Effect of Thiol Reagents on Phosphoinositide Hydrolysis in Rat Brain Synaptoneurosomes

Vignes, Michel; Guiramand, Janique; Sassetti, Isabelle; Récasens, Max

doi:10.1111/j.1460-9568.1993.tb00500.x

Cited by 16 publications

(4 citation statements)

References 25 publications

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“…Mercury is known to inhibit enzyme activity by binding to sulfhydryl groups in cysteine or methionine residues often present at active sites. 25 Experiments with β-glucuronidase indicated that enzyme activity was unaffected in the presence of 100-fold higher molar concentration of mercury chloride than used for sample preservation (data not shown), consistent with the fact that the active site of this enzyme contains a glutamate residue, not a sulfhydryl group. 26 In the case of the effluent, there are indications that extracellular β-glucuronidase is produced by bacteria in wastewater during activated sludge treatment.…”

Section: Resultsmentioning

confidence: 57%

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Analysis of Steroid Conjugates in Sewage Influent and Effluent by Liquid Chromatography−Tandem Mass Spectrometry

2005

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Environmental endocrine disruptors such as estrone (E1) and beta-estradiol (E2) are excreted in human urine primarily as water-soluble glucuronides and sulfates that can dissociate in wastewater treatment systems to the more active free estrogens. Measurement of the distribution and fate of the steroid conjugates and the corresponding free estrogens in treatment plants and receiving waters is critical for understanding the reproductive and developmental effects of these substances on aquatic organisms. A sensitive method to measure steroid estrogen conjugates in matrix-rich sewage influents and effluents (method detection limits ranged from 0.04 to 0.28 ng/L) has been developed using HPLC tandem mass spectrometry with electrospray ionization. The method employs extensive sample purification by selective extraction from an Oasis HLB solid-phase cartridge followed by separation by anion exchange chromatography. This purification scheme, combined with a stable isotope dilution approach, was used to overcome problems of matrix suppression of ionization and permitted selective and sensitive detection of six target conjugates of E1 and E2. Accurate quantitation was highly dependent on the method of sample preservation. Acidification of each sample (pH 2.0) was effective in preventing enzymatic or chemical decomposition of steroid conjugates in all sample types, whereas glucuronide conjugates were hydrolyzed in the presence of mercury and formalin preservatives. Measured concentrations of steroid sulfates in the influent to a sewage treatment plant were approximately 100 times greater than that of the respective steroid glucuronides, suggesting that the preponderance of glucuronides had dissociated prior to reaching the treatment plant. A small percentage of the steroid sulfates persisted through biological treatment of sewage and was measured in the effluent. Steroid conjugates that survive decomposition or bypass biological treatment of municipal wastewater are released into surface waters and may serve as a source of free steroids.

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Section: Resultsmentioning

confidence: 57%

“…The inability of mercury to preserve glucuronide conjugates is consistent with the structure of glucuronidase enzymes that are likely responsible for the observed transformations. Mercury is known to inhibit enzyme activity by binding to sulfhydryl groups in cysteine or methionine residues often present at active sites …”

Section: Resultsmentioning

confidence: 99%

Analysis of Steroid Conjugates in Sewage Influent and Effluent by Liquid Chromatography−Tandem Mass Spectrometry

2005

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“…p-Chloromercuribenzoic acid (PCMB), an anionic probe of moderate size, caused the oocytes to swell and burst at a concentration of 0.2 mM (data not shown). This was attributed to the entry of PCMB into the oocytes by non-ionic diffusion and the subsequent inhibition of energy metabolism, since several steps in glycolysis and oxidative phosphorylation are known to be sensitive to sulphydryl reagents [25][26][27]. By contrast, p-chloromercuribenzosulfonate (PCMBS), a bulky anion believed to be membrane impermeant [14,28], did not cause the oocytes to burst and had little or no effect on wild-type CFTR-mediated Cl -currents, even at concentrations as high as 1.3 mM.…”

Section: +mentioning

confidence: 99%

Intracellular Cysteines of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulate Channel Gating

