Different effects of endothelin-3 on the Ca2+ discharge induced by agonists and Ca(2+)-ATPase inhibitors in human platelets

1. The present study demonstrates that endothelin-3 (ET-3), previously shown to attenuate thrombin-evoked aggregation of human platelets, delayed the dose-dependent aggregatory response to thapsigargin (Tg). As this Ca(2+)-ATPase inhibitor induces platelet activation in part through the depletion of internal Ca(2+)-stores, we examined the influence of ET-3 on Ca2+ discharge from internal pools. 2. Cytosolic Ca2+ concentration was evaluated with Fura-2 in the absence of Ca2+ influx. Platelet preincubation for 15 min with 5 x 10(-7) M ET-3 decreased the Ca2+ release evoked by thrombin and U46619, a thromboxane-mimetic. However, ET-3 did not affect Ca2+ movements induced by 1 microM ADP. Addition of Tg (0.5 to 5 microM) to resting platelets induced a cytosolic [Ca2+] rise with concentration-dependent increase of the initial rate and decrease of the time to reach the peak. ET-3 slowed down these dose-dependent effects with a more marked influence on the responses induced by low concentrations of Tg. 3. ET-3 did not modify the Ca2+ response to another Ca(2+)-ATPase inhibitor, 2,5-di-(tert-butyl)-1,4-benzohydroquinone(tBuBHQ). The thromboxane A2 receptor antagonist, SQ 29548, reduced by 53% the calcium signal evoked by 1 microM Tg, which became similar to that induced by 15 microM tBuBHQ. Under these conditions, the ET-3 effects were suppressed. A subsequent addition of thrombin induced a substantial further Ca2+ increase which was again sensitive to ET-3. 4. ET-3 attenuates Ca2+ mobilization from an internal pool dependent on the stimulation of thrombin and thromboxane A2 receptors and insensitive to the direct effect of Ca2+-ATPase inhibitors. The small but significant inhibitory effect of ET-3 leads us to propose that endothelin-3 acts as a modulator of platelet activation.     

Agonist and antagonist effects of apomorphine enantiomers on 5-HT3 receptors

Human platelets express several sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) isoenzymes: SERCA2b of 100 kDa apparent molecular mass and two distinct enzymes of 97 kDa, one of them identified as being the SERCA3a isoform. The molecular identity of the third enzyme specifically recognized by the PL/IM430 monoclonal antibody has remained elusive. First, the study of the 3'-end part of platelet SERCA3 mRNA, by means of RT-PCR amplification using sets of primers covering the N-3 to N (ultimate) exons of the human SERCA3 sequence, revealed the presence of two distinct mRNA sequences, SERCA3a and a longer variant. Second, this additional sequence was identified as SERCA3b and found to refer to the insertion of a new exon of 73 bp, located at bp 349 from the beginning of the intronic sequence, linking the penultimate (N-1) exon to the last exon (N) of the human SERCA3 gene. Third, a relationship between the expression of this SERCA3b mRNA and the PL/ IM430 recognizable SERCA protein was observed. SERCA3b mRNA was found to be absent in epithelial HeLa cells not recognized by the PL/IM430 antibody and the expression of this SERCA3b RNA species correlated with that of the SERCA protein recognized by PL/IM430 which was down-modulated in the platelet precursor megakaryocytic CHRF 288-11 cell line as well as upon in vitro lymphocyte activation. Taken together, these results strongly support the notion of the presence of the SERCA3b protein in human cells by showing SERCA3b mRNA in platelets and the fact that the protein corresponding to this mRNA species is very likely the 97 kDa protein recognized by the PL/IM430 antibody.     

