Oxidative damage to sarcoplasmic reticulum Ca2+-ATPase AT submicromolar iron concentrations: evidence for metal-catalyzed oxidation

The sarcoplasmic reticulum (SR) calcium ATPase carries out active Ca2+ pumping at the expense of ATP hydrolysis. We have previously described the inhibition of SR ATPase by oxidative stress induced by the Fenton reaction (Fe2+ + H2O2 --> HO. + HO- + Fe3+). Inhibition was not related to peroxidation of the SR membrane nor to oxidation of ATPase thiols, and involved fragmentation of the ATPase polypeptide chain. The present study aims at further characterizing the mechanism of inhibition of the Ca2+-ATPase by oxygen reactive species at Fe2+ concentrations possibly found in pathological conditions of iron overload. ATP hydrolysis by SR vesicles was inhibited in a dose-dependent manner by micromolar concentrations of Fe2+, H2O2, and ascorbate. Measuring the rate constants of inactivation (k inact) at different Fe2+ concentrations in the presence of saturating concentrations of H2O2 and ascorbate (100 microM each) revealed a saturation profile with half-maximal inactivation rate at ca. 2 microM Fe2+. Inhibition was not affected by addition of 200 microM Ca2+ to the medium, indicating that it was not related to iron binding to the high affinity Ca2+ binding sites in the ATPase. Furthermore, inhibition was not prevented by the water-soluble hydroxyl radical scavengers mannitol or dimethylsulfoxide, nor by butylated hydroxytoluene (a lipid peroxidation blocker) or dithiothreitol (DTT). However, when Cu2+ was used instead of Fe2+ in the Fenton reaction, ATPase inhibition could be prevented by DTT. We propose that functional impairment of the Ca2+-pump may be related to oxidative protein fragmentation mediated by site-specific Fe2+ binding at submicromolar or low micromolar concentrations, which may occur in pathological conditions of iron overload.     

Identification of oxidation-sensitive peptides within the cytoplasmic domain of the sarcoplasmic reticulum Ca2+-ATPase

We have examined the oxidative sensitivity of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (SR) membranes, exposing isolated SR membranes to the thermolabile water soluble free radical initiator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Incubation with up to 702 microM AAPH-derived radicals results in a concentration- and time-dependent inhibition of calcium-dependent ATPase activity correlating with the loss of monomeric Ca2+-ATPase polypeptides, and the concomitant appearance of higher molecular weight species. However, no oxidant-induced protein fragmentation is detected. The observed formation of oxidant-induced bityrosine accounts for the intermolecular Ca2+-ATPase cross-links, as well as intramolecular cross-links. The oxidation of sulfhydryl groups to disulfides as another possible source of intermolecular cross-links has been ruled out after examination of SDS -PAGE performed under both reducing and non-reducing conditions. Exposure of the SR membranes to AAPH-derived radical species results in a small degree of lipid peroxidation that is not correlated with enzyme inactivation, suggesting that modification of membrane-spanning peptides is not related to enzyme inactivation. Six cytoplasmic peptides have been identified that are modified by exposure to AAPH or, alternatively, to hydrogen peroxide, suggesting that these regions of the Ca2+-ATPase are generally sensitive to oxidants. These oxidized peptides were identified after separation by reversed-phase HPLC followed by N-terminal sequencing and amino acid analysis as corresponding to the following sequences of the Ca2+-ATPase: (i) Glu121 to Lys128, (ii) His190 to Lys218, (iii) Asn330 to Lys352, (iv) Gly432 to Lys436, (v) Glu551 to Arg604, and (vi) Glu657 to Arg671. The Glu551 to Arg604 peptide, located within the nucleotide binding domain, was found to participate in the formation of intermolecular bityrosine cross-links with the identical Glu551 to Arg604 peptide from a neighboring Ca2+-ATPase polypeptide chain.     

