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PubMed1977

Mercury and organomercurial resistances determined by plasmids in Pseudomonas.

Mercury and organomercurial resistance determined by genes on ten Pseudomonas aeruginosa plasmids and one Pseudomonas putida plasmid have been studied with regard to the range of substrates and the range of inducers. The plasmidless strains were sensitive to growth inhibition by Hg(2+) and did not volatilize Hg(0) from Hg(2+). A strain with plasmid RP1 (which does not confer resistance to Hg(2+)) similarly did not volatilize mercury. All 10 plasmids determine mercury resistance by way of an inducible enzyme system. Hg(2+) was reduced to Hg(0), which is insoluble in water and rapidly volatilizes from the growth medium. Plasmids pMG1, pMG2, R26, R933, R93-1, and pVS1 in P. aeruginosa and MER in P. putida conferred resistance to and the ability to volatilize mercury from Hg(2+), but strains with these plasmids were sensitive to and could not volatilize mercury from the organomercurials methylmercury, ethylmercury, phenylmercury, and thimerosal. These plasmids, in addition, conferred resistance to the organomercurials merbromin, p-hydroxymercuribenzoate, and fluorescein mercuric acetate. The other plasmids, FP2, R38, R3108, and pVS2, determined resistance to and decomposition of a range of organomercurials, including methylmercury, ethylmercury, phenylmercury, and thimerosal. These plasmids also conferred resistance to the organomercurials merbromin, p-hydroxymercuribenzoate, and fluorescein mercuric acetate by a mechanism not involving degradation. In all cases, organomercurial decomposition and mercury volatilization were induced by exposure to Hg(2+) or organomercurials. The plasmids differed in the relative efficacy of inducers. Hg(2+) resistance with strains that are organomercurial sensitive appeared to be induced preferentially by Hg(2+) and only poorly by organomercurials to which the cells are sensitive. However, the organomercurials p-hydroxymercuribenzoate, merbromin, and fluorescein mercuric acetate were strong gratuitous inducers but not substrates for the Hg(2+) volatilization system. With strains resistant to phenylmercury and thimerosal, these organomercurials were both inducers and substrates.

Chemical Phenomena↗
PubMed1991

[Intracellular Ca2+ mobilization and its regulation in platelet activation].

It has been generally accepted that platelets play etiological roles for the development of atherosclerosis and arterial thrombosis. Platelet activation may be dependent upon the cytosolic free Ca2+ concentration ([Ca2+]i), and regulated by PGI2 and endothelium-derived relaxing factor (EDRF) released by vascular endothelium. We have studied here the effect of endothelial cells (EC) on platelet activation and intracellular Ca2+ mobilization. Effluent of non-stimulated EC column inhibited thrombin-induced platelet aggregation and intracellular Ca2+ mobilization. An addition of this effluent to platelet suspension leaded to increase in intraplatelet cyclic AMP (cAMP) which was inhibited by the treatment of indomethacin to EC, suggesting that this effect was involved in PGI2 released by EC. On the other hand, effluent of thimerosal-stimulated EC column inhibited platelet aggregation and increase in [Ca2+]i stimulated with thrombin, and leaded to increase in intraplatelet cyclic GMP (cGMP). But the treatment of indomethacin to EC had no effect of this inhibition. The effect of thimerosal-stimulated EC was inhibited by the addition of 1-NG-monomethylarginine (NMA), EDRF/NO inhibitor, suggesting that EDRF released by thimerosal-stimulated EC produced an increase in cGMP and inhibited platelet activation. Although forskolin-induced in cAMP caused a marked prevention of inositol 1, 4, 5-trisphosphate (IP3) production stimulated with thrombin, 8-bromo cGMP and EDRF-induced increase in cGMP had no effect of IP3 production. An increase in cAMP and cGMP was considered to inhibit intracellular Ca2+ mobilization by different mechanisms in platelets.

Blood Platelets↗
PubMed1988

Sulfhydryl reagents as model substances for eicosanoid research.

