calcium ATPase

By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induce...

Several studies have reported that amorphous nano-silica particles (nano-SPs) modulate calcium flux, although the mechanism remains incompletely understood. We thus analyzed the relationship between calcium flux and particle surface properties and determined the calcium flux route. Treatment of Balb/c 3T3 fibroblasts with nano-SPs with a diameter of 70 nm (nSP70) increased cytosolic calcium concentration, but that with SPs with a diameter of 300 or 1000 nm did not. Surface modification of nSP70 with a carboxy group also did not modulate calcium flux. Pretreatment with a general calcium entry blocker almost completely suppressed calcium flux by nSP70. Preconditioning by emptying the endoplasmic reticulum (ER) calcium stores slightly suppressed calcium flux by nSP70. These results indicate that nSP70 mainly modulates calcium flux across plasma membrane calcium channels, with subsequent activation of the ER calcium pump, and that the potential of calcium flux by nano-SPs is determined by the particle surface charge.

This article discusses how changes in luminal calcium concentration affect calcium release rates from triad-enriched sarcoplasmic reticulum vesicles, as well as single channel opening probability of the ryanodine receptor/calcium release channels incorporated in bilayers. The possible participation of calsequestrin, or of other luminal proteins of sarcoplasmic reticulum in this regulation is addressed. A comparison with the regulation by luminal calcium of calcium release mediated by the inositol 1,4,5-trisphosphate receptor/calcium channel is presented as well.

creased the dissociation constants for calcium, which suggests competition between the monovalent cations and calcium for the binding sites. At higher concentrations of monovalent cations, ouabain increased the apparent affinity of binding sites for calcium. Extrapolation to physiological cation concentrations revealed that the ouabain-induced increase in apparent affinity for calcium may be as much as 2-to 3-fold. These results suggest: (1) calcium binds to phospholipids associated with Na 1 ,K-ATPase; (2) ouabain interaction with Na + ,K + -ATPase induces a perturbation that is transmitted to adjacent phospholipids, altering their affinity for calcium; and (3) at physiological concentrations of Na * or K'. or both, ouabain interaction with Na + ,K'-ATPase may lead to an increased pool of membrane-bound calcium.

Calcium binding at 0 degrees C to a purified sheep kidney Na+,K+-ATPase was described by linear Scatchard plots. Binding at saturating free calcium was 65-80 nmol/mg of protein, or 30-40 mol of calcium/mol of enzyme. Aqueous emulsions of lipids extracted from Na+,K+-ATPase yielded dissociation constants and maximum calcium-binding values that were similar to those for native Na+,K+-ATPase. Phospholipase A treatment markedly reduced calcium binding. Pretreatment of native Na+,K+-ATPase with ouabain increased the dissociation constant for calcium binding from 131 +/- 7 to 192 +/- 7 muM without altering maximum calcium binding. Ouabain pretreatment did not affect calcium binding to extracted phospholipids, ouabain-insensitive ATPases, or heat denatured Na+,K+-ATPase, Na+ and K+ (5-20 mM) increased the dissociation constants for calcium, which suggests competition between the monovalent cations and calcium for the binding sites. At higher concentrations of monovalent cations, ouabain in...

Noncompetitive inhibitors of sarco-and endoplasmic reticulum calcium-ATPase (SERCA) have important therapeutic value in the treatment of cancer, due to their ability to induce apoptosis in cancer cells in a proliferation-independent manner. Thapsigargin (TG) and its analogues are one such class of inhibitors that bind to a hydrophobic pocket located in the transmembrane region of SERCA near the biomembrane surface and interfere with calcium transport. The binding free energies of thapsigargin-based inhibitors of SERCA were computed using a novel linear interaction energy (LIE) method with a surface generalized Born (SGB) continuum solvation model. A training set of 20 TG analogues was used to build a binding affinity model for estimating the free energy of binding for 18 new inhibitors with a root-mean-square (rms) error of 1.36 kcal/mol with respect to experimental data. For 15 out of the 18 inhibitors in the test set, the rms error was 1.02 kcal/mol, which is on the order of the accuracy level achieved by highly rigorous free energy of perturbation (FEP) or thermodynamic integration (TI) methods. On the basis of the analysis of the binding cavity at the interface of the membrane surface and the cytoplasmic region, we propose that side chains of TG derivatives at the O-8 position orient toward the cytoplasmic region through a hydrophobic channel. On the basis of this insight, four analogues of varying side chain length at the O-8 position with a charged moiety at the end were designed, tested with LIE methodology, and then validated experimentally for their SERCA inhibition activity. Low levels of rms error for the majority of inhibitors establish the structure-based LIE method as an efficient tool for generating more potent and specific inhibitors of SERCA by testing rationally designed lead compounds based on thapsigargin derivatization.

Prior studies identified phosphoenzyme intermediates in the turnover of sodium-and potassium-activated adenosinetriphosphatase [ (Na,K) ATPase] from several sources and of the calcium-activated adenosinetriphosphatase [ (Ca)-ATPase] of skeletal muscle sarcoplasmic reticulum. In both cases, the transphosphorylation is to a @-aspartyl carboxyl group at the active site. We now report observation of a K+-sensitive phosphorylated intermediate of purified (Na,-K)ATPase from the salt gland of the duck using high-field 31P nuclear magnetic resonance. Addition of ATP to a suspension of this enzyme in the presence of Mg2+ and Na+ produced a resonance at about +17 ppm relative to 85% phosphoric acid. Addition of inorganic phosphate and Mg2+ to (Na,K)ATPase also produced a resonance at about +17 Substantial evidence has accumulated indicating that the active transport of sodium and potassium across cell membranes involves transphosphorylation from ATP to the transport enzyme complex of sodium-and potassium-activated adenosinetriphosphatase [ (Na,K)ATPase] at a 8-aspartyl site . Prior conclusions have been based on the 32P content of enzyme preparations phosphorylated from radiolabeled ATP or inorganic phosphate followed by acid or detergent denaturation . Observation of one or more phosphorylated intermediates in (Na,K)ATPase by 31P nuclear magnetic resonance (NMR) would provide an opportunity for further characterization of intermediates through direct observation of active enzyme during catalysis. Phosphorylated intermediates have previously been observed by 31P NMR in phosphoglucomutase (Mildvan, 1973) and alkaline phosphatase (Brock & Sheard, 1975; Hull et al., 1976; Chlebowski et al., 1976). These are soluble enzymes, obtainable in high concentrations, and both the enzyme and its linkage to phosphate are relatively stable. Active (Na,K)-ATPase is a membrane-bound enzyme isolated with high phospholipid content (Goldman & Albers, 1973). Both the enzyme itself and its phosphorylated intermediate are unstable at 20 OC. Samples at a sufficient concentration of active sites for NMR study are viscous. The phosphorylation sites may