Nitric oxide inhibits capacitative cation influx in human platelets by promoting sarcoplasmic/endoplasmic reticulum Ca2+-ATPase-dependent refilling of Ca2+ stores

Nitric oxide (NO) is a potent inhibitor of thrombin-induced increase in cytoplasmic free Ca2+ concentration and aggregation in platelets, but the precise mechanism of this inhibition is unclear. To measure Ca2+/Mn2+ influx in intact platelets and to monitor Ca2+ uptake into the stores in permeabilized platelets, fura-2 was used. In intact platelets, maximal capacitative Ca2+ and Mn2+ influx developed rapidly (within 30 s) after fast release of Ca2+ from the stores with thrombin (0.5 U/mL) or slowly (within 5 to 10 minutes) following passive Ca2+ leak caused by inhibition of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) with 30 micromol/L 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ). NO (1 micromol/L) inhibited capacitative Ca2+ and Mn2+ influx independently of the time after thrombin application. In contrast, the effect of NO on BHQ-induced Ca2+ and Mn2+ influx was observed only during the first few minutes after BHQ application and completely disappeared when capacitative cation influx reached its maximum. In Ca2+-free medium, NO reduced the peak Ca2+ rise caused by thrombin and significantly promoted Ca2+ back-sequestration into the stores. Both effects disappeared in the presence of BHQ. Inhibition of guanylate cyclase with H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (10 micromol/L) attenuated but did not prevent the effects of NO on cytoplasmic free Ca2+ concentration. Inhibition of Ca2+ uptake by mitochondria did not change the effects of NO. In permeabilized platelets, NO accelerated back-sequestration of Ca2+ into the stores after inositol-1,4,5-trisphosphate-induced Ca2+ release or after addition of Ca2+ (1 micromol/L) in the absence of inositol-1,4,5-trisphosphate. The effect of NO depended on the initial rate of Ca2+ uptake and on the concentration of ATP and was abolished by BHQ, indicating the direct involvement of SERCA. These data strongly support the hypothesis that NO inhibits store-operated cation influx in human platelets indirectly via acceleration of SERCA-dependent refilling of Ca2+ stores.     

Ca2+ in the dense tubules: a model of platelet Ca2+ load

In this work, we explored the relationship between the freely exchangeable Ca2+ (FECa2+) in the dense tubules (DT) and the sarco(endo)plasmic reticulum (SER) Ca2+-ATPase (SERCA) in circulating human platelets and examined the relationship between blood pressure (BP) and these platelet parameters. Studying platelets from 32 healthy men, we showed that the maximal reaction velocity (Vmax) of the SERCA significantly correlated with FECa2+ in the DT and with the protein expressions of SERCA 2 and 3. BP positively correlated with both the Vmax of the SERCA (r=.462, P=.010) and the FECa2+ sequestered in the DT (r=.492, P=.005). The relationships between these platelet Ca2+ parameters and BP were in part confounded by increased levels of serum triglycerides and diminished HDL cholesterol with a higher BP. No correlation was observed between the resting cytosolic Ca2+ and BP. Collectively, these findings indicate that (1) an increase in the cellular Ca2+ load in platelets is expressed by a higher activity of the SERCA and an increase in the expressions of SERCA 2 and 3 proteins, coupled with an increase in the FECa2+ in the DT, and (2) a higher BP is associated with an increase in platelet Ca2+ load in human beings, expressed by a rise in the FECa2+ in the DT and the upregulation of SERCA activity.     

Cyclopiazonic acid and thapsigargin induce platelet aggregation resulting from Ca2+ influx through Ca2+ store-activated Ca2+-channels

The effects of cyclopiazonic acid and thapsigargin, selective inhibitors of the endoplasmic reticulum Ca2+-ATPase pump, on the platelet aggregation were investigated using washed rat platelets prepared by chromatography on Sepharose 2B columns. In Ca2+-free medium, cyclopiazonic acid and thapsigargin did not induce aggregation, but in the presence of 1 mM Ca2+, platelet aggregation was induced in a concentration-dependent manner. Cyclopiazonic acid- and thapsigargin-induced platelet aggregation was blocked by 1 mM Ni2+ but not by 100 microM indomethacin or 1 microM nifedipine. In aequorin-loaded platelets, cyclopiazonic acid and thapsigargin caused sustained elevation of the cytosolic Ca2+ concentration, an effect which was blocked by Ni2+, a non-selective Ca2+ channel blocker and SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenyl]-1H-imidazole hydrochloride), a putative receptor-operated Ca2+ channel antagonist. The above results indicated that both cyclopiazonic acid and thapsigargin induced platelet aggregation and elevation of cytosolic Ca2+ concentration, that extracellular Ca2+ was essential for cyclopiazonic acid- and thapsigargin-induced platelet aggregation, and that platelet aggregation may be associated with Ca2+ influx through Ca2+ store-activated Ca2+ channels.     

