Presence Of N-ethylmaleimide Research Articles

Fully automated immunoassay for cholesterol uptake capacity to assess high-density lipoprotein function and cardiovascular disease risk

High-density lipoprotein (HDL) cholesterol efflux capacity (CEC), which is a conventional metric of HDL function, has been associated with coronary heart disease risk. However, the CEC assay requires cultured cells and takes several days to perform. We previously established a cell-free assay to evaluate cholesterol uptake capacity (CUC) as a novel measure of HDL functionality and demonstrated its utility in coronary risk stratification. To apply this concept clinically, we developed a rapid and sensitive assay system based on a chemiluminescent magnetic particle immunoassay. The system is fully automated, providing high reproducibility. Measurement of CUC in serum is completed within 20 min per sample without HDL isolation, a notably higher throughput than that of the conventional CEC assay. CUC decreased with myeloperoxidase-mediated oxidation of HDL or in the presence of N-ethylmaleimide, an inhibitor of lecithin: cholesterol acyltransferase (LCAT), whereas CUC was enhanced by the addition of recombinant LCAT. Furthermore, CUC correlated with CEC even after being normalized by ApoA1 concentration and was significantly associated with the requirement for revascularization due to the recurrence of coronary lesions. Therefore, our new assay system shows potential for the accurate measurement of CUC in serum and permits assessing cardiovascular health.

N-Ethylmaleimide influenced the evaluation of disulfide cross-links in the oxidized myofibrillar proteins using the non-reducing SDS-PAGE

The present study aimed to investigate the effect of N-Ethylmaleimide (NEM) on the evaluation of disulfide formation in the oxi dized myofibrillar proteins during the sample preparation of the non-reducing SDS-PAGE procedure. For this purpose, extracted myofibrillar proteins were oxidized firstly via a Fenton oxidation reaction, and non-oxidized proteins were used as a control. Before running SDS-PAGE, in the sample preparation, these oxidized and non-oxidized proteins were prepared according to the three dif ferent sample preparation methods with or without the presence of N-Ethylmaleimide or β-mercaptoethanol. Results showed that oxidized proteins treated with NEM regardless of sample preparation methods presented attenuated bands of myosin heavy chain monomer in the non-reducing SDS-PAGE gels, suggesting that the disulfide bonds formed as a result of protein oxidation could be preserved by NEM during sample preparation. Meanwhile, a possible mechanism for the effect of NEM was proposed.

Coexistence of D3 R typical and atypical signaling in striatonigral neurons during dopaminergic denervation. Correlation with D3 nf expression changes.

Dopamine D3 R are widely expressed in basal ganglia where interact with D1 R. D3 R potentiate cAMP accumulation and GABA release stimulated by D1 R in striatonigral neurons through "atypical" signaling. During dopaminergic denervation, D3 R signaling changes to a "typical" in which antagonizes the effects of D1 R, the mechanisms of this switching are unknown. D3 nf splice variant regulates membrane anchorage and function of D3 R and decreases in denervation; thus, it is possible that D3 R signaling switching correlates with changes in D3 nf expression and increases of membranal D3 R that mask D3 R atypical effects. We performed experiments in unilaterally 6-hydroxydopamine lesioned rats and found a decrease in mRNA and protein of D3 nf, but not of D3 R in the denervated striatum. Proximity ligation assay showed that D3 R-D3 nf interaction decreased after denervation, whereas binding revealed an increased Bmax in D3 R. The new D3 R antagonized cAMP accumulation and GABA release stimulated by D1 R; however, in the presence of N-Ethylmaleimide (NEM), to block Gi protein signaling, activation of D3 R produced its atypical signaling stimulating D1 R effects. Finally, we investigated if the typical and atypical effects of D3 R modulating GABA release are capable of influencing motor behavior. Injections of D3 R agonist into denervated nigra decreased D1 R agonist-induced turning behavior but potentiated it in the presence of NEM. Our data indicate the coexistence of D3 R typical and atypical signaling in striatonigral neurons during denervation that correlated with changes in the ratio of expression of D3 nf and D3 R isoforms. The coexistence of both atypical and typical signaling during denervation influences motor behavior.

Tyrosinase from Penicillium chrysogenum: Characterization and application in phenol removal from aqueous solution.