The rate of eicosanoid synthesis is controlled by the availability of free arachidonic acid. Recently we showed that inhibition of reacylation of liberated fatty acids by the sulfhydryl group-blocking agent thimerosal increased prostaglandin as well as leukotriene synthesis. In order to gain further insight into the basic regulatory mechanisms controlling cellular eicosanoid production, the effects of different sulfhydryl reagents on prostaglandin E2 synthesis in the macrophage-like cell line P388D1 were investigated. As these cells exhibit an enormous acyl turnover of cellular phospholipids even under basal conditions, any interference with their fatty acid deacylation-reacylation cycle should lead to a change in intracellular concentrations of the eicosanoid precursor arachidonic acid. Therefore, arachidonic acid release from prelabelled cells as well as arachidonic acid incorporation into different phospholipid species under the influence of mercuric chloride (HgCl2), ethylmercurithiosalicylate (thimerosal), p-hydroxymercuribenzoic acid (PHMB), 0-(3-hydroxymercuri-3-methoxypropyl)carbamoyl-phenoxyacetic acid (mersalyl), N-ethylmaleimide (NEM), and ethacrynic acid were examined. Additionally, direct effects of those substances on corresponding enzyme activities, i.e. phospholipase A2 (PLA2), acyl-CoA synthetase, and lysophospholipid acyltransferase (LAT), on glutathione levels, and on cell viability were estimated. The results demonstrate that the organic mercury compound thimerosal was the most effective in enhancing free arachidonic acid levels, and concomitantly prostaglandin E2 synthesis in P388D1 cells, as result of a rather selective inhibition of the fatty acid reacylating enzyme LAT.

Acyltransferases↗
PubMed1978

Prevention of surface bacterial contamination of donor corneas.

A simple method has been developed to reduce contamination in postmortem donor human eyes in anticipation of corneal transplantation. In vivo investigation of albino rabbits demonstrates that vigorous saline solution irrigation is extremely effective in decreasing the surface bacterial counts of the postmortem eye. In vitro and in vivo studies show that Neosporin kills bacteria at room temperature and further show that a tenfold increase in the thimerosal concentration of the Neosporin will kill fungus. Postmortem eyes contaminated by pathogenic organisms can be effectively cleaned by a combination of saline solution irrigation and the new Neosporin-thimerosal solution. No substantial damage of the donor tissue was noted by scanning electron microscopy. Human eyes cultured before this procedure were all contaminated, but after cleansing and immersion, no bacterial or fungal growth occurred.

Animals↗
PubMed1980

Conjunctival hyperemia and corneal infiltrates with chemically disinfected soft contact lenses.

For three cosmetic soft contact lens wearers who chemically disinfected their lenses, conjunctival hyperemia and anterior stromal infiltrates of the cornea developed. When the conjunctival hyperemia and corneal infiltrates resolved, two of the three patients were challenged with their chemically disinfected soft contact lenses, with reappearance of the conjunctival and corneal reactions. Bacterial cultures of the conjunctiva, lids, lens cases, lens solutions, and eye cosmetics were not helpful. Conjunctival cultures for adenovirus and chlamytdial titers were negative. Giemsa stain of conjunctival scrapings showed a few mononuclear cells, polymorphonuclear leukocytes, and rare eosinophils but no inclusion bodies. Occlusive patch tests and intradermal tests showed positive delayed hypersensitivity reactions to the chemical disinfectants in all three patients. Occlusive patch tests with thimerosal suggested that this preservative was responsible for the reactions. Delayed hypersensitivity to thimerosal may play a role in the development of the conjunctival hyperemia and corneal infiltrates described in these patients.

Adolescent↗
PubMed2001

Impact of recommendations to suspend the birth dose of hepatitis B virus vaccine.