Differential effect of halothane and forskolin on platelet cytosolic Ca2+ mobilization and aggregation

BACKGROUND: Previous works have suggested that the impairment of platelet aggregation by halothane was partly related to a stimulation of cyclic adenosine monophosphate (cAMP) production, to an inhibitory effect on Ca2+ signaling, or both. Intracellular Ca2+ measurements therefore were undertaken, first to determine the critical steps in the platelet CaZ+ signaling cascade most likely to be affected by halothane or by an increase in cAMP production, and second to establish if the effect of halothane involves aggregation-related biochemical pathways triggered by an increase in internal Ca2+. METHODS: Human washed platelets were treated with halothane or forskolin for 5 min before application of either platelet-activating factor, thrombin, U46619, or thapsigargin. The cytosolic Ca2+ concentration ([Ca2+]i) was measured with the fluorescent Ca2+ indicator fura-2. Nephelometric measurements were also performed to assay the aggregation process. RESULTS: Our results indicate that pretreating platelets with halothane leads to a partial impairment of the [Ca2+]i increase induced either by U46619, thrombin, or platelet-activating factor, but this had no significant effect on the [Ca2+]i response triggered by thapsigargin. In addition, our results show that halothane inhibits platelet aggregation triggered by U46619, but not by thapsigargin. Conversely, forskolin completely inhibited the [Ca2+]i response to U46619 and thapsigargin and prevented platelet aggregation induced by both agonists. CONCLUSIONS: These results suggest that halothane and cAMP exert their effects on platelet aggregation and Ca2+ signaling through different mechanisms, and that halothane cannot impair platelet aggregation independently of phospholipase C stimulation.     

Mechanism of nitric oxide-induced vasodilatation: refilling of intracellular stores by sarcoplasmic reticulum Ca2+ ATPase and inhibition of store-operated Ca2+ influx

The precise mechanisms by which nitric oxide (NO) decreases free [Ca2+]i, inhibits Ca2+ influx, and relaxes vascular smooth muscle are poorly understood. In rabbit and mouse aorta, agonist-induced contractions and increases in [Ca2+]i were resistant to nifedipine, suggesting Ca2+ entry through non-L-type Ca2+ channels. Relaxations to NO were inhibited by thapsigargin (TG) or cyclopiazonic acid (CPA) indicating the involvement of sarcoplasmic reticulum ATPase (SERCA). Studies of the effect of NO on [Ca2+]i and the rate of Mn2+ influx with fura-2 fluorometry in rabbit aortic smooth muscle cells in primary culture were designed to test how SERCA is involved in mediating the response to NO. When cells were stimulated with angiotensin II (AII), NO accelerated the removal of Ca2+ from the cytoplasm, decreased [Ca2+]i, and inhibited Ca2+ and Mn2+ influx. Inhibition of SERCA abolished all the effects of NO. In contrast, inhibition of the Na+/Ca2+exchanger or the plasma membrane Ca2+ ATPase had no influence on the ability of NO to decrease [Ca2+]i. NO maximally decreased [Ca2+]i within 5 s, whereas significant inhibition of AII-induced Ca2+ and Mn2+ influx required more than 15 s. The inhibition of cation influx strictly depended on [Ca2+]o and functional SERCA, suggesting that during the delay before NO inhibits Ca2+ influx, the influx of Ca2+ and the uptake into intracellular stores are required. In the absence of [Ca2+]o, NO diminished the AII-induced [Ca2+]i transient by a SERCA-dependent mechanism and increased the amount of Ca2+ in the stores subsequently released by ionomycin. The present study indicates that the initial rapid decrease in [Ca2+]i caused by NO in vascular smooth muscle is accounted for by the uptake of Ca2+ by SERCA into intracellular stores. It is proposed that the refilling of the stores inhibits store-operated Ca2+ influx through non-L-type Ca2+ conducting ion channels and that this maintains the decrease in [Ca2+]i and NO-induced relaxation.     

