Inhibition of neutral Mg2+-dependent sphingomyelinase by ubiquinol-mediated plasma membrane electron transport

Summary.  Sphingomyelin is an abundant constituent of the plasma membranes of mammalian cells. Ceramide, its primary catabolic intermediate, has emerged as an important lipid signaling molecule. Previous work carried out by our group has documented that plasma membrane Mg²⁺-dependent neutral sphingomyelinase can be effectively inhibited by exogenous ubiquinol. In this work, we have tested whether or not plasma-membrane-associated electron transport can also achieve this inhibition through endogenous ubiquinol. Our results have shown that Mg²⁺-dependent neutral sphingomyelinase in isolated plasma membranes was inhibited by NAD(P)H under conditions where ubiquinone is reduced to ubiquinol. This inhibition was potentiated in the presence of an extra amount of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2). Depletion of plasma membranes from lipophilic antioxidants by solvent extraction abolished the inhibition by reduced pyridine nucleotides without affecting the sensitivity of the neutral sphingomyelinase to exogenous ubiquinol. Reconstitution of plasma membranes with ubiquinone restored the ability of NAD(P)H to inhibit the enzyme. Our results support that the reduction of endogenous ubiquinone to ubiquinol by NAD(P)H-driven electron transport may regulate the activity of the plasma membrane neutral sphingomyelinase. Received May 20, 2002; accepted September 20, 2002; published online May 21, 2003 RID="**" ID="**" Present address: Department of Biomedical Engineering, School of Medicine, University of Baltimore, Maryland, U.S.A. RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Edificio C-6, Campus Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.     

Evidence that the platelet plasma membrane does not contain a (Ca2+ + Mg2+)-dependent ATPase

The present study was designed to determine the subcellular distribution of the platelet (Ca2+ + Mg2+)-ATPase. Human platelets were surface labeled by the periodate-boro[3H]hydride method. Plasma membrane vesicles were then isolated to a purity of approx. 90% by a procedure utilizing wheat germ agglutinin affinity chromatography. These membranes were found to be 2.6-fold enriched in surface glycoproteins compared to an unfractionated vesicle fraction and almost 7-fold enriched compared to intact platelets. In contrast, the isolated plasma membranes showed a decreased specific activity of the (Ca2+ + Mg2+)-ATPase compared to the unfractionated vesicle fraction. This decrease in specific activity was found to be similar to that of an endoplasmic reticulum marker, glucose-6-phosphatase, and to that of a platelet inner membrane marker, phospholipase A2. We conclude, therefore, that the (Ca2+ + Mg2+)-ATPase is not located in the platelet plasma membrane but is restricted to membranes of intracellular origin.     

Existence of Mg2+-dependent, neutral sphingomyelinase in nuclei of rat ascites hepatoma cells

A sphingomyelinase, which specifically hydrolyzes sphingomyelin into ceramide and phosphocholine, was solubilized from nuclear matrix fraction of rat ascites hepatoma, AH7974 cells. The solubilized enzyme was subjected to Mono Q column chromatography in an FPLC system. The sphingomyelinase which was adsorbed on the column and eluted at 0.25-0.5 M NaCl was characterized. The enzyme required 10 mM MgCl2, 0.01% Triton X-100, 1 mM dithiothreitol, and a higher concentration of buffer than 1 M for its maximal activity, and the optimal pH was 6.7-7.2 in 2 M Tris/acetic acid or 7.5 in 2 M potassium acetate/acetic acid. N-Ethylmaleimide completely inhibited the enzyme activity at 0.2 mM. Therefore, this enzyme is classified as a Mg2+-dependent, neutral sphingomyelinase. The sphingomyelinase sedimented at 4.3S through a 10-30% glycerol gradient containing 2 M potassium acetate. This enzyme was highly specific to sphingomyelin and did not hydrolyze phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. Various characteristics of the nuclear sphingomyelinase were similar to those of the plasma membrane enzyme except its requirement for a high concentration of buffer and SH-reagent.     