The plasma membrane calcium ATPase pump (PMCA) is one of two major mechanisms known to be involved in extruding calcium from cells. The monoclonal antibody 5F10 was used to examine the distribution of PMCA in chick Edinger-Westphal neurons, a population of cholinergic preganglionic neurons whose cells bodies reside in the Edinger-Westphal nucleus in the brainstem and whose axons form synaptic terminals on parasympathetic neurons in the ciliary ganglion. Definitive PMCA immunoreactivity was undetectable in Edinger-Westphal cell bodies in the brainstem. In contrast, immunoreactivity for PMCA was robust in ciliary ganglia and resembled patterns of immunoreactivity for the synaptic vesicle antigen SV-2, suggesting that PMCA is expressed in Edinger-Westphal synaptic terminals. Moreover, PMCA immunoreactivity co-localized with immunoreactivity for enkephalin and substance P, two neuropeptides known to be expressed in Edinger-Westphal synaptic terminals. Fine structure studies revealed that PMCA immunoreactivity is associated with synaptic vesicles rather than the plasma membrane in Edinger-Westphal terminals. In immunodot assays, synaptic vesicles purified from Torpedo electric organ are also immunoreactive for PMCA as well as SV-2. Torpedo vesicles are negative for the sarcoplasmic/endoplasmic reticulum ATPase, suggesting that the observed PMCA immunoreactivity is not associated with smooth endoplasmic reticulum. Immunoblot analysis confirmed that 5F10 recognizes a protein with the correct molecular mass tbr PMCA in tissue homogenates of chick cerebellum, chick ciliary ganglia, and Torpedo synaptic vesicles. These findings describe a previously unrecognized location for PMCA in the membranes of cholinergic synaptic vesicles. Relevance to previous data and possible functions are discussed.

We have examined fura 2-loaded purified peroxisomes under confocal microscope to prove that this mammalian organelle is a store of intracellular calcium pool. Presence of calcium channel and vanadate sensitive Ca 2+ -ATPase in the purified peroxisomal membrane has been demonstrated. We have further observed that machineries to maintain calcium pool in this mammalian organelle are impaired during infection caused by Leishmania donovani. Results reveal that peroxisomes have a merit to play a significant role in the metabolism of intracellular calcium.

While several transgenic mouse models exhibit improved contractile characteristics in the heart, less is known about how these changes influence energy metabolism, specifically the balance between carbohydrate and fatty acid oxidation. In the present study we examine glucose and fatty acid oxidation in transgenic mice, generated to overexpress sarco(endo)plasmic reticulum calcium-ATPase (SERCA), which have an enhanced contractile phenotype. Energy substrate metabolism was measured in isolated working hearts using radiolabeled glucose and palmitate. We also examined oxygen consumption to see whether SERCA overexpression is associated with increased oxygen utilization. Since SERCA is important in calcium handling within the cardiac myocyte, we examined cytosolic calcium transients in isolated myocytes using indo-1, and mitochondrial calcium levels using pericam, an adenovirally expressed, mitochondrially targeted ratiometric calcium indicator. Oxygen consumption did not differ between...

Endoplasmic reticulum calcium homeostasis is involved in a multitude of signaling, as well as ''house-keeping'' functions that control cell growth, differentiation or apoptosis in every human/eukaryotic cell. Calcium is actively accumulated in the endoplasmic reticulum by Sarco/Endoplasmic Reticulum Calcium transport ATPases (SERCA enzymes). SERCAdependent calcium transport is the only calcium uptake mechanism in this organelle, and therefore the regulation of SERCA function by the cell constitutes a key mechanism to adjust calcium homeostasis in the endoplasmic reticulum depending on the cell type and its state of differentiation. The direct pharmacological modulation of SERCA activity affects cell differentiation and survival. SERCA expression levels can undergo significant changes during cell differentiation or tumorigenesis, leading to modified endoplasmic reticulum calcium storage. In several cell types such as cells of

Cell polarization enables restriction of signalling into microdomains. Polarization of lymphocytes following formation of a mature immunological synapse (IS) is essential for calcium-dependent T-cell activation. Here, we analyse calcium microdomains at the IS with total internal reflection fluorescence microscopy. We find that the subplasmalemmal calcium signal following IS formation is sufficiently low to prevent calcium-dependent inactivation of ORAI channels. This is achieved by localizing mitochondria close to ORAI channels. Furthermore, we find that plasma membrane calcium ATPases (PMCAs) are re-distributed into areas beneath mitochondria, which prevented PMCA up-modulation and decreased calcium export locally. This nano-scale distribution-only induced following IS formation-maximizes the efficiency of calcium influx through ORAI channels while it decreases calcium clearance by PMCA, resulting in a more sustained NFAT activity and subsequent activation of T cells.

Sarcolipin (SLN) is an integral membrane protein that is expressed in both skeletal and cardiac muscle, where it inhibits SERCA (calcium ATPase) by lowering its apparent Ca 2C affinity in a manner similar to that of its homologue phospholamban (PLN). We use solution NMR to map the structural changes occurring within SLN upon interaction with the regulatory target, SERCA, co-reconstituting the two proteins in dodecylphosphocholine (DPC) detergent micelles, a system that preserves the native structure of SLN and the activity of SERCA, with the goal of comparing these interactions with those of the previously studied PLN-SERCA complex. Our analysis of the structural dynamics of SLN in DPC micelles shows this polypeptide to be partitioned into four subdomains: a short unstructured N terminus (residues 1-6), a short dynamic helix (residues 7-14), a more rigid helix (residues 15-26), and an unstructured C terminus (residues 27-31). Upon addition of SERCA, the different domains behave according to their dynamics, molding onto the surface of the enzyme. Remarkably, each domain of SLN behaves in a manner similar to that of the corresponding domains in PLN, supporting the hypothesis that both SLN and PLN bind SERCA in the same groove and with similar mechanisms.