Sarcoplasmic reticular Ca2+ pump ATPase activity in congestive heart failure due to myocardial infarction

OBJECTIVE: Earlier studies have shown a depression in the sarcoplasmic reticular (SR) Ca2+ uptake and gene expression in Ca2+ pump ATPase protein in congestive heart failure subsequent to myocardial infarction. It is the objective of this study to understand further the mechanisms of depressed SR Ca2+ pump activity in the failing heart. METHODS: Heart failure in rats was induced by occluding the left coronary artery for 16 weeks and the viable left ventricle was processed for the isolation of SR membranes. Sham-operated animals were used as control. The characteristics of SR Ca2+ pump ATPase in the presence of different concentrations of K+, Ca2+ and ATP were examined and the purity of these membranes was monitored by determining the marker enzyme activities. In addition to measuring changes in cyclic adenosine monophosphate (cAMP) protein kinase and Ca(2+)-calmodulin induced phosphorylation, alterations in SR phospholipid composition as well as sulfhydryl (SH) group content were investigated. RESULTS: Ca(2+)-stimulated ATPase activity, unlike Mg(2+)-ATPase activity, was depressed in the left ventricular SR from failing hearts as compared to control. The decrease in Ca(2+)-stimulated ATPase activity was seen at different concentrations of Ca2+, K+ and ATP but no changes in the affinities of the enzyme for Ca2+ and ATP were evident. The SR Ca(2+)-stimulated ATPase activities in the presence of both cAMP-dependent protein kinase and Ca(2+)-calmodulin were markedly decreased in the failing hearts when compared to control preparations. Furthermore, the 32P incorporation in the presence of cAMP-dependent protein kinase or Ca(2+)-calmodulin was also reduced in the experimental heart SR membranes. The phospholipid composition of the SR membranes from the failing heart was markedly altered. No changes in SH-group or the degree of cross contamination with other membranes were apparent in the failing heart SR. CONCLUSIONS: These results suggest that abnormalities in membrane phospholipid composition and phosphorylation of the enzyme may partly explain the observed depression in SR Ca2+ pump ATPase activity in heart failure following myocardial infarction.     

A novel phasic contraction induced by dithiothreitol in frog skeletal muscle

1. Dithiothreitol (DTT), at 50-100 mM, induced a phasic reversible contraction of frog skeletal muscle. 2. Exposure of single fibers to nifedipine (20 microM), an L-type Ca2+ antagonist, blocked the twitch and tetanus tensions but never affected the DTT-induced contraction. 3. DTT also produced a phasic contraction in fibers where voltage sensors were inactivated in the presence of high K+ concentration (190 mM). 4. A fiber was mechanically skinned after observation of DTT-induced contraction. The skinned fiber contracted in response to a DTT concentration similar to that required to produce contraction in intact fibers before skinning. 5. In skinned fibers, DTT, at 100 or 200 mM, inhibited the accumulation of Ca2+ by SR, but not Ca2+ ATPase activity. 6. These results suggest that a high concentration of DTT triggers Ca2+ efflux from the SR through action on the Ca2+ release channel and/or closely associated proteins, such as triadin and FK-506 binding protein.     