The tyrosinase of Penicillium chrysogenum strain AUMC 14100 Accession No. MN219732 was purified to homogeneity and chemically modified by N-ethylmaleimide (NEM) and 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride, DC). The inactivation of the purified enzyme obeyed pseudo-first-order reaction kinetics in the presence of NEM and DC (1-5 mM). The rate constants of the enzyme inactivation by NEM and DC were calculated to be 0.083 mol/min and 0.0013 mol/min, respectively. The recovery of enzyme activity by the protective effect of substrate indicates a non-specific modification of the active center. The order of tyrosinase inactivation kinetics and the substrate protection revealed the essentiality of sulfhydryl and lysyl residues in the enzyme active site and its role in the enzyme catalysis. The immobilized tyrosinase on alginate showed a gradual increase in residual activity over the immobilization time until the fourth hour. The desorptivity of tyrosinase was gradually raised with higher sodium dodecyl sulfate (SDS) concentrations. The immobilized enzyme retained about 70% of its original activity after 8 repeated cycles. Thus, immobilized tyrosinase of Penicillium chrysogenum removed 75% of phenol after 8 cycles and thus seems likely to be a good candidate for phenol removal in aqueous solution.

Antiulcerative Activity of Milk Proteins Hydrolysates.

Several studies have shown the protective effect of dairy products, especially α-lactalbumin and derived hydrolysates, against induced gastric ulcerative lesions. The mucus strengthening represents an important mechanism in the defense of gastrointestinal mucosa. Previously, a hydrolysate from casein (CNH) and a hydrolysate from whey protein concentrate rich in β-lactoglobulin (WPH) demonstrated a stimulatory activity on mucus production in intestinal goblet cells. The aim of this work was to evaluate the possible antiulcerative activity of these two hydrolysates in an ethanol-induced ulcer model in rats. All tested samples significantly reduced the ulcerative lesions index (ULI), compared with the saline solution, using doses of 300 and 1000 mg kg-1 body weight with decreases up to 66.3% ULI. A dose-response relationship was found for both hydrolysates. The involvement of endogenous sulfhydryl (SH) groups and prostaglandins (PGs) in the antiulcerative activity was evaluated using their blockage. The antiulcerative activity of WPH showed a drastic decrease in presence of N-ethylmaleimide (from 41.4% to 9.2% ULI). However, the CNH antiulcerative properties were not significantly affected. The cytoprotective effect of WPH appears to depend on a PG-mediated mechanism. In conclusion, CNH and WPH demonstrated in vivo antiulcerative properties and represent a promising alternative as protectors of the gastric mucosa.

Molecular insights into the normal operation, regulation, and multisystemic roles of K+-Cl- cotransporter 3 (KCC3).

Long before the molecular identity of the Na+-dependent K+-Cl- cotransporters was uncovered in the mid-nineties, a Na+-independent K+-Cl- cotransport system was also known to exist. It was initially observed in sheep and goat red blood cells where it was shown to be ouabain-insensitive and to increase in the presence of N-ethylmaleimide (NEM). After it was established between the early and mid-nineties, the expressed sequence tag (EST) databank was found to include a sequence that was highly homologous to those of the Na+-dependent K+-Cl- cotransporters. This sequence was eventually found to code for the Na+-independent K+-Cl- cotransport function that was described in red blood cells several years before. It was termed KCC1 and led to the discovery of three isoforms called KCC2, KCC3, and KCC4. Since then, it has become obvious that each one of these isoforms exhibits unique patterns of distribution and fulfills distinct physiological roles. Among them, KCC3 has been the subject of great attention in view of its important role in the nervous system and its association with a rare hereditary sensorimotor neuropathy (called Andermann syndrome) that affects many individuals in Quebec province (Canada). It was also found to play important roles in the cardiovascular system, the organ of Corti, and circulating blood cells. As will be seen in this review, however, there are still a number of uncertainties regarding the transport properties, structural organization, and regulation of KCC3. The same is true regarding the mechanisms by which KCC3 accomplishes its numerous functions in animal cells.