Phospholamban (PLN) and sarcolipin (SLN) are two single-pass membrane proteins that regulate Ca 2+ -ATPase (SERCA), an ATP-driven pump that translocates calcium ions into the lumen of the sarcoplasmic reticulum, initiating muscle relaxation. Both proteins bind SERCA through intramembrane interactions, impeding calcium translocation. While phosphorylation of PLN at Ser-16 and/or Thr-17 reestablishes calcium flux, the regulatory mechanism of SLN remains elusive. SERCA has been crystallized in several different states along the enzymatic reaction coordinates, providing remarkable mechanistic information; however, the lack of high-resolution crystals in the presence of PLN and SLN limits the current understanding of the regulatory mechanism. This brief review offers a survey of our hybrid structural approach using solution and solid-state NMR methodologies to understand SERCA regulation from the point of view of PLN and SLN. These results have improved our understanding of the calcium translocation process and are the basis for designing new therapeutic approaches to ameliorate muscle malfunctions.

The effects of various concentrations of thapsigargin, a specific inhibitor of Ca2+-ATPase in the endoplasmic reticulum (ER) membrane, on calcium homeostasis in lymphoidal T cells (Jurkat) were investigated. Preincubation of these cells suspended in nominally calcium-free medium with 0.1 microM thapsigargin resulted in a complete release of Ca2+ from intracellular calcium stores. When the medium was supplemented with 3 mM CaCl2 the cells maintained constantly elevated level of cytosolic Ca2+. However, thapsigargin applied at lower concentration produced only a partial depletion of the stores. For example, in the cells pretreated with 1 nM thapsigargin and suspended in calcium-free medium approximately 75% of the calcium content was released from the intracellular stores. The addition of 3 mM CaCl2 to such cell suspension led to a transient increase in cytosolic calcium concentration, followed by a return to a lower steady-state. This phenomenon, related to the refilling of the ER by...

Targeting the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) offers a promising strategy for treating drug-resistant cancers. However, as SERCA activity is essential for all cells types, specific inhibitors such as thapsigargin (TG) possess general toxicity. We explored the potential for developing SERCA inhibitors controllable with light by synthesizing TG derivatives containing an azobenzene photoswitch. These compounds (AzTG derivatives) were assessed by their ability to inhibit SERCA ATPase activity. We have identified cis-active and trans-active derivatives and our results reveal a ~2-fold difference in IC50 in response to photoisomerization, demonstrating an effect of the incorporated photoswitch. Furthermore, X-ray structures of SERCA in complex with two AzTG derivatives were obtained, revealing two different binding modes for the photoswitch group of these compounds. Taken together, we have developed photo-activatable SERCA inhibitors that can serve as tools to study the fu...

The present paper reports data regarding the influence of aluminum, at micromolar concentrations, on intracellular 3q 2q Ž . 2q calcium homeostasis. Al modifies Ca uptake in the endoplasmic reticulum ER , accelerates Ca release from mitochondria and strongly inhibits Ca 2q -ATPase activity with a consequent high-level calcium accumulation inside the cell. These results suggest that Al 3q neurotoxicity may be related to an alteration of the intracellular calcium regulatory system.

The microsomal fraction of normal human skeletal muscle was subfractionated by isopycnic sucrose‐density centrifugation, using the procedure originally described by Saito et al.38 for rabbit fast muscle, and specific markers of the junctional face membrane of terminal cisternae (TC) (ryanodine receptor, high‐molecular‐weight feet proteins and membrane‐associated calcium‐binding protein calsequestrin), of the sarcoplasmic reticulum (SR) Ca‐pump membrane (chicken antibody to rabbit Ca‐ATPase), and of transverse tubules (TT) (dihydropiridine receptor, membrane cholesterol), respectively. The results show that isolated TC from human skeletal muscle share extensive morphological characteristics, protein composition, as well as Ca‐release properties with rabbit TC, as tested with an inhibitor (Ruthenium red) and an activator (doxorubicin) of SR Ca‐release. The Ca‐pump membrane of human muscle SR, in distinction to rabbit fast muscle SR, showed a relatively low specific activity of the Ca‐...

Background: Endoplasmic reticulum (ER) calcium storage and release play important roles in B lymphocyte maturation, survival, antigen-dependent cell activation and immunoglobulin synthesis. Calcium is accumulated in the endoplasmic reticulum (ER) by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). Because lymphocyte function is critically dependent on SERCA activity, it is important to understand qualitative and quantitative changes of SERCA protein expression that occur during B lymphoid differentiation and leukemogenesis. Methods: In this work we investigated the modulation of SERCA expression during the pharmacologically induced differentiation of leukemic precursor B lymphoblast cell lines that carry the E2A-PBX1 fusion oncoprotein. Changes of SERCA levels during differentiation were determined and compared to those of established early B lymphoid differentiation markers. SERCA expression of the cells was compared to that of mature B cell lines as well, and the effect of the direct inhibition of SERCA-dependent calcium transport on the differentiation process was investigated. Results: We show that E2A-PBX1 + leukemia cells simultaneously express SERCA2 and SERCA3-type calcium pumps; however, their SERCA3 expression is markedly inferior to that of mature B cells. Activation of protein kinase C enzymes by phorbol ester leads to phenotypic differentiation of the cells, and this is accompanied by the induction of SERCA3 expression. Direct pharmacological inhibition of SERCA-dependent calcium transport during phorbol ester treatment interferes with the differentiation process. Conclusion: These data show that the calcium pump composition of the ER is concurrent with increased SERCA3 expression during the differentiation of precursor B acute lymphoblastic leukemia cells, that a cross-talk exists between SERCA function and the control of differentiation, and that SERCA3 may constitute an interesting new marker for the study of early B cell phenotype.