Brominated detergents as tools to study protein-detergent interactions

In order to study protein-detergent short-range interactions, we analyzed the quenching by brominated detergents of reticulum sarcoplasmic (SR) Ca(2+)-ATPase intrinsic fluorescence. For this purpose, 7,8-dibromododecyl beta-maltoside and 2-O-(10,11-dibromoundecanoyl)sucrose, brominated analogs of two non-ionic detergents, the frequently used dodecylmaltoside and the newly synthesized 2-O-lauroylsucrose respectively, were prepared. Rayleigh scattering measurements showed that the brominated detergents efficiently and rapidly solubilized SR vesicles like their non-brominated analogs although at slightly higher concentrations. Similarly, each analog had a slightly higher critical micellar concentration than its parent detergent. The partition coefficient K (expressed as the ratio of the molar fraction of detergent in the SR lipid phase to that in the aqueous phase, at pH 7.5 and 20 degrees C) was similar for brominated and non-brominated dodecyl maltoside (3.5-4 x 10(5)) and slightly lower for dibromoundecanoylsucrose (approximately 10(5)) than for lauroylsucrose (approximately 2 x 10(5)). At detergent concentrations too low to solubilize the membrane, the brominated detergents rapidly inserted (within seconds) into SR vesicles. In this concentration range, Ca(2+)-ATPase fluorescence quenching steadily increased with detergent concentration. When the membrane was saturated with detergent, the residual fluorescence was about half of its initial value, indicating significant protein-detergent, contacts, possibly due to a slightly higher affinity of Ca(2+)-ATPase for these detergents than for phospholipids. For higher detergent concentrations, solubilizing the membrane, the fluorescence continued to decrease with detergent concentration, with no evidence for a dramatic change in the average hydrophobic environment of the protein during the transition from bilayers to a soluble state. For still higher detergent concentrations, above that necessary for membrane solubilization, the fluorescence was further quenched to a residual relative value of about 20%, corresponding to further delipidation of the protein surface, in agreement with previous results [de Foresta, B., le Maire, M., Orlowski, S., Champeil, P., Lund, S., M?ller, J.V., Michelangeli, F. & Lee, A.G. (1989) Biochemistry 28, 2558-2567]. Fluorescence quenching for solubilized Ca(2+)-ATPase was quickly reversed upon addition of excess non-brominated detergent. The effects of the four detergents on the Ca(2+)-ATPase hydrolysis of p-nitrophenyl phosphate were similar and correlated with the protein-detergent contacts evidenced above. In conclusion, both these brominated detergents appear to be promising tools to study protein-detergent interactions at the hydrophobic surface of a membrane protein, either in a membrane or in solubilized complexes.     

Irreversible inhibition of plasma membrane (Ca2+ + Mg2+)-ATPase and Ca2+ transport by 4-OH-2,3-trans-nonenal

4-OH-2,3-trans-nonenal (HNE), a major aldehydic lipid peroxidation product, has been shown to cause cellular toxicities and has been linked to a number of pathophysiological processes including atherogenesis. Specifically, in vitro exposure of erythrocyte plasma membrane preparations to HNE resulted in the inhibition of membrane transport function and integrity. To characterize the nature of the inhibitory effects of HNE on plasma membrane regulatory mechanisms, we investigated its effects on substrate and calmodulin (CaM) stimulation on erythrocyte Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities. Concentration-effect relationship analysis in erythrocyte membrane "ghosts" and inside-out vesicles (IOVs) yielded purely noncompetitive kinetics for Ca2+, ATP, and CaM activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport. Reductions of Vmax from direct addition of 0.1 mM HNE to the assay incubation mixtures ranged from 23 to 41%. Similarly, pretreatment with HNE of both membrane ghosts and IOVs resulted in a concentration-dependent inactivation of ATPase and transport activities without changes in affinity for Ca2+, ATP, or CaM. Conversely, pretreatment of CaM itself did not impair its ability to stimulate (Ca2+ + Mg2+)-ATPase activity threefold. Moreover, HNE-pretreated membranes exhibited unaltered acetylcholinesterase activity compared to sham-pretreated membranes. Together, these results suggest that HNE may structurally, and thus irreversibly, modify one or more functionally important sites on the transport protein itself.     