CONTEXT: In July 1999, due to concerns about thimerosal content, the American Academy of Pediatrics (AAP) and the Public Health Service (PHS) recommended suspending hepatitis B virus (HBV) vaccination at birth except for mothers who had positive or unknown hepatitis B surface antigen (HBsAg) status. In September 1999, the Centers for Disease Control and Prevention recommended that hospitals resume HBV vaccination at birth with a new thimerosal-free vaccine. Whether the 2 changes in recommendations within 3 months led to less-than-optimal compliance in hospital nurseries is unknown. OBJECTIVE: To determine hospital HBV vaccination policy before the recommendation for delay of HBV vaccination and 1 year later. DESIGN, SETTING, AND PARTICIPANTS: Survey of all 46 hospitals with obstetric services and neonatal nurseries in Cook County, Illinois. MAIN OUTCOME MEASURES: Hepatitis B virus immunization practices before July 1999 and in August 2000; hospital factors associated with routine HBV immunization and compliance with AAP and PHS recommendations. RESULTS: Before July 1999, 74% of surveyed hospital nurseries offered HBV vaccine to all neonates; only 39% did so in August 2000. Being located in the Chicago city limits (88% vs 57%; P =.02) and having an academic affiliation (93% vs 66%; P =.05) were positively associated with routine neonatal immunization before July 1999. Both academic affiliation and city location were associated with routine immunization in August 2000 (71% vs 25% [P =.003] and 60% vs 14% [P =.002], respectively) and with compliance with recommendations for suspension (57% vs 25% [P =.03] and 56% vs 10% [P =.001]). CONCLUSIONS: We documented a 35% decrease in hospital nurseries that routinely offered HBV immunization 1 year after the AAP and PHS recommendations were made. Special efforts may be required to make at-birth administration of HBV vaccination universal.

Guideline Adherence↗
PubMed2001

Porcine oocyte activation: differing roles of calcium and pH.

Intracellular pH has recently been shown to increase during parthenogenetic activation of the porcine oocyte. In the following set of experiments, intracellular pH was monitored during activation and pronuclear development was assessed following activation treatments with calcium, in the absence of calcium, and in oocytes loaded with the calcium chelator BAPTA-AM in calcium-free medium. Intracellular pH increase was not different among groups when treating with 7% ethanol or 50 microM calcium ionophore, or during treatment with thimerosal for 12 or 25 min. Activation with thimerosal (200 microM, 12 min) followed by 8 mM dithiothreitol (DTT, 30 min) resulted in a decreased pronuclear development in calcium-free medium with or without BAPTA-AM loaded oocytes as compared to controls. Activation with 50 microM calcium ionophore resulted in pronuclear development that was different between the calcium-free and BAPTA-AM loaded oocytes in calcium-free medium. Similar incidences of pronuclear formation were observed in all ethanol treatment groups. It was concluded that external calcium as well as large changes in intracellular free calcium are not necessary for the increase in intracellular pH, but normal intracellular calcium signaling is critical for normal levels of pronuclear development. Finally, oocytes were measured for intracellular pH changes for 30 min following subzonal sperm injection. Intracellular pH did not increase, although pronuclear formation was observed 6 hr post SUZI. This suggested that major differences were still present between sperm-induced and parthenogenetic activation of the porcine oocyte.

Animals↗
PubMed1999

Incompetence of preovulatory mouse oocytes to undergo cortical granule exocytosis following induced calcium oscillations.

Immature oocytes of many species are incompetent to undergo cortical granule (CG) exocytosis upon fertilization. In mouse eggs, CG exocytosis is dependent primarily on an inositol 1,4,5-trisphosphate (IP3)-mediated elevation of intracellular calcium ([Ca2+]i). While deficiencies upstream of [Ca2+]i release are known, this study examined whether downstream deficiencies also contribute to the incompetence of preovulatory mouse oocytes to release CGs. The experimental strategy was to bypass upstream deficiencies by inducing normal, fertilization-like [Ca2+]i oscillations in fully grown, germinal vesicle (GV) stage oocytes and determine if the extent of CG exocytosis was restored to levels observed in mature, metaphase II (MII)-stage eggs. Because IP3 does not stimulate a normal Ca2+ response in GV-stage oocytes, three alternate methods were used to induce oscillations: thimerosal treatment, electroporation, and sperm factor injection. Long-lasting oscillations from thimerosal treatment resulted in 64 and 10% mean CG release at the MII and GV stages, respectively (P < 0.001). Three electrical pulses induced mean [Ca2+]i elevations of approximately 730 and 650 nM in MII- and GV-stage oocytes, respectively, and 31% CG release in MII-stage eggs and 9% in GV-stage oocytes (P < 0.001). Sperm factor microinjection resulted in 86% CG release in MII-stage eggs, while similarly treated GV-stage oocytes exhibited < 1% CG release (P < 0.001). Taken together, these results demonstrate a deficiency downstream of [Ca2+]i release which is developmentally regulated in the 12 h prior to ovulation.