Enhanced myocardial contractility and increased Ca2+ transport function in transgenic hearts expressing the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase

In this study, we investigated whether the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca2+ transport pump (SERCA1a) can functionally substitute the cardiac SERCA2a isoform and how its overexpression affects cardiac contractility. For this purpose, we generated transgenic (TG) mice that specifically overexpress SERCA1a in the heart, using the cardiac-specific alpha-myosin heavy chain promoter. Ectopic expression of SERCA1a resulted in a 2.5-fold increase in the amount of total SERCA protein. At the same time, the level of the endogenous SERCA2a protein was decreased by 50%, whereas the level of other muscle proteins, including calsequestrin, phospholamban, actin, and tropomyosin, remained unchanged. The steady-state level of SERCA phosphoenzyme intermediate was increased 2.5-fold, and the maximal velocity of Ca2+ uptake was increased 1.7-fold in TG hearts, demonstrating that the overexpressed protein is functional. Although the basal cytosolic calcium signal was decreased by 38% in TG cardiomyocytes, the amplitude of cytosolic calcium signal was increased by 71.8%. The rate of calcium resequestration was also increased in TG myocytes, which was reflected by a 51.6% decrease in the normalized time to 80% decay of calcium signal. This resulted in considerably increased peak rates of myocyte shortening and relengthening (50.0% and 66.6%, respectively). Cardiac functional analysis using isolated work-performing heart preparations revealed significantly faster rates of contraction and relaxation in TG hearts (41.9% and 39.5%, respectively). The time to peak pressure and the time to half-relaxation were shorter (29.1% and 32.7%, respectively). In conclusion, our study demonstrates that the SERCA1a pump can functionally substitute endogenous SERCA2a, and its overexpression significantly enhances Ca2+ transport and contractile function of the myocardium. These results also demonstrate that the SERCA pump level is a critical determinant of cardiac contractility.     

Biochemical and functional heterogeneity of rat cerebrum microsomal membranes in relation to SERCA Ca(2+)-ATPases and Ca2+ release channels

Rat cerebrum microsomes were subfractionated on isopycnic linear sucrose (20-42%) density gradients. The Ca2+ loading/release properties and the distribution of intracellular Ca2+ store channels, inositol 1,4,5-trisphosphate (IP3) receptor and ryanodine (Ry) receptor, and SERCA pumps, were monitored in each subfraction by ligand binding and 45Ca2+ loading/release assays. Three different classes of vesicles were identified: (i) heavy density vesicles with high content of Ry receptors and Ca2+ pumps and high thapsigargin (TG)-sensitivity of Ca2+ loading; (ii) intermediate sucrose density vesicles with high content of IP3 receptor, high IP(S)3-sensitivity of Ca2+ loading and low content of Ry receptors; and (iii) light sucrose density vesicles with high content of Ry receptors, low content of IP3 receptors and low content of SERCA pumps highly sensitive to TG. Isolation of molecularly heterogeneous rat cerebrum microsomes and identification of specific Ca2+ loading/release properties support the presence of multiple, potentially active, heterogeneous rapidly exchanging Ca2+ stores in rat cerebrum.     

Measurement of resting cytosolic Ca2+ concentrations and Ca2+ store size in HEK-293 cells transfected with malignant hyperthermia or central core disease mutant Ca2+ release channels