Patients with chronic renal failure frequently develop cardiac hypertrophy and diastolic dysfunction; however, the mechanisms by which this occurs are still unclear. Male Sprague-Dawley rats were subjected to 5/6 nephrectomy and studied for their isolated myocyte function, calcium cycling, and gene expression of proteins important in calcium homeostasis after 4 wk. Comparable rats subjected to suprarenal aortic banding for the same duration were used for comparison. Rats subjected to 5/6 nephrectomy and aortic banding developed comparable hypertension; however, rats subjected to 5/6 nephrectomy experienced a greater degree of cardiac hypertrophy and downregulation of cardiac sodium potassium ATPase (Na ϩ /K ϩ-ATPase) activity than rats subjected to aortic band

In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca 2؉-ATPase (PMCA) to study the mechanism of calcium transport. To this end, we developed a procedure for measuring the occlusion of Ca 2؉ in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapid mixing device combined with a filtration chamber, allowing the isolation of the enzyme and quantification of retained calcium. Measurements of retained calcium as a function of the Ca 2؉ concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 ؎ 2.2 M, which agrees with the value found through measurements of PMCA activity in the absence of calmodulin. When enzyme phosphorylation and the retained calcium were studied as a function of time in the presence of La III (inducing accumulation of phosphoenzyme in the E 1 P state), we obtained apparent rate constants not significantly different from each other. Quantification of EP and retained calcium in steady state yield a stoichiometry of one mole of occluded calcium per mole of phosphoenzyme. These results demonstrate for the first time that one calcium ion becomes occluded in the E 1 P-phosphorylated intermediate of the PMCA.

The activity and protein expression of plasma membrane and sarco(endo)plasmic reticulum (Ca 2+-Mg 2+)ATPases and ryanodine receptors were investigated in surgically denervated rat vas deferens. The function of thapsigargin-sensitive but not thapsigargin-resistant (Ca 2+-Mg 2+) ATPase (from sarco(endo)plasmic reticulum and plasma membrane, respectively), evidenced by enzyme activity and Ca 2+ uptake experiments, was significantly depressed by 30-50% when compared to innervated vas. Western blots showed that such reduction in sarco(endo)plasmic reticulum (Ca 2+-Mg 2+)ATPase performance was accompanied by a decrement of similar magnitude in sarco(endo)plasmic reticulum (Ca 2+-Mg 2+) ATPase type 2 protein expression, without any significant change in plasma membrane (Ca 2+-Mg 2+)ATPase expression. Finally, [ 3 H]ryanodine binding revealed that the density of ryanodine binding sites was reduced by 45% after denervation without modification in affinity. The present findings demonstrate that sarco(endo)plasmic reticulum proteins involved in intracellular calcium homeostasis are clearly down-regulated and brings further evidence of a modified calcium translocation in denervated rat vas deferens.

In pulmonary arterial smooth muscle, Ca 2؉ release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) may induce constriction and dilation in a manner that is not mutually exclusive. We show here that the targeting of different sarcoplasmic/endoplasmic reticulum Ca 2؉-ATPases (SERCA) and RyR subtypes to discrete SR regions explains this paradox. Western blots identified protein bands for SERCA2a and SERCA2b, whereas immunofluorescence labeling of isolated pulmonary arterial smooth muscle cells revealed striking differences in the spatial distribution of SERCA2a and SERCA2b and RyR1, RyR2, and RyR3, respectively. Almost all SERCA2a and RyR3 labeling was restricted to a region within 1.5 m of the nucleus. In marked contrast, SERCA2b labeling was primarily found within 1.5 m of the plasma membrane, where labeling for RyR1 was maximal. The majority of labeling for RyR2 lay in between these two regions of the cell. Application of the vasoconstrictor endothelin-1 induced global Ca 2؉ waves in pulmonary arterial smooth muscle cells, which were markedly attenuated upon depletion of SR Ca 2؉ stores by preincubation of cells with the SERCA inhibitor thapsigargin but remained unaffected after preincubation of cells with a second SERCA antagonist, cyclopiazonic acid. We conclude that functionally segregated SR Ca 2؉ stores exist within pulmonary arterial smooth muscle cells. One sits proximal to the plasma membrane, receives Ca 2؉ via SERCA2b, and likely releases Ca 2؉ via RyR1 to mediate vasodilation. The other is located centrally, receives Ca 2؉ via SERCA2a, and likely releases Ca 2؉ via RyR3 and RyR2 to initiate vasoconstriction.

Wherever you travel through the cytoplasm of the cells you will find organelles with internal [Ca 2+ ] levels higher than in the surrounding cytosol. This is particularly true of the endoplasmic reticulum (ER) (or sarcoplasmic reticulum (SR) in muscle cells); such organelles serve as the main sources of releasable Ca 2+ for cytosolic cellular signalling. Calcium pumps of the SERCA family (sarcoplasmic and endoplasmic reticulum calcium ATP-ases) import calcium into the organelle lumen. The other mechanism that is responsible for the steady state calcium level within the lumen of ER or SR is a calcium leak that balances the influx created by the pumps. The leak remains the most enigmatic of the processes involved in calcium regulation. The molecular nature of the leak mechanism is not known. The basal leak is a relatively slow process, which is difficult to investigate and which is easily outmatched (both in the amplitude of calcium responses and in attractiveness to experimenters) by substantially faster second messenger-induced release. Nevertheless, information on the properties of the calcium leak, although thinly scattered through the pages of PubMed, has been slowly accumulating. In this review we will discuss the properties of the calcium leak and speculate about possible mechanisms, which could mediate this process.

Background Peripheral insulin resistance and compromised insulin secretion from pancreatic β-cells are significant factors and pathogenic hallmarks of diabetes mellitus (DM). NF-κβ/TLR-4 and SERCA/Ca 2+ pathways have been identified as potential pathways regulating insulin synthesis by preserving pancreatic β-cell functioning. The current study aimed to evaluate the therapeutic effect of aged garlic extract (AGE) against DM in a streptozotocin (STZ)-induced rat model with particular emphasis on pancreatic β-cell functioning. Methods AGE was characterized by gas chromatography-mass spectrometry (GC-MS), Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) to evaluate its physio-chemical characteristics followed by in-vitro anti-diabetic and antioxidant potential. This was followed by the induction of DM in laboratory animals for investigating the therapeutic action of AGE by evaluating the role of NF-κβ/TLR-4 and the SERCA/Ca 2+ pathway. The parameters assessed in the present experimental setup encompassed antioxidant parameters, metabolic indicators, insulin concentration, intracellular calcium levels, apoptotic markers (CCK-8 and Caspase Glo-8), and protein expression (P-62 and APACHE-II). Results AGE characterization by SEM, GC-MS, and X-ray diffraction (XRD) revealed the presence of phenylalanine, alliin, S-allylmercaptocysteine (SAMC), tryptophan, 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid as major bioactive constituents of AGE. Metabolic studies, including intraperitoneal glucose tolerance test (IPGTT), revealed significantly lower blood glucose levels in the AGE group compared to the disease control group. In contrast, the intraperitoneal insulin tolerance test (ITT) exhibited no significant difference in insulin sensitivity between the AGE