Characterization of Ca(2+) flows essential in ornithine decarboxylase induction by L-asparagine in rat hepatoma cells using Ca(2+) flow inhibitors

L-Asparagine stimulates bi-directional Ca(2+) flows and induces ornithine decarboxylase in Reuber H-35 hepatoma cells. Previously it has been shown that these effects are completely, but reversibly inhibited by lanthanum chloride. In this study we examined the role(s) of Ca(2+) flows using more specific Ca(2+) flow inhibitors. It was shown that ornithine decarboxylase induction was inhibited by CdCl(2) and verapamil at concentrations above 1 mu M and 100 mu M respectively, but was unaffected by as much as 300 mu M NiCl(2), 1 mM nifedipine, or 10 mu M omega-conotoxin. Enzyme induction was blocked by the Ca(2+)-ATPase pump antagonists vanadate and Compound 48/80 in a dose-dependent manner. These results, taken together with the observations that extracellular Ca(2+) is essential for enzyme induction but a substantial elevation of cytoplasmic [Ca(2+)] is not, suggest that Ca(2+) inflow independent of the receptor-activated Ca(2+) channels, and the Ca(2+)-ATPase mediated Ca(2+) out-flow, are both important factors in the action of L-asparagine.     

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.     

Transmembrane Ca2+ gradient-mediated phosphatidylcholine modulating sarcoplasmic reticulum C(a2+)-ATPase

The sarcoplasmic reticulum (SR) C(a2+)-ATPase was purified and reconstituted into the sealed phospholipids vesicles with or without transmembrane Ca2+ gradient. The role of phospholipids, especially phosphatidylcholine (PC), in the modulation of C(a2+)-ATPase by transmembrane Ca2+ gradient was investigated. The results are as follows. (i) Incubated with phospholipids, the enzyme activity of the delipidated C(a2+)-ATPase is inhibited by Ca2+ and the highest inhibition is observed in the presence of PC. (ii) When there exists a transmembrane Ca2+ gradient (higher Ca2+ concentration inside vesicles, 1,000 mumol/L:50 mumol/L, similar to the physiological condition), the inhibition of C(a2+)-ATPase by transmembrane Ca2+ gradient can be only observed in the vesicles containing PC:PE, but not in those containing PS:PE or PG:PE. The highest inhibition is obtained at a 50:50 molar ratio of PC:PE (iii) By comparing the effects of PC differing in acyl chains, higher inhibition of C(a2+)-ATPase is observed in vesicles containing DPPC:PE and DOPC:PE, while no inhibition in DMPC:PE vesicles (iv) If the transmembrane Ca2+ gradient is in the inverse direction, the enzyme activity of C(a2+)-ATPase is inhibited whenever reconstituted with acidic or neutral phospholipids.     

Bacterial metabolism, cytokine mRNA transcription and viability of bovine alveolar macrophages infected with Mycobacterium bovis BCG or virulent M. bovis

The effect of amino acid peroxides, relatively stable products of irradiation of amino acid solutions, on erythrocyte components was studied. Interaction of proline, lysine, valine, and leucine peroxides (100-300 mu M) with erythrocyte membranes brought about a decrease of membrane protein -SH group content and of activities of (Na+, K+)-ATPase and Ca2+ -ATPase, and induced aggregation of membrane proteins, due mainly to the formation of interpeptide disulfides. Interaction of amino acid peroxides with hemoglobin brought about hemoglobin oxidation to methemoglobin. The effects of amino acid peroxides are similar to those of t-butyl hydroperoxide. These results indicate that peroxides of amino acid and proteins, which can also be formed under physiological conditions, may be mediators of the cellular action of reactive oxygen species.     

Calcium content of the sarcoplasmic reticulum in isolated ventricular myocytes from patients with terminal heart failure