Animals↗
PubMed1992

Elevated glucose alters A23187-induced release of arachidonic acid from porcine aortic endothelial cells by enhancing reacylation.

Cultured porcine aortic endothelial cells were conditioned in normal (5.2 mM) and elevated (15.6 mM) glucose, prelabeled with [14C]arachidonic acid and stimulated with ionophore A23187. Elevated glucose cultures released less radiolabeled products and less [14C]arachidonic acid. Analysis of cellular lipids revealed that elevated glucose reduced net loss of radiolabel from diacylphosphatidylethanolamine, did not affect early phosphatidylinositol hydrolysis, and increased net loss from diacylphosphatidylcholine and alkenylacylphosphatidylethanolamine. Uptake of radiolabel upon stimulation was examined to measure the role of reacylation on the diminished net release of radiolabel in elevated glucose cultures. Enhanced acylation of [3H]arachidonic acid into cellular lipids, especially PI, was observed in stimulated and resting cultures with elevated glucose. Further, pretreatment of the cultures with an acyltransferase inhibitor, thimerosal, prior to A23187 stimulation in radiolabeled cultures, abolished the effects of glucose on eicosanoid and arachidonic acid release. Differences in the ionophore-induced net loss of radiolabel from diacylphosphatidylethanolamine and phosphatidylinositol of the two glucose treatments were diminished by thimerosal exposure, while net loss of radiolabel from diacylphosphatidylcholine and alkenylacylphosphatidylethanolamine were unaffected. The data indicate that elevated glucose alters deacylation and enhances reacylation of arachidonic acid into endothelial cells and particularly into phosphatidylinositol. Enhanced reacylation may explain some of the altered lipid pathways that have been observed in experiments that elevate glucose concentrations or involve diabetes.

Acylation↗
PubMed1993

Sulfhydryl oxidation induces rapid and reversible closure of the ATP-regulated K+ channel in the pancreatic beta-cell.

Effects of sulfhydryl modification on the ATP regulated K+ channel (KATP channel) in the pancreatic beta-cell were studied, using the patch clamp technique. Application of the sulfhydryl oxidizing agents thimerosal and 2,2'-dithio-bis(5-nitropyridine) (DTBNP), in micromolar concentrations, caused complete inhibition of the KATP channel, in inside-out patches. The inhibition was rapid and was reversed by the disulfide reducing agents dithiothreitol and cysteine. Thimerosal, which is poorly membrane permeable, inhibited channel activity, only when applied to the intracellular face of the plasma membrane. In contrast, DTBNP, which is highly lipophilic, caused closure of the KATP channel and consequent depolarization of the membrane potential, also when applied extracellularly. Our results indicate the presence of accessible free SH groups on the cytoplasmic side of the KATP channel in the pancreatic beta-cell. These SH groups are essential for channel function and it is possible that thiol-dependent redox mechanisms can modulate KATP channel activity.

Adenosine Triphosphate↗
PubMed1993

A comparison of two in vitro mammalian cell cytogenetic assays for the detection of mitotic aneuploidy using 10 known or suspected aneugens.

Two in vitro cytogenetic assays were evaluated for their ability to detect aneugenic and polyploidy-inducing agents using a battery of 10 known or suspected aneugens supplied as part of the EEC 4th Environmental Research and Development Programme. The compounds tested were colchicine, vinblastine, chloral hydrate, thiabendazole, hydroquinone, thimerosal, cadmium chloride, econazole nitrate, pyrimethamine and diazepam. The cell division aberration assay employed a differential chromosome/spindle staining procedure to detect perturbations of the mitotic division apparatus. This assay was carried out in two pulmonary-derived Chinese hamster cell lines; the immortal DON:Wg3h culture and a low passage LUC2 culture. The second assay involved quantification of metaphase chromosomes, for which only the LUC2 cell line was used, due to the stability of its diploid karyotype. All the chemicals induced spindle disturbances in the immortal line. In addition, all the compounds except cadmium chloride yielded positive results in the LUC2 culture, although many were not as potent. In the low passage line, 8 of the compounds (colchicine, vinblastine, chloral hydrate, thiabendazole, thimerosal, econazole nitrate, pyrimethamine and diazepam) induced aneuploidy and/or tetraploidy. Cadmium chloride was negative in the chromosome enumeration assay and hydroquinone yielded inconclusive results. The study of cell division aberrations was much less time-consuming and technically complex than the counting of metaphase chromosomes. In addition, it provided a degree of mechanistic understanding of the mode of action of some aneugenic and polyploidy-producing agents. However, the enumeration of chromosomes provides a more definitive data set for the evaluation of a chemical's aneugenic potential.