In higher eukaryotes, the sarco-endoplasmic reticulum (ER) Ca 2+-ATPase (SERCA) is characterized for its high sensitivity to low concentrations of thapsigargin (TG), a very specific inhibitor. In contrast, SERCA-like enzymes with different sensitivities to TG have been reported in trypanosomatids. Here, we characterized a SERCA-like enzyme from Trypanosoma evansi and evaluated its interaction with TG. Confocal fluorescence microscopy using BODIPY FL TG and specific anti-SERCA antibodies localized the T. evansi SERCA-like enzyme in the ER and confirmed its direct interaction with TG. Moreover, the use of either 1 µM TG or 25 µM 2',5'-di (tert-butyl)-1,4-benzohydroquinone prevented the reuptake of Ca 2+ and consequently produced a small increase in the parasite cytosolic calcium concentration in a calcium-free medium, which was released from the ER pool. A 3035 bp-sequence coding for a protein with an estimated molecular mass of 110.2 kDa was cloned from T. evansi. The corresponding gene product contained all the invariant residues and conserved motifs found in other P-type ATPases but lacked the calmodulin binding site. Modeling of the three-dimensional structure of the parasite enzyme revealed that the amino acid changes found in the TG-SERCA binding pocket do not compromise the interaction between the enzyme and the inhibitor. Therefore, we concluded that T. evansi possesses a SERCA-like protein that is inhibited by TG.

RESUMEN Se describe la estandarización de un ensayo de actividad para la ATPasa de calcio del eritrocito. De acuerdo con el ensayo, la actividad máxima de la enzima corresponde a 3.79 micromoles de fosfato por miligramo de proteína de membrana por hora y la actividad basal representa aproximadamente el 10% de la actividad máxima. El comportamiento de la curva de actividad contra el tiempo es lineal durante los primeros 30 minutos y se presenta una alta correlación entre aumento en la actividad de la enzima y concentración de Calmodulina. Gracias al ensayo es posible cuantificar Calmodulina en concentraciones menores a I^iM.

Two P-type Ca transporters, the plasma membrane Ca-ATPase (PMCA) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA), play a crucial role in maintaining Ca homeostasis, controlling contractility and contributing to excitably and cell signalling in smooth muscle cells. There is considerable structural homology between the two Ca-ATPases; they both have transmembrane spanning regions, have similar ATP-phosphorylated intermediaries, counter transport protons and are regulated by several second messengers. They both also exist in several isoforms and have many splice variants, which presumably impart some of their tissue specific functions. We describe the relative contribution of PMCA and the Na-Ca exchanger to Ca efflux in relaxation to smooth muscle, including recent data from transgenic mice, which has begun to elucidate the specific contributions of individual isoforms to Ca signalling. We then consider Ca release and uptake into the SR in smooth muscle. Experiments investigating the distribution of SERCA in smooth muscle cells have provided new insights into control of SR luminal Ca, and the effects of SR Ca load on signalling, is discussed. This is followed by a detailed consideration of the interactions between the surface membrane and SR membrane pumps, exchangers and ion channels in smooth muscle, along with their distribution to caveolae and cholesterol-rich membrane domains. Where relevant the importance of these functions to health and disease are noted. We conclude that the dynamic changes in splice variants expressed, constituents of membrane microdomains and environment of the sub-sarcolemmal space, close to the SR, need to be the focus of future research, so that the full importance of Ca transporters to smooth muscle signalling cascades can be better understood.

The crustacean hepatopancreas is an epithelial-lined, multifunctional organ that, among other activities, regulates the flow of calcium into and out of the animal's body throughout the life cycle. Transepithelial calcium flow across this epithelial cell layer occurs by the combination of calcium channels and cation exchangers at the apical pole of the cell and by an ATP-dependent, calcium ATPase in conjunction with a calcium channel and an Na 1 /Ca 21 antiporter in the basolateral cell region. The roles of intracellular organelles such as mitochondria, lysosomes, and endoplasmic reticulum (ER) in transepithelial calcium transport or in transient calcium sequestration are unclear, but may be involved in transferring cytosolic calcium from one cell pole to the other. The ER membrane has a complement of ATP-dependent calcium ATPases (SERCA) and calcium channels that regulate the uptake and possible transfer of calcium through this organelle during periods of intense calcium fluxes across the epithelium as a whole. This investigation characterized the mechanisms of calcium transport by lobster hepatopancreatic ER vesicles and the effects of drugs and heavy metals on them. Kinetic constants for 45 Ca 21 influx under control conditions were K n m 5 10.3871.01 mM, J max 5 14.7571.27 pmol/mg protein  sec, and n 5 2.5370.46. The Hill coefficient for 45 Ca 21 influx under control conditions, approximating 2, suggests that approximately two calcium ions were transported for each transport cycle in the absence of ATP or the inhibitors. Addition of 1 mM ATP to the incubation medium significantly (Po0.01) elevated the rate of 45 Ca 21 influx at all calcium activities used and retained the sigmoidal nature of the transport relationship. The kinetic constants for 45 Ca 21 influx in the presence of 1 mM ATP were K n m 5 12.7670.91 mM, J max 5 25.4671.45 pmol/mg protein  sec, and n 5 1.9570.15. Kinetic analyses of ER 65 Zn 21 influx resulted in a sigmoidal relationship between transport rate and zinc activity under control conditions (K n m 5 38.6370.52 mM, J max 5 19.3570.17 pmol/mg protein  sec, n 5 1.8170.03). The Addition of 1 mM ATP enhanced 65 Zn 21 influx at each zinc activity, but maintained the overall sigmoidal nature of the kinetic relationship. The kinetic constants for zinc influx in the presence of 1 mM ATP were K n m 5 34.5972.31 mM, J max 5 26.0971.17 pmol/mg protein  sec, and n 5 1.9670.17. Both sigmoidal and ATP-dependent calcium and zinc influxes by ER vesicles were reduced in the presence of thapsigargin and vanadate. This investigation found that lobster hepatopancreatic ER exhibited a thapsigargin-and vanadate-inhibited, SERCA-like, calcium ATPase. This transporter displayed cooperative calcium transport kinetics (Hill coefficient, n$2.0) and was inhibited by the heavy metals zinc and copper, suggesting that the metals may reduce the binding and transport of calcium when they are present in the cytosol.