Systolic [Ca2+]i-transients have been shown to be depressed in isolated ventricular myocytes from patients with terminal heart failure compared to controls. Experiments were performed in human ventricular cells to investigate whether this reduced systolic [Ca2+]i-transient may be due to a decreased Ca(2+)-content of the sarcoplasmic reticulum (SR). Single myocytes were isolated from left ventricular myocardium of patients with terminal heart failure undergoing cardiac transplantation. These results were compared to those obtained from cells of healthy donor hearts that were not suitable for transplantation for technical reasons. [Ca2+]i-transients were recorded from isolated cells under voltage clamp perfused internally with the Ca(2+)-indicator fura-2. The Ca(2+)-content of the SR was estimated by rapid extracellular application of caffeine (10 mM) to open the Ca(2+)-release channel of the SR and comparison of the caffeine-induced [Ca2+]i-transients in cells from patients with heart failure and from controls without heart failure. Upon steady-state depolarizations to +10 mV (maximum of the Ca(2+)-current), [Ca2+]i-transients in cells from patients with heart failure were significantly smaller than in myocytes from undiseased hearts (333 +/- 26 v 596 +/- 80 nM, P < 0.05). Application of caffeine caused a [Ca2+]i-transient that was always larger than during depolarization. Caffeine-induced [Ca2+]i-transients were significantly smaller in cells from diseased hearts compared with controls (970 +/- 129 v 2586 +/- 288 nM, P < 0.01). A positive correlation was found between left ventricular ejection fraction and caffeine-induced [Ca2+]i-transients in these cells. It is concluded, that depressed [Ca2+]i-transients in myocytes from patients with heart failure may be caused by a decreased Ca(2+)-content of the SR possibly due to an altered Ca(2+)-ATPase activity in these hearts. It is not necessary to postulate an additional defect of the Ca(2+)-release function of the SR to account for the alterations of intracellular (Ca2+]i-handling.     

The role of ganglioside GM3 in the modulation of conformation and activity of sarcoplasmic reticulum Ca(2+)-ATPase

Rabbit sarcoplasmic reticulum does contain trace amounts of gangliosides, and the main species is GM3. Incorporation of GM3 into the SR vesicles or addition of it to the soybean phospholipid used for reconstitution of proteoliposomes obviously increased ATP hydrolysis, as well as, Ca2+ uptake activity of sarcoplasmic reticulum Ca(2+)-ATPase. Conformation changes of Ca(2+)-ATPase induced by GM3 were also observed by circular dichroism, intrinsic fluorescence and fluorescence quenching measurements.     

Biochemical pathways of cell damage during the oxygen paradox of the rat heart

1. The standard O2-paradox has been studied in the Langendorff-perfused rat heart. 2. Perfusion of glucose-free saline under anoxia did not cause release of creatine kinase (CK) although, it is suggested, there was a progressive rise in [Ca2+]i. 3. Ca(2+)-depletion after anoxia caused CK release. 4. Prolonged anoxic perfusion (55 min) produced a markedly reduced release of CK on Ca(2+)-depletion because, it is suggested, of the reduction in substrates for the release mechanism. 5. No protection against the O2-paradox was found with oxygen radical scavengers and inhibitors. 6. Lowering [Ca2+]o during reoxygenation to 0.1 mM did not reduce CK release. 7. Neither 1 mM amiloride (Na+/H+ antiporter inhibitor) nor 2 x 10(-6) M 1-(5-isoquinolinesulphonyl) piperazine (protein kinase C inhibitor) reduced CK release, unlike their effects in the Ca(2+)-paradox. 8. An hypothesis for events in the O2-paradox in presented: anoxia causes a loss of Ca(2+)-homeostasis and a rise in [Ca2+]i thereby activating a transmembrane NAD(P) oxido-reductase/diaphorase (stage 1); the return of O2 synergistically activates this molecular complex and causes CK release (stage 2).     

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

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

Mechanism of intracellular calcium homeostasis in heat-exposed cardiomyocytes of rats

The activity of Ca(2+)-ATPase and calcium content were measured in sarcoplasmic reticulum (SR) and mitochrondria of myocardium from 40 adult female Wistar rats after exposure to 37 degrees C, 39 degrees C, 41 degrees C and 43 degrees C for 40 min respectively in vitro. The 45Ca uptake, intracellular free Ca2+ concentration ([Ca2+]i) and the relatively active calmodulin content were also observed in cultured cardiomyocytes from 120 neonatal Wistar rats under the same condition of heat exposure. The results showed that (1) the activity of Ca(2+)-ATPase of SR and mitochrondria of myocardium decreased after heat exposure, (2) the calcium content of SR and mitochrondria also showed a tendency of decrease with increase of exposure temperature, (3) the 45Ca uptake and mean [Ca2+]i increased, whereas the calmodulin content decreased. It is suggested that disturbances of intracellular calcium homeostasis may be responsible for cardiac functional disorder after heat exposure.     