Aneuploidy↗
PubMed1987

The effects of different inactivating agents on the potency, toxicity and stability of pertussis vaccine.

The effect of heat (56 degrees C for 10 min), formaldehyde (0.1% at 37 degrees for 24h), glutaraldehyde (0.05% at room temperature for 10 min), thimerosal (0.02% at 37 degrees C for 24h), acetone-I (three treatments at room temperature) and acetone-II (three treatments at room temperature and fourth treatment at 37 degrees C), when used as inactivating agents in the preparation of pertussis suspension, was studied with regard to potency, toxicity and stability. Five batches each of Bordetella pertussis strains 134 and 509 were used for the study. The thimerosal inactivated pertussis (TIP) preparation was 1.5-2 times more potent than the heat inactivated pertussis (HIP) preparation. The potency values of the formaldehyde inactivated pertussis (TIP) and glutaraldehyde inactivated pertussis (GIP) preparations were similar to those of the HIP preparation, while the potencies of the acetone-I treated pertussis (A(I)TP) and acetone-II treated pertussis (A(II)TP) preparations were about half those of the HIP preparation. The FIP preparation was the least toxic showing maximum weight gain in the mouse weight-gain test (MGWT), while the TIP preparation did not pass the MWGT. The weight gains shown the GIP, A(I)TP and A(II)TP preparations were greater than those shown by the HIP preparation. The potency of pertussis component in the adsorbed diphtheria-pertussis-tetanus (DPT) vaccine was stable at 4-8 degrees C and 25 degrees C for three months for all types of pertussis vaccine. There was about 54-65% loss in the potency of the samples after three months at 35 degrees C. The inactivating agents used in the manufacture of pertussis preparations had no effect on the stability of the vaccine.

Acetone↗
PubMed2004

Immunizations, immunology, and autism.

Public fears of rising rates of children being diagnosed with autistic spectrum disorders has led to a fear that immunizations, specifically the measles-mumps-varicella vaccine (MMR), may trigger autism. This article reviews theories of immunization as a risk factor for autism, including thimerosal exposure. We also review theories of autoimmunity as a predisposing genetic risk in autistic patients. We summarize from multiple population-based studies and extensive review committee reports that neither immunization nor thimerosal exposure has been conclusively linked to autism. Current treatments for autoimmunity in autism are reviewed and summarized as being only anecdotally effective, with no controlled studies to conclusively determine effectiveness. The goal of this article is to allow child neurologists to effectively counsel parents of autistic patients about vaccination risks and treatment options in presumed cases of autoimmune dysfunction.

Autistic Disorder↗
PubMed2006

Dose and Hg species determine the T-helper cell activation in murine autoimmunity.