Epithelial cells of the gut, gills, antennal glands and integument regulate calcium concentrations in crustaceans during the molt cycle. A cellular calcium transport model has been proposed suggesting the presence of calcium pumps, cation antiporters and calcium channels in transporting epithelial membranes that regulate the movements of this cation across the cell layer. Basolateral calcium transport during postmolt appears mainly regulated by the low affinity NCX antiporter, while calcium regulating 'housekeeping' activities of these cells in intermolt are controlled by the high affinity calcium ATPase (PMCA). A model is proposed for the involvement of the epithelial ER in the massive transepithelial calcium fluxes that occur during premolt and postmolt. This model involves the endoplasmic reticulum SERCA and RyR proteins and proposed cytoplasmic unstirred layers adjacent to apical and basolateral plasma membranes where calcium activities may largely exceed those in the bulk cytoplasmic phase. A result of the proposed transepithelial calcium transport model is that large quantities of calcium can be moved through these cells by these processes without affecting the low, and carefully controlled, bulk cytoplasmic calcium activities.

Gene Expression in Human Atrial Fibrillation. Introduction: Atrial fibrillation (AF) leads to a loss of atrial contraction within hours to days. During persistence of AF, cellular dedifferentiation and hypertrophy occur, eventually resulting in degenerative changes and cell death. Abnormalities in the calcium handling in response to tachycardia‐induced intracellular calcium overload play a pivotal role in these processes. Methods and Results: The purpose was to investigate the mRNA expression of proteins and ion channels influencing the calcium handling in patients with persistent AF. Right atrial appendages were obtained from 18 matched controls in sinus rhythm (group 1) and 18 patients with persistent AF undergoing elective cardiac surgery. Previous duration of AF was ≥ 6 months in 9 (group 2) and > 6 months in 9 patients (group 3). In a single semiquantitative polymerase chain reaction, the mRNA of interest and of glyceraldehydes‐3‐phosphate dehydrogenase, were coamplified and...

A large number of Ca 2þ -signaling proteins have been previously identified and characterized in Saccharomyces cerevisiae but relatively few have been discovered in filamentous fungi. In this study, a detailed, comparative genomic analysis of Ca 2þ -signaling proteins in Neurospora crassa, Magnaporthe grisea, and S. cerevisiae has been made. Our BLAST analysis identified 48, 42, and 40 Ca 2þ -signaling proteins in N. crassa, M. grisea, and S. cerevisiae, respectively. In N. crassa, M. grisea, and S. cerevisiae, 79, 100, and 13% of these proteins, respectively, were previously unknown. For N. crassa, M. grisea, and S. cerevisiae, respectively, we have identified: three Ca 2þ -permeable channels in each species; 9, 12, and 5 Ca 2þ /cation-ATPases; eight, six, and four Ca 2þ -exchangers; four, four, and two phospholipase CÕs; one calmodulin in each species; and 23, 21, and 29 Ca 2þ /calmodulin-regulated proteins. Homologs of a number of key proteins involved in the release of Ca 2þ from intracellular stores, and in the sensing of extracellular Ca 2þ , in animal and plant cells, were not identified. The greater complexity of the Ca 2þ -signaling machinery in N. crassa and M. grisea over that in S. cerevisiae probably reflects their more complex cellular organization and behavior, and the greater range of external signals which filamentous fungi have to respond to in their natural habitats. To complement the data presented in this paper, a comprehensive web-based database resource () of all Ca 2þ -signaling proteins identified in N. crassa, M. grisea, and S. cerevisiae has been provided.

We studied the effects of caffeine on calcium transport by subcellular organelles isolated from rabbit myocardium. Caffeine increased myofibrillar basic and calcium-activated ATPase activity at 20 mM but not at lower concentrations. Mitochondrial and sarcoplasmic reticulum (SR) calcium accumulation was measured both by dual wavelength spectrophotometry with the calcium-sensitive dye, murexide, and by Millipore filtration with 45Ca. In mitochondria, caffeine impaired phosphate-assisted calcium transport but did not alter the closely related parameters of oxygen uptake, P/O ratio (nmol adenosine diphosphate consumed/n ats oxygen consumed, state 3 respiration) or limited calcium loading. In SR, caffeine impaired calcium accumulation. New methods were used to characterize calcium accumulation in the absence of oxalate according to first order reaction kinetics. Caffeine increased the rate constant while decreasing the calcium accumulated. It also increased the associated calcium-activat...

Many cell types encode signals in the frequency component of calcium oscillations. In non-excitable cells calcium oscillations are induced by calcium influx from inositol trisphosphate receptors (IP3R) or/and ryanodine receptors (RyR). The frequency of Calcium oscillations induced in such cases is primarily by the way intracellular stores like endoplasmic reticulum, sarco-endoplasmic reticulum are refilled. Sarco-endoplasmic reticulum calcium ATPase (SERCA) is the major pathway by which this refilling process is effected. Most of the mathematical models proposed thus far used Michaelis-Menten kinetics to formulate SERCA pump. In this paper more emphasis has been given over the mathematical formulation of SERCA pump. In particular reverse quasi steady-state approximation (rQSSA) is used to formulate SERCA pump. Comprehensive comparative analysis is performed with the previous SERCA pump formulation. An apparent increase in frequency and amplitude of calcium oscillation is observed.

Background. Chronic renal failure has been referred to as a state of cellular calcium toxicity. The aim of this study was to investigate the status of free cytosolic calcium ([Ca 2+ ] i), intracellular calcium reserves and the capacitative calcium entry in peripheral blood mononuclear cells (PBMCs) of early-stage chronic kidney disease (CKD) patients, and to determine the effect of vitamin D 3 supplementation on these parameters. Methods. The study involved 44 patients with CKD stages 2-3; 27 of them were treated with cholecalciferol (5000 IU/week) for 12 months. [Ca 2+ ] i was measured using Fluo-3 AM fluorimetry. Intracellular calcium reserves were emptied by the application of thapsigargin (Tg), a specific inhibitor of endoplasmic reticulum Ca 2+-ATPase. 2-Aminoethyl-diphenyl borate (2APB) was used to examine the capacitative calcium entry. Results. [Ca 2+ ] i of CKD patients was substantially higher in comparison with healthy subjects: 123 (115-127) versus 102 (98-103) nmol/l, P < 0.001. The calcium concentration of Tg-sensitive stores and the capacitative calcium entry were also significantly increased in CKD patients. After the 12-month vitamin D 3 supplementation, there was a marked decrease in [Ca 2+ ] i [105 (103-112) nmol/l, P < 0.001 versus baseline], independently of the increase in 25(OH)D 3 or the decrease in PTH levels. No significant changes in intracellular calcium reserves and the capacitative calcium entry were found. Conclusions. Our results demonstrate that (1) [Ca 2+ ] i , intracellular calcium stores and the capacitative calcium entry were significantly increased already in early stages of CKD; (2) long-term vitamin D 3 supplementation normalized [Ca 2+ ] i without any effect on intracellular calcium reserves or the capacitative calcium entry.