Effects of amount and type of dietary fibre (soluble and insoluble) on short-term control of appetite

OBJECTIVE: The calcium (Ca) pump of cardiac sarcoplasmic reticulum (SR) membranes is vulnerable to oxidation and hence likely to be damaged by chlorinated compounds, specifically hypochlorite (NaOCl) and monochloramine (NH2Cl), the most potent oxidants produced upon neutrophil activation. This could occur during prolonged ischemia or myocardial infarction when tissue levels of catecholamines are high. Phospholamban (PLN), the phosphorylatable regulator of the Ca pump, plays a central role in the effects of beta-adrenergic agonists on the heart. The purpose of this study was to investigate a possible role of PLN in determining the pump's sensitivity to NaOCl and NH2Cl. METHODS: Ca-uptake and Ca(2+)-ATPase activities in purified phosphorylated and control canine cardiac microsomes, incubated at increasing concentrations of NaOCl or NH2Cl, were related to the extent of PLN phosphorylation by protein kinase A, which was quantitated by PhosphorImager analysis. RESULTS AND CONCLUSIONS: Our data indicate that microsomal phosphorylation protects the Ca pump fully against 10 microM NaOCl or NH2Cl, which inhibit Ca-uptake by 21-41% when assayed at 25 or 37 degrees C and saturating Ca2+ in unphosphorylated microsomes, and protects partially at higher oxidant concentrations. The protective effect of protein kinase A on Ca-uptake is proportional to the amount of phosphorylated PLN. No comparable protection against similar oxidative damage of the Ca pump is observed when light fast skeletal muscle microsomes, which lack PLN, are incubated under conditions favorable for phosphorylation nor when PLN's inhibition of the cardiac Ca pump is relieved by proteolytic cleavage of its cytoplasmic domain. Our findings contribute toward an understanding of possible endogenous protective mechanisms that may promote calcium homeostasis in myocardial cells in inflammatory states associated with neutrophil activation and may suggest an approach toward development of protective strategies against oxidative damage in the heart.     

Kinetic uniformity of the ATP hydrolysis reaction catalyzed by Mg2+-ATPase in smooth muscle cell membrane

The kinetic properties of Mg(2+)-ATPase (EC 3.6.1.3) from myometrium cell plasma membranes have been studied. Under conditions of enzyme saturation with ATP (0.5-1.0 mM) or Mg2+ (1.0-5.0 mM) the initial maximal rates of the Mg(2+)-dependent enzymatic ATP hydrolysis, V0 ATP and V0 Mg, are 27.4 +/- 3.3 and 25.2 +/- 4.1 mumol Pi/hour/mg of protein, respectively. The apparent Michaelis constant, Km, for ATP and of the apparent activation constant, K alpha, for Mg2+ are equal to 28.1 +/- 2.6 and 107.0 +/- 26.0 microM, respectively. The bivalent metal ions used at 1.0 mM suppress the Mg(2+)-dependent hydrolysis of ATP whose efficiency decreases in the following order: Cu2+ > Zn2+ = Ni2+ > Mn2+ > Ca2+ > Co2+. Alkalinization of the incubation medium from pH 6.0 to pH 8.0 stimulates the Mg(2+)-dependent hydrolysis of ATP. It has been found that Mg(2+)-ATPase has the properties of an H(+)-sensitive enzymatic sensor which is characterized by a linear dependence between the initial maximal rate of the reaction, V0, and the pH value. The feasible role of plasma membrane Mg(2+)-ATPase in some reactions responsible for the control of proton and Ca2+ homeostasis in myometrium cells has been investigated.     