The role of oxidative protein modification and the gluthatione system in modulation of the redox status of breast epithelial cells

The effects of the SH-group blocker N-ethylmaleimide (NEM) and thiol group protector 1,4-dithioerythritol (DTE) on the redox status of cells HBL-100 cells, oxidative modification of their proteins and the state of glutathione and thioredoxin systems have been investigated. Breast epithelial cells cultivated in the presence of NEM were characterized by decreased redox status, increased glutathione reductase activity, and increased concentrations of products of irreversible oxidative modification of protein and amino acids. Cultivation of HBL-100 cells in the presence of DTE resulted in a shift of the redox status towards reduction processes and increased reversible protein modification by glutathionylation. The proposed model of intracellular redox modulation may be used in the development of new therapeutic approaches to treat diseases accompanied by impaired redox homeostasis (e.g. oncologic, inflammatory, cardiovascular and neurodegenerative disease).

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein.

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP-Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

Structural Modifications of Gluten Proteins in Strong and Weak Wheat Dough During Mixing

ABSTRACTThe network‐forming attributes of gluten have been investigated for decades, but no study has comprehensively addressed the differences in gluten network evolution between strong and weak wheat types (hard and soft wheat). This study monitored changes in SDS protein extractability, SDS‐accessible thiols, protein surface hydrophobicity, molecular weight distribution, and secondary structural features of proteins during mixing to bring out the molecular determinants of protein network formation in hard and soft wheat dough. Soft wheat flour and dough exhibited greater protein extractability and more accessible thiols than hard wheat flour and dough. The addition of the thiol‐blocking agent N‐ethylmaleimide (NEM) resulted in similar results for protein extractability and accessible thiols in hard and soft wheat samples. Soft wheat dough had greater protein surface hydrophobicity than hard wheat and exhibited a larger decrease in surface hydrophobicity in the presence of NEM. Formation of high‐molecular‐weight (HMW) protein in soft wheat dough was primarily because of formation of disulfides among low‐molecular‐weight (LMW) proteins, as indicated by the absence of changes in protein distribution when NEM was present, whereas in hard wheat dough the LMW fraction formed disulfide interaction with the HMW fraction. Fourier transform infrared spectroscopy indicated formation of β‐sheets in dough from either wheat type at peak mixing torque. Formation of β‐sheets in soft wheat dough appears to be driven by hydrophobic interactions, whereas disulfide linkages stabilize secondary structure elements in hard wheat dough.

Are disulphide bonds formed during acid gelation of preheated milk?

Summary The role of thiol/disulphide exchanges during acid gelation of preheated milk was studied with milk samples with or without N-ethylmaleimide (NEM), a thiol-blocking agent, and acidified to pH 4 by the addition of glucono-delta-lactone at 20 °C. Active or total thiol groups, particle size with light scattering measurements in a dissociating solvent or by SDS-agarose electrophoresis were determined on acidified milk samples. Diffusing wave spectroscopy and rheology in low strain were applied during acidification of sample, while rheology in large strain was applied on final acid gels. The only effect of the presence of NEM was a reduced firmness of acid gels as measured at large strain and a reduced tendency to form large aggregates at pH<5.5. In conclusions, thiol/disulphide exchanges during acidification of milk played only a minor role in the building of acid gel networks from heated milk.

Analysis of Cationic Amino Acid Transport Activity in Canine Lens Epithelial Cells

Cationic amino acid transport activity in a canine lens epithelial cells (LEC) line was investigated. The transporter activity of arginine was 0.424 ± 0.047 nmol/mg protein min, while the presence of N-ethylmaleimide, an inhibitor of the canine cationic amino acid transporter (CAT), reduced transport activity by 30%. A full-length cDNA sequence of canine CAT1 was 2558 bp long and was predicted to encode the 629 amino acid polypeptides. The deduced amino acid sequence of canine CAT1 showed similarities of 92.1% and 88.6% to those of the human and mouse, respectively. Western blot analysis detected a band at 70 kDa in a membrane protein sample of LEC. RT-PCR analysis confirmed that CAT1 was ubiquitously detected in all tissues examined.