The plasma membrane (Mg2+)-dependent adenosine triphosphatase from the human erythrocyte is not an ion pump

Summary The plasma membrane (Mg²⁺)-dependent adenosine triphosphatase ((Mg²⁺)-ATPase) from human erythrocytes has been tested for its ability to transport ions. Using a preparation of inside-out vesicles loaded with the pH-sensitive fluorescence probe 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS), we have demonstrated the absence of proton movement during (Mg²⁺)-ATPase activity. From the rate of ATP hydrolysis and the passive proton permeability of these vesicles, an upper limit of 0.03 H⁺ transported per ATP hydrolyzed was calculated. To verify that proton pumping could be detected in this system, the intravesicular pH was monitored during (Ca²⁺)-dependent adenosine triphosphatase ((Ca²⁺)-ATPase) activity. Proton efflux associated with (Ca²⁺)-ATPase activity was observed (in agreement with a recent report of proton pumping by a reconstituted erythrocyte (Ca²⁺)-ATPase (Niggli, V., Sigel, E., Carafoli, E. (1982)J. Biol. Chem. 257:2350–2356)) and was shown to be stimulated by calmodulin. The ability of the (Mg²⁺)-ATPase to pump²⁸Mg²⁺,³⁵SO ₄ ^2– and⁸⁶Rb⁺ was also tested, with the results leading to the conclusion that the human erythrocyte enzyme does not function as an ion transport system.     

Cholera toxin blocks glucagon-mediated inhibition of the liver plasma membrane (Ca2+-Mg2+)-ATPase

We have previously shown that liver plasma membrane (Ca2+-Mg2+)-ATPase activity is inhibited by glucagon. To investigate the possible involvement of a GTP-binding (G) protein in this regulation, we have examined the effects of pertussis toxin and cholera toxin on inhibition of (Ca2+-Mg2+)-ATPase by glucagon. Treatment of liver plasma membranes with pertussis toxin did not affect the sensitivity of (Ca2+-Mg2+)-ATPase to the hormone. In contrast, treatment of plasma membranes or prior injection of animals with cholera toxin prevented inhibition of the (Ca2+-Mg2+)-ATPase by glucagon. Even though adenylate cyclase activity was increased by cholera toxin treatment, addition of cyclic AMP did not mimic the effect of cholera toxin in blocking glucagon-mediated inhibition of (Ca2+-Mg2+)-ATPase activity. These data suggest that a cholera toxin-sensitive protein, perhaps Gs or a Gs-like protein, is involved in the regulation of liver (Ca2+-Mg2+)-ATPase activity. The results emphasize the possible role of Gs-like proteins in regulation of enzymes other than adenylate cyclase and suggest that the study of (Ca2+-Mg2+)-ATPase may provide a useful enzymatic system to examine such regulation.     

Phospholipid dependence of the neutral sphingomyelinase in rat liver plasma membranes

Investigations have been carried out on the influence of the phospholipid composition of rat liver plasma membranes and of their physico-chemical properties on the activity of membrane-bound neutral sphingomyelinase. The membrane phospholipid composition was modified by the incorporation of different phospholipids into the membrane bilayer by means of lipid transfer proteins, n-butanol delipidation or exogenous sphingomyelinase (Staphylococcus aureus) treatment. The results indicate that the activity of neutral sphingomyelinase in liver plasma membranes depends upon phosphatidyl choline presence in the membrane bilayer and not upon membrane fluidity.     

Effect of polyamines on Mg(2+)-dependent ATP-hydrolase activity in plasmatic membrane of myometrium cells

