA2^+/− keratinocytes showed reduced initial increases in intracellular concentration of calcium in response to ATP, a G-protein coupled receptor agonist, and higher store-operated Ca²⁺ entry with the treatment of thapsigargin, an inhibitor of SERCA, compared to wild type kerationcytes. Protein expressions of plasma membrane Ca²⁺ ATPases, NFATc1, phosphorylated ERK, JNK, and phospholipase γ1 were increased in SERCA2^+/− keratinocytes. Using the gene fishing system, we first found in SERCA2^+/− keratinocytes that gene level of tumor-associated calcium signal transducer 1, crystalline αB, procollagen XVIII α1, and nuclear factor I-B were increased. Expression of involucrin, a marker of keratinocyte differentiation, was decreased in SERCA2^+/− keratinocytes. These results suggest that the alterations of Ca²⁺ signaling by SERCA2 haploinsufficiency alternate the gene expression of tumor induction and differentiation in keratinocytes.     

Role of platelet plasma membrane Ca-ATPase in health and disease

Platelets have essential roles in both health and disease. Normal platelet function is required for hemostasis. Inhibition of platelet function in disease or by pharmacological treatment results in bleeding disorders. On the other hand, hyperactive platelets lead to heart attack and stroke. Calcium is a major second messenger in platelet activation, and elevated intracellular calcium leads to hyperactive platelets. Elevated platelet calcium has been documented in hypertension and diabetes; both conditions increase the likelihood of heart attack and stroke. Thus, proper regulation of calcium metabolism in the platelet is extremely important. Plasma membrane Ca(2+)-ATPase (PMCA) is a major player in platelet calcium metabolism since it provides the only significant route for calcium efflux. In keeping with the important role of calcium in platelet function, PMCA is a highly regulated transporter. In human platelets, PMCA is activated by Ca(2+)/calmodulin, by cAMP-dependent phosphorylation and by calpain-dependent removal of the inhibitory peptide. It is inhibited by tyrosine phosphorylation and calpain-dependent proteolysis. In addition, the cellular location of PMCA is regulated by a PDZ-domain-dependent interaction with the cytoskeleton during platelet activation. Rapid regulation by phosphorylation results in changes in the rate of platelet activation, whereas calpain-dependent proteolysis and interaction with the cytoskeleton appears to regulate later events such as clot retraction. In hypertension and diabetes, PMCA expression is upregulated while activity is decreased, presumably due to tyrosine phosphorylation. Clearly, a more complete understanding of PMCA function in human platelets could result in the identification of new ways to control platelet function in disease states.     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.     

Store-operated Ca2+ entry in astrocytes: different spatial arrangement of endoplasmic reticulum explains functional diversity in vitro and in situ

Ca(2+) signaling is the astrocyte form of excitability and the endoplasmic reticulum (ER) plays an important role as an intracellular Ca(2+) store. Since the subcellular distribution of the ER influences Ca(2+) signaling, we compared the arrangement of ER in astrocytes of hippocampus tissue and astrocytes in cell culture by electron microscopy. While the ER was usually located in close apposition to the plasma membrane in astrocytes in situ, the ER in cultured astrocytes was close to the nuclear membrane. Activation of metabotropic receptors linked to release of Ca(2+) from ER stores triggered distinct responses in cultured and in situ astrocytes. In culture, Ca(2+) signals were commonly first recorded close to the nucleus and with a delay at peripheral regions of the cells. Store-operated Ca(2+) entry (SOC) as a route to refill the Ca(2+) stores could be easily identified in cultured astrocytes as the Zn(2+)-sensitive component of the Ca(2+) signal. In contrast, such a Zn(2+)-sensitive component was not recorded in astrocytes from hippocampal slices despite of evidence for SOC. Our data indicate that both, astrocytes in situ and in vitro express SOC necessary to refill stores, but that a SOC-related signal is not recorded in the cytoplasm of astrocytes in situ since the stores are close to the plasma membrane and the refill does not affect cytoplasmic Ca(2+) levels.     