Malignant hyperthermia (MH) and central core disease (CCD) mutations were introduced into full-length rabbit Ca2+ release channel (RYR1) cDNA, which was then expressed transiently in HEK-293 cells. Resting Ca2+ concentrations were higher in HEK-293 cells expressing homotetrameric CCD mutant RyR1 than in cells expressing homotetrameric MH mutant RyR1. Cells expressing homotetrameric CCD or MH mutant RyR1 exhibited lower maximal peak amplitudes of caffeine-induced Ca2+ release than cells expressing wild type RyR1, suggesting that MH and CCD mutants might be "leaky." In cells expressing homotetrameric wild type or mutant RyR1, the amplitude of 10 mM caffeine-induced Ca2+ release was correlated significantly with the amplitude of carbachol- or thapsigargin-induced Ca2+ release, indicating that maximal drug-induced Ca2+ release depends on the size of the endoplasmic reticulum Ca2+ store. The content of endogenous sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b), measured by enzyme-linked immunosorbent assay, 45Ca2+ uptake, and confocal microscopy, was increased in HEK-293 cells expressing wild type or mutant RyR1, supporting the view that endoplasmic reticulum Ca2+ storage capacity is increased as a compensatory response to an enhanced Ca2+ leak. When heterotetrameric (1:1) combinations of MH/CCD mutant and wild type RyR1 were expressed together with SERCA1 to enhance Ca2+ reuptake, the amplitude of Ca2+ release in response to low concentrations of caffeine and halothane was higher than that observed in cells expressing wild type RyR1 and SERCA1. In Ca2+-free medium, MH/CCD mutants were more sensitive to caffeine than wild type RyR1, indicating that caffeine hypersensitivity observed with a variety of MH/CCD mutant RyR1 proteins is not dependent on extracellular Ca2+ concentration.     

Association between ligand-induced conformational changes of integrin IIbbeta3 and IIbbeta3-mediated intracellular Ca2+ signaling

Platelet IIbbeta3 is a prototypic integrin and plays a critical role in platelet aggregation. Occupancy of IIbbeta3 with multivalent RGD ligands, such as fibrinogen, induces both expression of ligand-induced binding sites (LIBS) and IIbbeta3 clustering, which are thought to be necessary for outside-in signaling. However, the association between LIBS expression and outside-in signaling remains elusive. In this study, we used various IIbbeta3-specific peptidomimetic compounds as a monovalent ligand instead of fibrinogen and examined the association between LIBS expression and outside-in signaling such as IIbbeta3-mediated intracellular Ca2+ signaling. Using a set of monoclonal antibodies (MoAbs) against LIBS, we showed that antagonists can be divided into two groups. In group I, antagonists can induce LIBS on both IIb and beta3 subunits. In group II, antagonists can induce LIBS on the IIb subunit, but not on the beta3 subunit. Inhibition studies suggested that group I and group II antagonists interact with distinct but mutually exclusive sites on IIbbeta3. Neither group I nor group II antagonist increased intracellular Ca2+ concentrations ([Ca2+]i) in nonactivated platelets. All antagonists at nanomolar concentrations abolished the increase in [Ca2+]i in 0.03 U/mL thrombin-stimulated platelets, which is dependent on both fibrinogen-binding to IIbbeta3 and platelet-aggregation. However, only group I antagonists at higher concentrations dose-dependently augmented the [Ca2+]i increase, which is due to aggregation-independent thromboxane A2 production. This increase in [Ca2+]i was not observed in thrombasthenic platelets, which express no detectable IIbbeta3. Thus, only the group I antagonists, albeit a monovalent ligand, can initiate IIbbeta3-mediated intracellular Ca2+ signaling in the presence of thrombin stimulation. Our findings strongly suggest the association between beta3 LIBS expression and IIbbeta3-mediated intracellular Ca2+ signaling in platelets.     

Intracellular Ca2+ elevation and contraction due to prostaglandin F2alpha in rat aorta

Prostaglandin F2alpha was tested to determine (a) whether its effect on intracellular Ca2+ levels ([Ca2+]i) and force in vascular smooth muscle was mediated through activation of the thromboxane A2 and/or prostaglandin receptor, and (b) the relative roles of Ca2+ influx via L-type and non-L-type Ca2+ channels in prostaglandin receptor-mediated contraction. [Ca2+]i and force were measured simultaneously in fura-2-loaded rat aortic strips. The thromboxane A2 receptor antagonist, SQ29548 ([1S]-1a,2b(5Z),3b,4a-7-(3-[2-[(phenylamino)carbonyl] hydrazinomethyl)-7-oxobicyclo-[2.2.1]hept-2-yl-5-heptenoic acid), prevented the prostaglandin F2alpha-induced plateau [Ca2+]i elevation and force by 80-90%, while abolishing these responses due to the thromboxane A2 receptor agonist, U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy prostaglandin F2alpha). Prostaglandin F2alpha (+ SQ29548)-induced plateau [Ca2+]i elevation and force were not inhibited by verapamil. Ni2+, a non-selective cation channel blocker, in the presence of verapamil, abolished the prostaglandin F2alpha (+ SQ29548)-elevated [Ca2+]i, while the contraction was only partially inhibited. These results suggest that, in rat aorta, (1) elevated [Ca2+]i and force due to high prostaglandin F2alpha concentrations largely results from thromboxane A2 receptor activation, and (2) the prostaglandin component of the prostaglandin F2alpha-induced contraction is dependent on Ca2+ influx via non-L-type channels.     