Influence of aliphatic polyamines of spermine and spermidine on the enzymatic activity of the ouabain-sensitive Na+,K(+)-ATPase and the ouabain-resistant basal Mg(2+)-ATPase (specific activity--10.6 +/- 0.9 and 18.1 +/- 1.2 microM P(i)/hour on 1 mg of protein accordingly, n = 7) has been studied in the experiments carried out with the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution. It was found, that the polyamine spermine in concentration of 1 and 10 mM activated the Na+,K(+)-ATPase by 54 and 64% on the average relative to control value. Spermidine also stimulated the Na+,K(+)-ATPase activity, however it did it less efficiently than spermine: by 8 and 20% on the average at concentration of 1 and 10 mM, accordingly. Similarly, polyamines had affect on the basal Mg(2+)-ATPase: spermine in concentration of 1 and 10 mM activated it by 26 and 39% relative to control value; spermidine in concentration of 1 and 10 mM activated it by 10 and 32% relative to control. The magnitudes of the apparent activation constant K(a) of spermine were 0.35 +/- 0.07 and 0.10 +/- 0.02 mM for Na+,K(+)-ATPase and basal Mg(2+)-ATPase, accordingly (M +/- m, n = 5). It is supposed, that the obtained experimental data can be useful in the further research of the membrane mechanisms underlying of the cationic exchange in the smooth muscles, in particular, when investigating the role of the plasmatic membrane in providing electromechanical coupling in them, and also in regulation of ionic homeostasis in the smooth muscle cells.     

Differential localization and operation of distinct Mg(2+) transporters in apical and basolateral sides of rat liver plasma membrane

Upon activation of specific cell signaling, hepatocytes rapidly accumulate or release an amount of Mg(2+) equivalent to 10% of their total Mg(2+) content. Although it is widely accepted that Mg(2+) efflux is Na(+)-dependent, little is known about transporter identity and the overall regulation. Even less is known about the mechanism of cellular Mg(2+) uptake. Using sealed and right-sided rat liver plasma membrane vesicles representing either the basolateral (bLPM) or apical (aLPM) domain, it was possible to dissect three different Mg(2+) transport mechanisms based upon specific inhibition, localization within the plasma membrane, and directionality. The bLPM possesses only one Mg(2+) transporter, which is strictly Na(+)-dependent, bi-directional, and not inhibited by amiloride. The aLPM possesses two separate Mg(2+) transporters. One, similar to that in the bLPM because it strictly depends on Na(+) transport, and it can be differentiated from that of the bLPM because it is unidirectional and fully inhibited by amiloride. The second is a novel Ca(2+)/Mg(2+) exchanger that is unidirectional and inhibited by amiloride and imipramine. Hence, the bLPM transporter may be responsible for the exchange of Mg(2+) between hepatocytes and plasma, and vice versa, shown in livers upon specific metabolic stimulation, whereas the aLPM transporters can only extrude Mg(2+) into the biliary tract. The dissection of these three distinct pathways and, therefore, the opportunity to study each individually will greatly facilitate further characterization of these transporters and a better understanding of Mg(2+) homeostasis.     

Identification of Mg2+ -dependent neutral sphingomyelinase 1 as a mediator of heat stress-induced ceramide generation and apoptosis

Neutral sphingomyelinases (SMases) are involved in the induction of ceramide-mediated proapoptotic signaling under heat stress conditions. Although ceramide is an important mediator of apoptosis, the neutral SMase that is activated under heat stress has not been identified. In this study, we cloned an Mg(2+)-dependent neutral SMase from a zebrafish embryonic cell cDNA library using an Escherichia coli expression-cloning vector. Screening of the clones using an SMase activity assay with C(6)-7-nitro-2-1,3-benzoxadiazol-4-yl-sphingomyelin as the substrate resulted in the isolation of one neutral SMase cDNA clone. This cDNA encoded a polypeptide of 420 amino acids (putative molecular weight: 46,900) containing two predicted transmembrane domains in its C-terminal region. The cloned neutral SMase 1 acted as a mediator of stress-induced apoptosis. Bacterially expressed recombinant neutral SMase 1 hydrolyzed [choline-methyl-(14)C]sphingomyelin optimally at pH 7.5 in the presence of an Mg(2+) ion. In zebrafish embryonic cells, the endogenous SMase enzyme was localized in the microsomal fraction. In FLAG-tagged SMase-overexpressing cells, neutral SMase 1 colocalized with a Golgi marker in a cytochemical analysis. Inactivation of the enzyme by an antisense phosphorothioate oligonucleotide repressed the induction of ceramide generation, caspase-3 activation, and apoptotic cell death by heat stress. Thus, neutral SMase 1 participates in an inducible ceramide-mediating, proapoptotic signaling pathway that operates in heat-induced apoptosis in zebrafish embryonic cells.     