Rheological properties of acid-induced soy protein-stabilized emulsion gels in the absence and presence of N-ethylmaleimide

The storage modulus (G′) and fracture properties of the non-treated and NEM-treated emulsion gels were investigated in the absence and presence of unadsorbed soy protein aggregates (USPA). In the absence of USPA, a decrease in the G′ of emulsion gels was observed after NEM treatment. However, in the presence of USPA, the addition of NEM only slightly decreased the G′ (p < 0.05). For both non-treated and NEM-treated emulsions, a ∼63-folds higher G′ value was obtained after the USPA addition. These results revealed the presence of sulphydryl group – disulfide bond interchange reactions at ambient temperature and under acidic conditions. In the absence of USPA, the sulphydryl group – disulfide bond interchange reactions was the main interactions responsible for the higher G′ values of non-treated emulsion gels, but for the emulsions with USPA presented, the large quantity of non-covalent interactions (e.g. hydrophobic group & hydrogen bonds) is the main interactions responsible for the aggregation and gelation of emulsion droplets. In the presence of USPA, the sulphydryl group – disulfide bond interchange reactions formed in the non-treated emulsion gels did not increase the final G′ but increased the stability of network. A power law relation was observed between the USPA concentration and the final G′, as well as between the oil volume fraction and the fracture stress/strain.

Modulation of the ribonucleotide reductase M1–gemcitabine interaction in vivo by N-ethylmaleimide

Ribonucleotide reductase M1 (RRM1) is the regulatory subunit of the holoenzyme that catalyzes the conversion of ribonucleotides to 2′-deoxyribonucleotides. Its function is indispensible in cell proliferation and DNA repair. It also serves as a biomarker of therapeutic efficacy of the antimetabolite drug gemcitabine (2′,2′-difluoro-2′-deoxycytidine) in various malignancies. However, a mechanistic explanation remains to be determined. This study investigated how the alkylating agent N-ethylmaleimide (NEM) interacts with the inhibitory activity of gemcitabine on its target protein RRM1 in vivo. We found, when cells were treated with gemcitabine in the presence of NEM, a novel 110kDa band, along with the 90kDa native RRM1 band, appeared in immunoblots. This 110kDa band was identified as RRM1 by mass spectrometry (LC–MS/MS) and represented a conformational change resulting from covalent labeling by gemcitabine. It is specific to gemcitabine/NEM, among 11 other chemotherapy drugs tested. It was also detectable in human tumor xenografts in mice treated with gemcitabine. Among mutations of seven residues essential for RRM1 function, C218A, C429A, and E431A abolished the conformational change, while N427A, C787A, and C790A diminished it. C444A was unique since it was able to alter the conformation even in absence of gemcitabine treatment. We conclude that the thiol alkylator NEM can stabilize the gemcitabine-induced conformational change of RRM1, and this stabilized RRM1 conformation has the potential to serve as a specific biomarker of gemcitabine’s therapeutic efficacy.

Insulin‐stimulated L‐arginine transport requires SLC7A1 gene expression and is associated with human umbilical vein relaxation

Insulin causes endothelium-derived nitric oxide (NO)-dependent vascular relaxation, and increases L-arginine transport via cationic amino acid transporter 1 (hCAT-1) and endothelial NO synthase (eNOS) expression and activity in human umbilical vein endothelium (HUVEC). We studied insulin effect on SLC7A1 gene (hCAT-1) expression and hCAT-transport activity role in insulin-modulated human fetal vascular reactivity. HUVEC were used for L-arginine transport and L-[(3) H]citrulline formation (NOS activity) assays in absence or presence of N-ethylmaleimide (NEM) or L-lysine (L-arginine transport inhibitors). hCAT-1 protein abundance was estimated by Western blot, mRNA quantification by real time PCR, and SLC7A1 promoter activity by Luciferase activity (-1,606 and -650 bp promoter fragments from ATG). Specific protein 1 (Sp1), and total or phosphorylated eNOS protein was determined by Western blot. Sp1 activity (at four sites between -177 and -105 bp from ATG) was assayed by chromatin immunoprecipitation (ChIP) and vascular reactivity in umbilical vein rings. Insulin increased hCATs-L-arginine transport, maximal transport capacity (V(max) /K(m) ), and hCAT-1 expression. NEM and L-lysine blocked L-arginine transport. In addition, it was trans-stimulated (∼7.8-fold) by L-lysine in absence of insulin, but unaltered (~1.4-fold) in presence of insulin. Sp1 nuclear protein abundance and binding to DNA, and SLC7A1 promoter activity was increased by insulin. Insulin increased NO synthesis and caused endothelium-dependent vessel relaxation and reduced U46619-induced contraction, effects blocked by NEM and L-lysine, and dependent on extracellular L-arginine. We suggest that insulin induces human umbilical vein relaxation by increasing HUVEC L-arginine transport via hCATs (likely hCAT-1) most likely requiring Sp1-activated SLC7A1 expression.