Fluorescence probe study of the lumenal Ca2+ of the sarcoplasmic reticulum vesicles during Ca2+ uptake and Ca2+ release

A limited amount of information is available about the lumenal Ca2+ kinetics of the sarcoplasmic reticulum (SR). Incubation of mag-fura-2AM permitted to incorporate a sufficient amount of the probe into the SR vesicles, as determined by Mn2+ quenching. Rapid changes in the lumenal [Ca2+] ([Ca2+]lum) during Ca2+ uptake and release could be monitored by following the signal derived from the lumenal probe while clamping the extra-vesicular Ca2+ ([Ca2+]ex) at various desired levels with a BAPTA/Ca buffer. Changes in the [Ca2+]lum during uptake and release show the characteristics intrinsic to the SR Ca2+ pump (the [Ca2+]ex-dependence of the activation and inhibition by thapsigargin) and the Ca2+ release channel (blocking by ruthenium red), respectively. A new feature revealed by the [Ca2+]lum measurement is that during the uptake reaction the free [Ca2+]lum showed a significant oscillation. Several pieces of evidence suggest that this is due to some interactions between the Ca2+ pump and lumenal proteins.     

Temporal alterations in mRNA levels for proteinases and inhibitors and their potential regulators in the healing medial collateral ligament

Thrombin elicits responses in platelets such as shape change, aggregation, arachidonate liberation and secretion of the contents of three storage granules, processes that coincide with serine/threonine and tyrosine phosphorylation of numerous proteins, hydrolysis of polyphosphoinositides and mobilisation of Ca2+ within the cell. However, the significance of these parallel signal transduction processes has not been clearly elucidated in the light of the prevalent autocrine stimulation in platelets: a great amplification of the thrombin signal through secreted ADP, by production of thromboxane A2 from the liberated arachidonic acid, by the close cell contact produced by aggregation caused by exposure of integrin receptors that become ligated by fibrinogen and other platelet-produced factors. In the present communication five pathways of autocrine stimulation have been prevented by appropriate inhibitors. Under these conditions thrombin stimulated platelet secretion with little tyrosine phosphorylation, except for a 125-130 kDa protein that was tyrosine-phosphorylated in response to one of the inhibitors, the peptide Arg-Gly-Asp-Ser (RGDS) used to block aggregation. In sharp contrast, collagen elicits massive tyrosine phosphorylation and platelet secretion in the absence of autocrine stimulation. When the thrombin-induced tyrosine phosphorylations was corrected for RGDS-induced phosphorylation, the presence of inhibitors of autocrine stimulation reduced the thrombin-induced phosphorylation by 97%. Our results strongly suggests that tyrosine phosphorylation is not part of the signal transduction pathway initiated by thrombin per se, but it represents an integral part of signal transduction initiated by collagen.     

Release-activated Ca2+ transport in neurons of frog sympathetic ganglia

Frog sympathetic ganglion neurons exhibit a novel Ca2+ uptake mechanism, release-activated calcium transport or RACT, which is manifest in both cytosolic and store [Ca2+] signals as greatly accelerated Ca2+ uptake after Ca2+ release from internal stores. RACT is activated by Ca2+ release but not by Ca2+ entry and serves to selectively refill Ca2+ stores after release. RACT lowers cytosolic [Ca2+] with a rate constant about 1.6 times that of the SERCA pump with empty ER. RACT is thapsigargin-insensitive, was eliminated by ryanodine, but was not affected by blocking mitochondrial or plasma membrane Ca2+ transport. A Ca2+ flux model with RACT in the ER membrane reproduced the cytosolic and store [Ca2+] responses to all stimuli.     