Tricyclic antidepressants inhibit the Ca(2+)-dependent ATPase activity from plasma membrane.

Tricyclic antidepressants are moderately potent inhibitors of the plasma membrane Ca(2+)-ATPase activity measured in erythrocyte ghosts. For the calmodulin-activated activity, half-maximal inhibition was observed in the presence of 0.25 mM clomipramine. Desipramine, imipramine, and trimipramine show half-maximal inhibition in the range of 0.8 to 1 mM. The inhibition dependence on clomipramine concentration is the same whether the enzyme is activated by exogenous calmodulin or by tryptic digestion. A similar behavior was observed for desipramine. The inhibition mechanisms utilized by clomipramine and desipramine are different. The clomipramine effect is associated with the Ca(2+)-bound enzyme conformation and can be attributed to a decrease in the rate of phosphorylation by ATP. The desipramine effect appears more related to the Ca(2+)-free conformation, since the partial reaction involved in the release of inorganic phosphate is perturbed by this drug. There is also little or no effect of tricyclics on the enzyme's affinity for ligand (Ca(2+) or ATP) binding.     

Selection of novel Mg(2+)-dependent self-cleaving ribozymes.

Four RNA motifs are known that catalyse site-specific cleavage in the presence of Mg2+ ions, all discovered in natural RNAs. In a single in vitro selection experiment we have isolated representatives of five novel classes of Mg(2+)-dependent ribozymes. Small versions of three of these showed that a very simple internal loop type of secondary structure is responsible for the activity. One of these was synthesized in a bimolecular form, and compared directly with the hammerhead ribozyme; for the new ribozyme, the cleavage step of the reaction is much faster than the spontaneous rate of phosphodiester bond cleavage, yet substantially slower than that for the hammerhead. The results suggest that many more Mg(2+)-dependent self-cleaving RNA sequences can be found.     

p-Nitrophenylphosphatase activity catalyzed by plasma membrane (Ca(2+) + Mg(2+)ATPase: correlation with structural changes modulated by glycerol and Ca(2+)

The plasma membrane (Ca²⁺+Mg²⁺)ATPase hydrolyzes pseudo-substrates such as p-nitrophenylphosphate. Except when calmodulin is present, Ca²⁺ ions inhibit the p-nitrophenylphosphatase activity. In this report it is shown that, in the presence of glycerol, Ca²⁺ strongly stimulates phosphatase activity in a dose-dependent manner. The glycerol- and Ca²⁺-induced increase in activity is correlated with modifications in the spectral center of mass (average emission wavenumber) of the intrinsic fluorescence of the enzyme. It is concluded that the synergistic effect of glycerol and Ca²⁺ is related to opposite long-term hydration effects on the substrate binding domain and the Ca²⁺ binding domain.     

Plasma membrane Mg(2+)-ATPase of Pachysolen tannophilus: characterization and role in alcohol tolerance.

Following cell fractionation in sucrose density gradients, plasma membrane Mg(2+)-ATPase from Pachysolen tannophilus was studied. The ATPase displayed an apparent Km for ATP of 1.42 mM and was inhibited by high concentrations of Mg2+. The inhibitory effects of ethanol, 1-propanol, 1-butanol, and benzyl alcohol on Mg(2+)-ATPase were evaluated, and the concentration of each alcohol that inhibited ATPase activity by 50% (IC50) was determined. The IC50 decreased as the chain length of the alcohol increased. Moreover, the IC50 for ATPase activity was similar to the IC50 for growth rate, suggesting an association between impaired growth and ATPase inhibition. Almost complete inhibition of ATPase activity occurred at temperatures approaching 60 degrees C, and the optimal temperature was around 44 degrees C for ATPase from both control and ethanol-treated cells. Inclusion of 50 mM MgCl2 or CaCl2 in the medium did not rescue cells from the deleterious effects of ethanol.     

Plasma membrane Mg(2+)-ATPase of Pachysolen tannophilus: characterization and role in alcohol tolerance.