Deubiquitylase, DeSUMOylase, and DeISGylase Activity Microarrays for Assay of Substrate Preference and Functional Modifiers

Microarray-based proteomics expanded the information potential of DNA arrays to the level of protein translation and interaction, but so far, not much beyond. Although enzymatic activity from immobilized proteins has been reliably studied using surface plasmon resonance, a microarray of catalytically competent enzymes would facilitate high throughput, parallel study of their function. The ability to localize activity from soluble substrates has frustrated development of such an array. Here, we report the novel use of previously developed, highly specific suicide substrates for three families of enzymes: deubiquitylases, deSUMOylases, and deISGylases. We show specificity of each family to its cognate substrate, and demonstrate utility of the array in a secondary screen of small molecule inhibitors.

Role of Plasma S-Nitrosothiols in Regulation of Blood Pressure in Anesthetized Rabbits with Special References to Hypotensive Effects of Acetylcholine and Nitrovasodilators

The regulatory role of plasma nitrosothiols (R-SNOs) under steady-state conditions and their possible contribution to pharmacological vasodilation were systematically examined in anesthetized rabbits. Nitrosocystein (Cys-NO), S-nitrosoglutathione (G-SNO), and S-nitrosoalbumin (Alb-SNO) were determined by HPLC-Saville's method with respective sensitivities of 1, 1, and 5 nM. These R-SNOs were not detected under steady-state conditions even in the presence of N-ethylmaleimide, a thiol protective agent used to prevent transnitrosation of R-SNOs. Development of plasma Alb-SNO below 300 nM was observed after intravenous injection (i.v.) of nitric oxide (NO) solution (0.1 to 3 ml/g), NOC7 (an NO releasing agent, above 1 µg/kg), and a low dose of Alb-SNO (10 nmol/kg). However, blood pressure was not significantly reduced by NO solution or Alb-SNO. Intravenous injection of a high dose of Alb-SNO (300 nmol/kg) significantly reduced blood pressure with the appearance not only Alb-SNO in micromolar level in plasma, but also G-SNO in lesser degree. Conversely, the hypotensive effect of Cys-NO (300 nmol/kg, i.v.) and G-SNO (300 nmol/kg, i.v.) accompanied development of Alb-SNO (micromolar level), but not Cys-NO or G-SNO in plasma. R-SNOs were not found in plasma during profound hypotension induced by acetylcholine (10 and 30 µg/kg/min, continuous i.v.), glyceryl trinitrate (100 µg/kg, i.v.), sodium nitroprusside (100 µg/kg, i.v.), and isosorbide dinitrate (300 µg/kg, i.v.). These results indicate that R-SNOs do not play an important role under unstimulated condition. In addition, plasma R-SNOs may not be involved in pharmacological vasodilation where contributions of NO or R-SNOs are suggested.

A novel disulphide switch mechanism in Ero1α balances ER oxidation in human cells

Oxidative maturation of secretory and membrane proteins in the endoplasmic reticulum (ER) is powered by Ero1 oxidases. To prevent cellular hyperoxidation, Ero1 activity can be regulated by intramolecular disulphide switches. Here, we determine the redox-driven shutdown mechanism of Ero1alpha, the housekeeping Ero1 enzyme in human cells. We show that functional silencing of Ero1alpha in cells arises from the formation of a disulphide bond-identified by mass spectrometry--between the active-site Cys(94) (connected to Cys(99) in the active enzyme) and Cys(131). Competition between substrate thiols and Cys(131) creates a feedback loop where activation of Ero1alpha is linked to the availability of its substrate, reduced protein disulphide isomerase (PDI). Overexpression of Ero1alpha-Cys131Ala or the isoform Ero1beta, which does not have an equivalent disulphide switch, leads to augmented ER oxidation. These data reveal a novel regulatory feedback system where PDI emerges as a central regulator of ER redox homoeostasis.

Human Herpesvirus 8 K1 Blocks Fas in Cells and Mice by Binding and Blocking Fas.