The plasma membrane Ca 2+-ATPase (PMCA) is responsible for the fine, long-term regulation of the cytoplasmic calcium concentration by extrusion of this cation from the cell. Although the general kinetic mechanisms for the action of both, well coordinated hydrolytic activity and calcium transport are reasonably understood in the majority of cell types, due to the complex physiologic and biochemical characteristics shown by the hepatocyte, the study of this enzyme in this cell type has become a real challenge. Here, we review the various molecular aspects known to date to be associated with liver PMCA activity, and outline the strategies to follow for establishing the role of this enzyme in the overall physiology of the hepatocyte. In this way, we first concentrate on the basic biochemical aspects of liver cell PMCA, and place an important emphasis on expression of its molecular forms to finally focus on the critical hormonal regulation of the enzyme. Although these complex aspects have been studied mainly under normal conditions, the significance of PMCA in the calcium homeostasis of an abnormal liver cell is also reviewed.

Preeclampsia (PE) represents a major factor for maternal and perinatal morbidity and mortality [1] and it affects 7-10% of pregnancies worldwide [2]. PE is characterized by maternal syndromes such as gestational hypertension, proteinuria, oedema and foetal syndromes like reduced amniotic fluid, abnormal oxygenation and, in 30% of cases, intrauterine growth retardation (IUGR). The precise factors involved in the pathogenesis of PE remain unclear and it is considered as a multisystem disorder [1]. PE is principally characterized by impaired placentation and would be the result of an impaired differentiation of villous trophoblasts, which leads to an abnormal remodelling of the spiral arteries [3]. Diabetes, obesity, hypertension, renal disease, primiparity, maternal age, sperm and oocyte donation are all known predisposing factors for PE [1]. The oxidative stress, resulting from deficient remodelling of spiral arteries, is an important consequence of PE. It induces the placenta

In higher eukaryotes, the sarco-endoplasmic reticulum (ER) Ca 2+-ATPase (SERCA) is characterized for its high sensitivity to low concentrations of thapsigargin (TG), a very specific inhibitor. In contrast, SERCA-like enzymes with different sensitivities to TG have been reported in trypanosomatids. Here, we characterized a SERCA-like enzyme from Trypanosoma evansi and evaluated its interaction with TG. Confocal fluorescence microscopy using BODIPY FL TG and specific anti-SERCA antibodies localized the T. evansi SERCA-like enzyme in the ER and confirmed its direct interaction with TG. Moreover, the use of either 1 µM TG or 25 µM 2',5'-di (tert-butyl)-1,4-benzohydroquinone prevented the reuptake of Ca 2+ and consequently produced a small increase in the parasite cytosolic calcium concentration in a calcium-free medium, which was released from the ER pool. A 3035 bp-sequence coding for a protein with an estimated molecular mass of 110.2 kDa was cloned from T. evansi. The corresponding gene product contained all the invariant residues and conserved motifs found in other P-type ATPases but lacked the calmodulin binding site. Modeling of the three-dimensional structure of the parasite enzyme revealed that the amino acid changes found in the TG-SERCA binding pocket do not compromise the interaction between the enzyme and the inhibitor. Therefore, we concluded that T. evansi possesses a SERCA-like protein that is inhibited by TG.

Recent investigations on specialized epithelia suggest a role of the smooth endoplasmic reticulum Ca 2+-ATPase (SERCA) in epithelial Ca 2+-transport (Franklin et al., 2001; Hagedorn and Ziegler, 2002). These epithelia are involved in quick mineralisation processes in which epithelial Ca 2+-transport predominantly follows a transcellular route (transport through the cells) (Hubbard, 2000; Roer, 1980; Wheatly, 1997; Ziegler, 2002), rather than a paracellular pathway in which Ca 2+ would move extracellularly across the apical cell contacts and between the epithelial cells along its electrochemical gradient (Bronner, 1991). In the transcellular pathway, Ca 2+ enters the cells passively across the plasma membrane at one side of the cells and is actively extruded on the other side (Ahearn and Franco, 1993; Ahearn and Zhuang, 1996; Roer, 1980). The most critical step in transcellular epithelial Ca 2+-transport, however, is the Ca 2+ transport within the cells, through the cytoplasm from one side to the plasma membrane on the opposite side. Sustained elevated concentrations of Ca 2+ within the cytoplasm would interfere with the multiple regulatory functions of cytosolic free Ca 2+ signals. In addition, such elevated concentrations of ionized calcium can lead to cell damage and even cell death (Berridge, 1993). How such a toxic rise is prevented during epithelial Ca 2+-transport is still unknown. Simkiss (1996) proposed a model in which organelles, e.g. the smooth endoplasmic reticulum (SER), function as a transient calcium store allowing vectorial bulk flow through epithelial cells without toxic effects. Within cells the SER generally functions as source and sink for cytosolic Ca 2+ signals, with the inositol 1,4,5 trisphosphate (IP3)-receptors and/or ryanodine-receptors releasing Ca 2+ and the SERCA pumping Ca 2+ back in a regulated fashion (Hussain and Inesi, 1999). Previous investigations on the anterior sternal epithelium (ASE) of Porcellio scaber (Hagedorn and Ziegler, 2002; Ziegler, 2002) suggest that the SER contributes to transcellular calcium transport. Like most crustaceans, P. scaber has a calcified cuticle, which is moulted regularly to allow for growth of the animal. During premoult the ASE transports Ca 2+ , originating from the posterior cuticle to form large CaCO3 deposits located within the ecdysial gap of the first four anterior sternites (Messner, 1965

This study aimed to investigate the mechanism of active calcium transport in the chick embryonic chorioallantoic membrane (CAM) by assessing the functional involvement of three previously identified, putative components of the transport pathway. These components are a calcium-binding protein (CaBP), Ca2+-activated ATPase and carbonic anhydrase. Using specific reagents, including antibodies and enzyme inhibitors in vivo and in vitro in CAM calcium uptake assays, it was shown that these biochemically identified components were all functionally involved. The results of these studies also indicate that active calcium uptake by the CAM requires the presence of the CaBP on the cell surface in a laterally mobile manner, while carbonic anhydrase appeared to be a cytosolic component. We further analysed the subcellular location of the calcium-uptake activity by gel filtration and density-gradient fractionation of cell-free microsomes of the CAM and the results suggest that this activity is a...