Inorganic mercury (mercuric chloride--HgCl(2)) induces in mice an autoimmune syndrome (HgIA) with T cell-dependent polyclonal B cell activation and hypergammaglobulinemia, dose- and H-2-dependent production of autoantibodies targeting the 34 kDa nucleolar protein fibrillarin (AFA), and systemic immune-complex deposits. The organic mercury species methylmercury (MeHg) and ethylmercury (EtHg--in the form of thimerosal) induce AFA, while the other manifestations of HgIA seen after treatment with HgCl(2) are present to varying extent. Since these organic Hg species are converted to the autoimmunogen Hg(2+) in the body, their primary autoimmunogen potential is uncertain and the subject of this study. A moderate dose of HgCl(2) (8 mg/L drinking water--internal dose 148 micro gHg/kg body weight [bw]/day) caused the fastest AFA response, while the induction was delayed after higher (25 mg/L) and lower (1.5 and 3 mg/L) doses. The lowest dose of HgCl(2) inducing AFA was 1.5 mg/L drinking water which corresponded to a renal Hg(2+) concentration of 0.53 micro g/g. Using a dose of 8 mg HgCl(2)/L this threshold concentration was reached within 24 h, and a consistent AFA response developed after 8-10 days. The time lag for the immunological part of the reaction leading to a consistent AFA response was therefore 7-9 days. A dose of thimerosal close to the threshold dose for induction of AFA (2 mg/L drinking water--internal dose 118 micro gHg/kg bw per day), caused a renal Hg(2+) concentration of 1.8 micro g/g. The autoimmunogen effect of EtHg might therefore be entirely due to Hg(2+) formed from EtHg in the body. The effect of organic and inorganic Hg species on T-helper type 1 and type 2 cells during induction of AFA was assessed as the presence and titre of AFA of the IgG1 and IgG2a isotype, respectively. EtHg induced a persistent Th1-skewed response irrespectively of the dose and time used. A low daily dose of HgCl(2) (1.5-3 mg/L) caused a Th1-skewed AFA response, while a moderate dose (8 mg/L) after 2 weeks resulted in a balanced or even Th2-skewed response. Higher daily doses of HgCl(2) (25 mg/L) caused a balanced Th2-Th1 response already from onset. In conclusion, while metabolically formed Hg(2+) might be the main AFA-inducing factor also after treatment with EtHg, the quality of the Hg-induced AFA response is modified by the species of Hg as well as the dose.

Animals↗
PubMed2006

Contributions of extracellular and intracellular Ca2+ to regulation of sperm motility: Release of intracellular stores can hyperactivate CatSper1 and CatSper2 null sperm.

In order to fertilize, mammalian sperm must hyperactivate. Hyperactivation is triggered by increased flagellar Ca(2+), which switches flagellar beating from a symmetrical to an asymmetrical pattern by increasing bending to one side. Thimerosal, which releases Ca(2+) from internal stores, induced hyperactivation in mouse sperm within seconds, even when extracellular Ca(2+) was buffered with BAPTA to approximately 30 nM. In sperm from CatSper1 or CatSper2 null mice, which lack functional flagellar alkaline-activated calcium currents, 50 microM thimerosal raised the flagellar bend amplitudes from abnormally low levels to normal pre-hyperactivated levels and, in 20-40% of sperm, induced hyperactivation. Addition of 1 mM Ni(2+) diminished the response. This suggests that intracellular Ca(2+) is abnormally low in the null sperm flagella. When intracellular Ca(2+) was reduced by BAPTA-AM in wild-type sperm, they exhibited flagellar beat patterns more closely resembling those of null sperm. Altogether, these results indicate that extracellular Ca(2+) is required to supplement store-released Ca(2+) to produce maximal and sustained hyperactivation and that CatSper1 and CatSper2 are key elements of the major Ca(2+) entry pathways that support not only hyperactivated motility but possibly also normal pre-hyperactivated motility.

Animals↗
PubMed1997

Effect of membrane-permeable sulfhydryl reagents and depletion of glutathione on calcium mobilisation in human platelets.

Exposure to peroxides is known to increase the sensitivity of platelets towards activation by agonists. Similar platelet-activating effects are induced by sulfhydryl reagents that evoke Ca2+-induced Ca2+ release (CICR) by stimulating the Ca2+-releasing property of the inositol-1,4,5-trisphosphate receptor. We questioned whether these compounds may act by mobilising intracellular calcium in platelets by altering the intracellular glutathione redox state. Using FURA2-loaded, aspirin-treated platelets, Ca2+ signals were studied following exposure to the membrane-permeable sulfhydryl reagents, thimerosal and disulfiram, the glutathione peroxidase substrate, tert-butyl hydroperoxide, and the inhibitor of glutathione reductase, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In single platelets monitored by fluorescence imaging techniques, thimerosal and disulfiram elicited repetitive spiking in [Ca2+]i after variable lag times, indicating that these compounds stimulated CICR. BCNU caused [Ca2+]i spiking of only low amplitude, whereas tert-butyl hydroperoxide was inactive. In platelets in suspension devoid of extracellular CaCl2, the sulfhydryl reagents, at concentrations which decreased glutathione by 25%, strongly increased the Ca2+ responses of agonists that stimulated phospholipase C (thrombin) or acted independently of phospholipase C stimulation (thapsigargin). However, Ca2+ release was only slightly promoted by concentrations of BCNU that resulted in substantial depletion of the glutathione level. Tert-butyl hydroperoxide was without effect on glutathione, but partially inhibited Ca2+ mobilisation with these agonists. It is concluded that, in platelets, the potent CICR-promoting effects of sulfhydryl reagents are not solely due to their reaction with intracellular glutathione, but that extensive reduction in glutathione content is associated with Ca2+ mobilisation and CICR.