Response of the electrochromic dye,merocyanine 540, to membrane potential in rat liver mitochondria

Summary We have previously shown that pertussis toxin (PTX) stimulates delayed-onset, [Ca^2–] _a -dependent catecholamine (CA) release from bovine chromaffin cells. We now show that this effect of PTX is inhibited in part (50%) by dihydropyridine Ca^2–-channel antagonists niludipine and nifedipine, and is potentiated by the dihydropyridine Ca²⁺-channel agonist Bay K-8644. We and others have shown that pretreatment of chromaffin cells with PTX results in enhanced catecholamine secretion in response to high [K^–] _a , nicotine and muscarine, and here we extend these observations by showing that toxin pretreatment also enhances the secretory response to [Ba²⁺] _a . All these data are consistent with the concept that PTX may act on Ca^2– channels. To examine the possibility of a direct action of the toxin on the voltage-gated L-type Ca²⁺ channel known to be present in these cells, we studied the effects of the toxin on whole cell Ca²⁺ currents. We found and report here that spontaneous electrical activity was considerably increased in PTX-treated cells. Our measurements of whole cell inward Ca²⁺ currents indicate that the underlying mechanism is a marked shift of the activation curve of the L-type Ca²⁺ current along the voltage axis towards more negative potentials. While treatment of the cells with PTX had no effect on L-type Ca²⁺-channel conductance (6 nS/cell at 2.6mm [Ca²⁺] _a ). PTX evoked the activation of a new class of Ca²⁺-selective channels (5 pS in 25mm [Ca²⁺]_pipet), which are rather insensitive to membrane potential. We have termed theseG-type calcium channels. These data suggest that treatment with PTX not only increases the probability of L-type Ca²⁺-channel activation at more negative potentials, but also increases the probability of opening of an entirely new, voltage-independent, Ca²⁺ channel. These actions of PTX should promote Ca²⁺ entry and might explain the stimulation by the toxin of CA secretion from medullary chromaffin cells in culture.     

Calcium mobilizing hormones activate the plasma membrane Ca2+ pump of pancreatic acinar cells

Summary ⁴⁵Ca fluxes and free-cytosolic Ca²⁺ ([Ca²⁺] _i ) measurements were used to study the effect of Ca²⁺-mobilizing hormones on plasma membrane Ca²⁺ permeability and the plasma membrane Ca²⁺ pump of pancreatic acinar cells. We showed before (Pandol, S.J., et al., 1987.J. Biol. Chem. 262:16963–16968) that hormone stimulation of pancreatic acinar cells activated a plasma membrane Ca²⁺ entry pathway, which remains activated for as long as the intracellular stores are not loaded with Ca²⁺. In the present study, we show that activation of this pathway increases the plasma membrane Ca²⁺ permeability by approximately sevenfold. Despite that, the cells reduce [Ca²⁺]i back to near resting levels. To compensate for the increased plasma membrane Ca²⁺ permeability, a plasma membrane Ca²⁺ efflux mechanism is also activated by the hormones. This mechanism is likely to be the plasma membrane Ca²⁺ pump. Activation of the plasma membrane Ca²⁺ pump by the hormones is time dependent and 1.5–2 min of cell stimulation are required for maximal Ca²⁺ pump activation. From the effect of protein kinase inhibitors on hormone-mediated activation of the pump and the effect of the phorbol ester 12-0-tetradecanoyl phorbol, 13-acetate (TPA) on plasma membrane Ca⁺ efflux, it is suggested that stimulation of protein kinase C is required for the hormone-dependent activation of the plasma membrane Ca²⁺ pump.     

Relationship between plasma membrane Ca2+-ATPase activity and acrosome reaction in guinea pig sperm

The results obtained by biochemical measurement demonstrated for the first time that significant decrease of the plasma membrane Ca²⁺-ATPase activity occurred during capacitation and acrosome reaction of guinea pig sperm. Ethaorynic acid, one kind of Ca²⁺-ATPase antagonists, inhibited the plasma membrane Ca²⁺-ATPase activity, but calmodulin (50μg/mL) and trifluoperazine (200- 500μmol/L) did not, suggesting that calmodulin is not involved in ATP-driven Ca²⁺ efflux from sperm. However, calmodulin is involved in the control of Ca²⁺ influx. TFP, one kind of calmodulin antagonists, accelerated the acrosome reaction and Ca²⁺ uptake into sperm cells significantly. Ca²⁺-ATPase antagonists, quercetin, sodium orthovandate, furosemide and ethacrynic acid promoted the acrosome reaction, but inhibited Ca2+ uptake, which cannot be explained by their inhibitory effects on the plasma membrane Ca²⁺-ATPase activity. It is speculated that this phenomenon might be caused by simultaneous inhibitions of the activities of Ca²⁺-ATPase present in the plasma membrane, the outer acrosome membrane and the outer mitochondrion membrane resulting in Ca²⁺ accumulation in the cytoplasm, which in turn blocks further Ca2+ entry through some negative feedback mechanism(s). The inhibitory effect of Ca²⁺-ATPase antagonist on glycolytic activity may also be the reason for Ca²⁺ accumulation in cytoplasm and inhibition of Ca²⁺ uptake.