Combining rapid filtration and rapid acid quenching, we have directly measured, at pH 7.0 and 5°C, the association and dissociation rate constants of Mg · ATP binding to the sarcoplasmic reticulum (SR) ATPase in the presence of 50 μM calcium and 5 mM MgCl2 (3–4x 106 M−1· s−1 and 9 s−1 respectively). Therefore, we have determined the true affinity for Mg · ATP (Kd= 3 μM) in the presence of calcium, which can not be measured at equilibrium because of spontaneous and fast phosphorylation. At low concentrations, Mg · ATP binding is the rate limiting step in the phosphorylation process, and Mg · ATP dissociation is slower than dephosphorylation.The kinetics of Ca2+ binding measured by rapid filtration are biphasic, reflecting a two‐step mechanism, both steps being accelerated by Mg · ATP. Combining rapid filtration and rapid monitoring of the intrinsic fluorescence of SR Ca2+ ‐ATPase, we showed that rate constants for calcium binding are always lower than those of Mg · ATP binding to an ...

Leaves from oak (Q uercus robur) contain an acyltransferase th a t catalyzes th e conversion o f ß-glucogallin (1-O-galloyl-ß-D-glucose) to 1,6-di-O-galloylglucose, an in term ed iate in the biosynthesis o f gallotannins. S u b strate specificity studies revealed th a t this enzym e w as also active w ith several structurally related 1-O-phenylcarboxyl-ß-D-glucoses; ap p reciab le reactio n rates, how ever, were observed only in the fo rm atio n o f 1,6-di-O-protocatechuoyl-ß-D-glucose. T his to d a te unknow n ester, as well as its digalloyl analog, w as synthesized using acy ltran sfer ase im m obilized on phenyl-Sepharose, and ch aracterized by UV an d 'H N M R spectroscopy. In ad d itio n , the 1,2-di-O-ß-D-glucose esters o f benzoic an d anisic acid were o b tain ed in this investigation.

Leaves from oak (Quercus robur) contain an acyltransferase that catalyzes the conversion of β-glucogallin (1-O-galloyl-β-D-glucose) to 1,6-di-O-galloylglucose, an intermediate in the biosynthesis of gallotannins. Substrate specificity studies revealed th at this enzyme was also active with several structurally related 1-O-phenylcarboxyl-β-D-glucoses; appreciable reaction rates, however, were observed only in the form ation of 1,6-di-O-protocatechuoyl-β-D-glucose. This to date unknown ester, as well as its digalloyl analog, was synthesized using acyltransferase immobilized on phenyl-Sepharose, and characterized by UV and 1H NMR spectroscopy. In addition, the 1,2-di-O-β-D-glucose esters of benzoic and anisic acid were obtained in this investigation.

After treatment of sarcoplasmic reticulum Ca 2؉-ATPase with proteinase K (PK) in the presence of Ca 2؉ and a protecting non-phosphorylated ligand (e.g. adenosine 5-(␤,␥-methylenetriphosphate), we were able to prepare in high yield an ATPase species that only differs from intact ATPase because of excision of the MAATE 243 sequence from the loop linking the A domain with the third transmembrane segment. The PK-treated ATPase was unable to transport Ca 2؉ and to catalyze ATP hydrolysis, but it could bind two calcium ions with high affinity and react with ATP to form a classical ADPsensitive phosphoenzyme, Ca 2 E1P, with occluded Ca 2؉. The ability of Ca 2 E1P to become converted to the Ca 2؉free ADP-insensitive form, E2P, was strongly reduced, as was the ability of PK-treated ATPase to react with orthovanadate or to form an E2P intermediate from inorganic phosphate in the absence of Ca 2؉. PK-treated ATPase also reacted with thapsigargin to form a complex with altered properties, and the tryptic cleavage "T2" site in the A domain was no longer protected in the absence of Ca 2؉. It is probable that disrupting the C-terminal link of the A domain with the transmembrane region severely compromises reorientation of A and P domains and the functionally critical cross-talk of these domains with the membrane-bound Ca 2؉ ions.

The effects of various concentrations of thapsigargin, a specific inhibitor of Ca2+-ATPase in the endoplasmic reticulum (ER) membrane, on calcium homeostasis in lymphoidal T cells (Jurkat) were investigated. Preincubation of these cells suspended in nominally calcium-free medium with 0.1 microM thapsigargin resulted in a complete release of Ca2+ from intracellular calcium stores. When the medium was supplemented with 3 mM CaCl2 the cells maintained constantly elevated level of cytosolic Ca2+. However, thapsigargin applied at lower concentration produced only a partial depletion of the stores. For example, in the cells pretreated with 1 nM thapsigargin and suspended in calcium-free medium approximately 75% of the calcium content was released from the intracellular stores. The addition of 3 mM CaCl2 to such cell suspension led to a transient increase in cytosolic calcium concentration, followed by a return to a lower steady-state. This phenomenon, related to the refilling of the ER by...

Deregulated or enhanced calcium ion (Ca 2+) influx across an unstable sarcolemma has been proposed to directly affect cardiac hypertrophic remodelling, vascular proliferative diseases and degenerative muscle disorders. Aberrant intracellular handling is partly due to a defect in Sarcoplasmic Reticulum (SR) function. Decreased Ca 2+ uptake in cardiac, vascular and skeletal myocytes is associated with a decrease in the expression and activity of the fast sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA2a or SERCA1a isoforms). SERCA2a gene transfer was successfully used in heart failure; this approach holds further therapeutic promises in vascular proliferative diseases and dystrophin-deficient muscular diseases. The growing family of human SERCA isoforms comprises at least 14 mRNA and proteins with different functional characteristics and cell-specific expression. This review focuses on the biological role and therapeutic potential of different isoforms of SERCA in the physiology and pathology of cardiac, vascular and skeletal muscle cells.