Rho guanosine triphosphatase mediates the selective stabilization of microtubules induced by lysophosphatidic acid

We have examined the ryanodine receptor, Ca(2+)-ATPase, calsequestrin and phospholamban mRNA levels in the left ventricles of pacing-induced heart failure and norepinephrine infusion dogs. The heart failure dogs showed a decrease in the levels of ryanodine receptor and Ca(2+)-ATPase mRNAs. Norepinephrine infusion caused a reduction of Ca(2+)-ATPase mRNA but no change in ryanodine receptor mRNA. There was a corresponding reduction of the immunoreactive Ca(2+)-ATPase protein levels in both heart failure and norepinephrine infusion animals compared to controls. In contrast, the mRNAs of calsequestrin and phospholamban were unchanged in dogs with either congestive heart failure or norepinephrine infusion. Thus, since norepinephrine infusion and congestive heart failure produced similar reductions of Ca(2+)-ATPase mRNA and protein, we postulate that the down-regulation of Ca(2+)-ATPase in congestive heart failure may be caused, at least in part, by sympathetic stimulation that occurs in heart failure.     

Inhibition of plasma membrane Ca(2+)-ATPase activity by volatile anesthetics

BACKGROUND: The precise sites and mechanisms of action of volatile anesthetics remain unknown. Recently, several integral membrane proteins have been suggested as potential targets to which anesthetics can bind at hydrophobic regions. Impairment of cell Ca2+ homeostasis has been postulated as one of the possible mechanisms of anesthetic action. To test these hypotheses, the authors selected the human erythrocyte Ca(2+)-ATPase as a model membrane protein. This enzyme is an integral membrane protein that is instrumental in maintaining Ca2+ homeostasis in the cell in which it is the sole Ca(2+)-transporting system. Thus, any functional alteration of the Ca(2+)-ATPase by anesthetics may lead to serious perturbations in Ca(2+)-regulated processes in the cell. METHODS: The Ca(2+)-ATPase activity was measured as a function of increased concentration of four volatile anesthetics: halothane, isoflurane, enflurane, and desflurane. RESULTS: All four anesthetics significantly inhibited the Ca(2+)-ATPase activity in a dose-dependent manner. The half-maximal inhibition occurred at anesthetic concentrations from 0.3 to 0.7 vol% at 37 degrees C, which, except for desflurane, is a clinically relevant concentration range. The greater the clinical potency of the volatile anesthetics studied, the less was the concentration required to inhibit the Ca(2+)-ATPase activity. The inhibition was less at 25 degrees C than at 37 degrees C, which is consistent with direct interactions of the nonpolar interfaces of the enzyme with the nonpolar of the portions of the anesthetics. CONCLUSIONS: The authors' findings indicate that the Ca(2+)-ATPase is a suitable model for investigating the mechanism of action of volatile anesthetics on the integral membrane protein, and that this inhibition may be specific.     

Ca(2+)-ATPases in the cochlear duct

Differing levels of the Ca(2+)-ATPase enzymes that reside on the plasma membrane (PM) and on the endoplasmic reticulum (ER) were identified in individual rat cochlear tissues by the use of a semi-quantitative enzyme-linked immunosorbent assay (ELISA). Unlike other studies, a specific antibody to PM Ca(2+)-ATPase was used to detect significantly greater levels (about 2x) of PM Ca(2+)-ATPase in the stria vascularis (SV) than that in the spiral ligament (SL) and organ of Corti (OC) tissues. Similarly, levels of ER Ca(2+)-ATPase were also significantly higher in the SV than in the SL and OC tissues. The presence of ER Ca(2+)-ATPase in the tissues of the SV has not been demonstrated previously. Given the importance of Ca2+ homeostasis in the inner ear, the statistically significantly higher densities of both PM and ER Ca(2+)-ATPase measured in the SV relative to the SL and OC regions would indicate tissue-specific responses to fluctuations in systemic and local Ca2+ concentrations.