Impaired cardiac performance in heterozygous mice with a null mutation in the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) gene

The sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) gene encodes both SERCA2a, the cardiac sarcoplasmic reticulum Ca2+ pump, and SERCA2b, which is expressed in all tissues. To gain a better understanding of the physiological functions of SERCA2, we used gene targeting to develop a mouse in which the promoter and 5' end of the gene were eliminated. Mating of heterozygous mutant mice yielded wild-type and heterozygous offspring; homozygous mutants were not observed. RNase protection, Western blotting, and biochemical analysis of heart samples showed that SERCA2 mRNA was reduced by approximately 45% in heterozygous mutant hearts and that SERCA2 protein and maximal velocity of Ca2+ uptake into the sarcoplasmic reticulum were reduced by approximately 35%. Measurements of cardiovascular performance via transducers in the left ventricle and right femoral artery of the anesthetized mouse revealed reductions in mean arterial pressure, systolic ventricular pressure, and the absolute values of both positive and negative dP/dt in heterozygous mutants. These results demonstrate that two functional copies of the SERCA2 gene are required to maintain normal levels of SERCA2 mRNA, protein, and Ca2+ sequestering activity, and that the deficit in Ca2+ sequestering activity due to the loss of one copy of the SERCA2 gene impairs cardiac contractility and relaxation.     

Inhibition of skeletal muscle sarcoplasmic reticulum Ca2+-ATPase by nitric oxide

The effects of nitric oxide on the activities of thapsigargin-sensitive sarcoplasmic reticulum Ca2+-ATPase (SERCA) and Ca2+ uptake by sarcoplasmic reticulum (SR) membranes prepared from white skeletal muscle of rabbit femoral muscle were studied. Pretreatment of the SR preparations with nitric oxide at concentrations of up to 250 microM for 1 min decreased the SERCA activity concentration dependently, and also decreased their Ca2+ uptake. Both these effects of nitric oxide were reversible. Inhibitors of guanylyl cyclase and protein kinase G (PKG) had no significant effect on the nitric oxide-induced inhibitions of SERCA and Ca2+ uptake. Moreover, dithiothreitol did not reverse the inhibitory effects of nitric oxide on SERCA and Ca2+ uptake. These findings suggest that nitric oxide inhibits SERCA, mainly SERCA 1, of rabbit femoral skeletal muscle by an action independent of the cyclic GMP-PKG system or oxidation of thiols, and probably by a direct action on SERCA protein.     

Differential effects of extracellular Mg2+ on thrombin-induced and capacitative Ca2+ entry in human coronary arterial endothelial cells

Receptor-mediated and capacitative Ca2+ entry are the primary Ca2+ entry pathways in endothelial cells (ECs). The mechanisms for Ca2+ entry via these pathways have not been fully elucidated. In this study, the effect of low and high external Mg2+ concentrations on these Ca2+ entry pathways was examined in human coronary arterial ECs. External Mg2+ concentration did not affect cytosolic free Mg2+ concentration. After exposure to thrombin in Ca(2+)-free medium, addition of Ca2+ to the medium caused a rise in cytosolic free Ca2+ concentration ([Ca2+]i), indicating thrombin-induced Ca2+ influx. Thrombin-induced Ca2+ influx was inhibited by not only low but also high external Mg2+ concentrations. After depletion of endoplasmic Ca2+ stores by thapsigargin, addition of Ca2+ to the medium induced an increase in [Ca2+]i, indicating capacitative Ca2+ entry. Capacitative entry was found to be accelerated by low external Mg2+ and inhibited by high external Mg2+ concentration. Results suggest that receptor-mediated Ca2+ influx requires external Mg2+ but is inhibited by increased external Mg2+ concentrations and that capacitative Ca2+ entry is reduced by external Mg2+ in human coronary arterial ECs.