Blood Platelets↗
PubMed2000

Sulfhydryl oxidation overrides Mg(2+) inhibition of calcium-induced calcium release in skeletal muscle triads.

We studied the effect of oxidation of sulfhydryl (SH) residues on the inhibition by Mg(2+) of calcium-induced calcium release (CICR) in triad-enriched sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. Vesicles were either passively or actively loaded with calcium before eliciting CICR by dilution at pCa 4.6-4.4 in the presence of 1.2 mM free [ATP] and variable free [Mg(2+)]. Native triads exhibited a significant inhibition of CICR by Mg(2+), with a K(0.5) approximately 50 microM. Partial oxidation of vesicles with thimerosal produced a significant increase of release rate constants and initial release rates at all [Mg(2+)] tested (up to 1 mM), and shifted the K(0.5) value for Mg(2+) inhibition to 101 or 137 microM in triads actively or passively loaded with calcium, respectively. Further oxidation of vesicles with thimerosal completely suppressed the inhibitory effect of [Mg(2+)] on CICR, yielding initial rates of CICR of 2 micromol/(mg x s) in the presence of 1 mM free [Mg(2+)]. These effects of oxidation on CICR were fully reversed by SH reducing agents. We propose that oxidation of calcium release channels, by decreasing markedly the affinity of the channel inhibitory site for Mg(2+), makes CICR possible in skeletal muscle.

Adenosine Triphosphate↗
PubMed2000

Block of sodium channels by divalent mercury: role of specific cysteinyl residues in the P-loop region.

Divalent mercury (Hg(2+)) blocked human skeletal Na(+) channels (hSkM1) in a stable dose-dependent manner (K(d) = 0.96 microM) in the absence of reducing agent. Dithiothreitol (DTT) significantly prevented Hg(2+) block of hSkM1, and Hg(2+) block was also readily reversed by DTT. Both thimerosal and 2,2'-dithiodipyridine had little effect on hSkM1; however, pretreatment with thimerosal attenuated Hg(2+) block of hSkM1. Y401C+E758C rat skeletal muscle Na(+) channels (mu1) that form a disulfide bond spontaneously between two cysteines at the 401 and 758 positions showed a significantly lower sensitivity to Hg(2+) (K(d) = 18 microM). However, Y401C+E758C mu1 after reduction with DTT had a significantly higher sensitivity to Hg(2+) (K(d) = 0.36 microM) than wild-type hSkM1. Mutants C753Amu1 (K(d) = 8.47 microM) or C1521A mu1 (K(d) = 8.63 microM) exhibited significantly lower sensitivity to Hg(2+) than did wild-type hSkM1, suggesting that these two conserved cysteinyl residues of the P-loop region may play an important role in the Hg(2+) block of the hSkM1 isoform. The heart Na(+) channel (hH1) was significantly more sensitive to low-dose Hg(2+) (K(d) = 0.43 microM) than was hSkM1. The C373Y hH1 mutant exhibited higher resistance (K(d) = 1.12 microM) to Hg(2+) than did wild-type hH1. In summary, Hg(2+) probably inhibits the muscle Na(+) channels at more than one cysteinyl residue in the Na(+) channel P-loop region. Hg(2+) exhibits a lower K(d) value (<1. 23 microM) for inhibition by forming a sulfur-Hg-sulfur bridge, as compared to reaction at a single cysteinyl residue with a higher K(d) value (>8.47 microM) by forming sulfur-Hg(+) covalently. The heart Na(+) channel isoform with more than two cysteinyl residues in the P-loop region exhibits an extremely high sensitivity (K(d) < 0. 43 microM) to Hg(+), accounting for heart-specific high sensitivity to the divalent mercury.

2,2'-Dipyridyl↗