Background: Kaposi's sarcoma and primary effusion lymphoma are linked to human herpesvirus 8 (HHV-8 or Kaposi's sarcoma-associated virus) infection. Key to the pathogenesis of these cancers is the HHV-8 transmembrane oncoprotein K1. This viral protein, which immortalizes cells, produces lymphoproliferation and lymphomas through unknown mechanisms when expressed in mice.Results: We show here that K1 blocks Fas (CD95)-mediated apoptosis by binding the ectodomain of K1 to Fas in hematopoietic cancer cells (BJAB, U937 and THP-1). K1 bound to Fas in K1-transfected BJAB cells. To assess whether K1 and Fas formed complexes in cells, we chemically crosslinked proteins in transfected cells using 3,3′-dithiobissulfosuccinimidyl propionate in the presence of N-ethyl maleimide. Stabled K1-Fas complexes were present in intact cells despite blocking of free reactive cysteines. The selective ability of K1 to antagonize Fas-mediated apoptosis was strictly dependent on having its immunoreceptor tyrosine-based activation motif (ITAM). Using activating anti-Fas (CH-11) and nonactivating anti-Fas (B10) antibodies in a serial manner, we showed that K1 associated with Fas that was not activated, strongly suggesting that K1 prevents Fas from being activated. Mice transfected with K1 were protected from the apoptosis inducing effects of agonistic Jo2 anti-Fas antibody and tissues from these mice contained K1-Fas complexes. K1-transfected compared to vector-transfected mice had fewer terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive cells (13 ± 3% vs. 60 ± 7%, P=0.008), fewer activated caspase 3 positive cells (24 ± 5% vs. 88 ± 6%, P=0.006). Tissues had less hemorrhaging, and necrosis.Conclusion: These data support a mechanism of K1 direct binding to and blockage by K1 to Fas in suppression of apoptosis in an ITAM-dependent manner, which may be a key step in lymphoma development.

Both Sphingomyelin Synthases SMS1 and SMS2 Are Required for Sphingomyelin Homeostasis and Growth in Human HeLa Cells

Sphingomyelin (SM) is a vital component of cellular membranes in organisms ranging from mammals to protozoa. Its production involves the transfer of phosphocholine from phosphatidylcholine to ceramide, yielding diacylglycerol in the process. The mammalian genome encodes two known SM synthase (SMS) isoforms, SMS1 and SMS2. However, the relative contributions of these enzymes to SM production in mammalian cells remained to be established. Here we show that SMS1 and SMS2 are co-expressed in a variety of cell types and function as the key Golgi- and plasma membrane-associated SM synthases in human cervical carcinoma HeLa cells, respectively. RNA interference-mediated depletion of either SMS1 or SMS2 caused a substantial decrease in SM production levels, an accumulation of ceramides, and a block in cell growth. Although SMS-depleted cells displayed a reduced SM content, external addition of SM did not restore growth. These results indicate that the biological role of SM synthases goes beyond formation of SM.

Modulation of Voltage-Gated Ca2+Current in Vestibular Hair Cells by Nitric Oxide

The structural elements of the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signaling pathway have been described in the vestibular peripheral system. However, the functions of NO in the vestibular endorgans are still not clear. We evaluated the action of NO on the Ca(2+) currents in hair cells isolated from the semicircular canal crista ampullaris of the rat (P14-P18) by using the whole cell and perforated-cell patch-clamp technique. The NO donors 3-morpholinosydnonimine (SIN-1), sodium nitroprusside (SNP), and (+/-)-(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl-nicotinamide (NOR-4) inhibited the Ca(2+) current in hair cells in a voltage-independent manner. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) prevented the inhibitory effect of SNP on the Ca(2+) current. The selective inhibitor of the soluble form of the enzyme guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), also decreased the SNP-induced inhibition of the Ca(2+) current. The membrane-permeant cGMP analogue 8-Br-cGMP mimicked the SNP effect. KT-5823, a specific inhibitor of cGMP-dependent protein kinase (PGK), prevented the inhibition of the Ca(2+) current by SNP and 8-Br-cGMP. In the presence of N-ethylmaleimide (NEM), a sulfhydryl alkylating agent that prevents the S-nitrosylation reaction, the SNP effect on the Ca(2+) current was significantly diminished. These results demonstrated that NO inhibits in a voltage-independent manner the voltage-activated Ca(2+) current in rat vestibular hair cells by the activation of a cGMP-signaling pathway and through a direct action on the channel protein by a S-nitrosylation reaction. The inhibition of the Ca(2+) current by NO may contribute to the regulation of the intracellular Ca(2+) concentration and hair-cell synaptic transmission.