Following cell fractionation in sucrose density gradients, plasma membrane Mg(2+)-ATPase from Pachysolen tannophilus was studied. The ATPase displayed an apparent Km for ATP of 1.42 mM and was inhibited by high concentrations of Mg2+. The inhibitory effects of ethanol, 1-propanol, 1-butanol, and benzyl alcohol on Mg(2+)-ATPase were evaluated, and the concentration of each alcohol that inhibited ATPase activity by 50% (IC50) was determined. The IC50 decreased as the chain length of the alcohol increased. Moreover, the IC50 for ATPase activity was similar to the IC50 for growth rate, suggesting an association between impaired growth and ATPase inhibition. Almost complete inhibition of ATPase activity occurred at temperatures approaching 60 degrees C, and the optimal temperature was around 44 degrees C for ATPase from both control and ethanol-treated cells. Inclusion of 50 mM MgCl2 or CaCl2 in the medium did not rescue cells from the deleterious effects of ethanol.     

Reversible inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid of the plasma membrane (Ca(2+)+Mg2+)ATPase from kidney proximal tubules

Calcium accumulation by purified vesicles derived from basolateral membranes of kidney proximal tubules was reversibly inhibited by micromolar concentrations of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of anion transport. The inhibitory effect of this compound on Ca2+ uptake cannot be attributed solely to the inhibition of anion transport: (Ca(2+)+Mg2+)ATPase and ATP-dependent Ca2+ transport, respectively. The rate constant of EGTA-induced Ca2+ efflux from preloaded vesicles was not affected by DIDS, indicating that this compound does not increase the permeability of the membrane vesicles to Ca2+. In the presence of DIDS, the effects of the physiological ligands Ca2+, Mg2+, and ATP on (Ca(2+)+Mg2+)ATPase activity were modified. The Ca2+ concentration that inhibited (Ca(2+)+Mg2+)ATPase activity in the low-affinity range decreased from 91 to 40 microM, but DIDS had no effect on the Km for Ca2+ in the high-affinity, stimulatory range. Free Mg2+ activated (Ca(2+)+Mg2+)ATPase activity at a low Ca2+ concentration, and DIDS impaired this stimulation in a noncompetitive fashion. The inhibition by DIDS was eliminated when the free ATP concentration of the medium was raised from 0.3 to 8 mM, possibly due to an increase in the turnover of the enzyme caused by free ATP accelerating the E2----E1 transition, and leading to a decrease in the proportion of E2 forms under steady-state conditions. Alkaline pH totally abolished the inhibition of the (Ca(2+)+Mg2+)ATPase activity by DIDS, with a half-maximal effect at pH 8.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of Ca(2+)-activated Mg(2+)-dependent ATP-hydrolase activity of membrane microsomal fraction of loach embryo (Misgurnus fossilis L.)

The functional confirmation of availability of Ca2+ transport initially-active systems in the embryo cells of loach Misgurnus fossilis L. has been obtained. Using thapsigargin, the specific inhibitor of endoplasmic reticulum of Ca2+, Mg(2+)-ATPase, this enzyme activity was divided into thapsigargin-sensitive (actually endoplasmic reticulum Ca2+, Mg(2+)-ATPase) and thapsigargin-insensitive (plasma membrane Ca2+, Mg(2+)-ATPase) constituents. The Ca(2+)-independent Mg(2+)-dependent ATPase activity makes above 39.7% of the common Ca2+, Mg(2+)-ATPase activity of embryo loach. The periodic changes of Ca2+, Mg(2+)-ATPase activity (except for the changes of plasma membrane Ca2+, Mg(2+)-ATPase activity) were found out, which coincide with periodic [Ca2+]i oscillations during the synchronous divisions of loach blastomers embryos.     

Localization of neutral,magnesium-stimulated sphingomyelinase in plasma membrane of cultured neuroblastoma cells

A parallel is shown between the distribution of neutral sphingomyelinase and plasma membrane enzymes (5′-nucleotidase and (Na⁺ + K⁺)-activated ATPase) in cultured neuroblastoma cells. In contrast there is no evidence of localization in lysosomes (β-hexosaminidase and acid sphingomyelinase), mitochondria (carnitine palmitoyltransferase), or cytosol. Activity in the microsomal fraction is attributed primarily to plasma membrane contamination.