Ketchum¹,

Yue²,

Alessi³

et al. 2002

Cell Physiol Biochem

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The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette superfamily, is a cAMP-activated chloride channel. CFTR contains two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. We found that whole-cell CFTR-dependent Cl- currents in Xenopus laevis oocytes were sensitive to HgCl₂, suggesting that modification of endogenous cysteines alters channel activity. To understand better this phenomenon, site-directed mutagenesis was employed to generate both individual cysteine replacements and a version of the molecule with no cysteines in the hydrophobic sector. Each mutant displayed a forskolin/IBMX-activated Cl^- conductance similar to wild type, indicating that none of the cysteines located within the TMDs is essential. Subsequent single-channel analysis of inside-out patches excised from HEK293 cells expressing either cysteine-less or wild-type CFTR showed that intracellular application of a membrane impermeant sulphydryl reagent, p-chloromercuribenzosulfonate (PCMBS), significantly reduced open probability without affecting ion selectivity or conductance. The cysteine-less molecule also acquired a voltage-dependent sensitivity to extracellular PCMBS not observed in the wild type, perhaps due to a more flexible conformation that allowed PCMBS access to the intracellular surface. Together, these experiments suggest that endogenous intracellular cysteines, located primarily within the NBDs and/or R domain, influence channel gating.

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“…[Ca2+]i measurements were performed by loading synaptoneurosomes with fura2 (Grynkiewicz ef al., 1985), by incubation with 5 pM of fura2-AM and 0.02% p h o n i c during 30 min at 37°C as previously described (Vignes et al, 1993). The preparation was alternately excited at 340 and 380 nm using an optical chopper (300 Hz).…”

Section: [Cand+li Measurementsmentioning

confidence: 99%

Stimulation of Ca²⁺‐Activated Non‐specific Cationic Channels by Phospholipase C‐linked Glutamate Receptors in Synaptoneurosomes?

Vignes

Blanc

Récasens

1995

Eur J of Neuroscience

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The regulation of intracellular Ca2+ concentration ([Ca2+]i) by glutamate metabotropic receptors (mGluR) was studied in 8-day-old rat forebrain synaptoneurosomes using spectrofluorimetric methods. Here we demonstrate that metabotropic glutamate agonists induce in rat brain synaptoneurosomes a Ca2+ influx largely dependent upon the presence of Ca2+ in the external medium. The pharmacological profile of this influx is strongly correlated with the pharmacological profile of the activation of phosphoinositide hydrolysis, i.e. quisqualic acid >> 1S,3R-amino-1-dicarboxylate-1,3 cyclopentane approximately equal to glutamate. This metabotropic glutamate receptor-induced Ca2+ influx is insensitive to voltage-dependent Ca2+ channel antagonists and occurs through a Mn2+ impermeant pathway. The study of the rapid kinetics shows that this influx is triggered after a 300 ms delay compared with that elicited by depolarizing agents and Ca2+ ionophore A23187. In order to assess further if mGluR stimulate this influx through the recruitment of inositol triphosphate (IP3)-sensitive intracellular Ca2+ stores, we have tested the effect of thapsigargin on membrane potential and intracellular Ca2+ simultaneously. Thapsigargin induces a depolarization of the synaptoneurosomal membrane followed by a massive Ca2+ influx, occurring via a Mn2+ nonpermeant route. This depolarizing effect is sensitive to the presence of the intracellular Ca2+ chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetoxymethyl ester], and partially sensitive to extracellular Na+, but insensitive to the presence of extracellular Ca2+. Taken together, our data suggest that mGluR stimulate self-maintained increases of [Ca2+]i in rat forebrain synaptoneurosomes via the activation of a multistep mechanism, sequenced in the following steps: (i) mGluR-induced IP3 synthesis; (ii) IP3-stimulated intracellular Ca2+ release; (iii) Ca(2+)-activated non-specific cation channel, leading to local depolarization and a Ca2+ influx; and (iv) activation of Ca(2+)-sensitive phospholipase C.

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Effect of Thiol Reagents on Phosphoinositide Hydrolysis in Rat Brain Synaptoneurosomes

Cited by 16 publications

References 25 publications

Analysis of Steroid Conjugates in Sewage Influent and Effluent by Liquid Chromatography−Tandem Mass Spectrometry

Analysis of Steroid Conjugates in Sewage Influent and Effluent by Liquid Chromatography−Tandem Mass Spectrometry

Intracellular Cysteines of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulate Channel Gating

Stimulation of Ca²⁺‐Activated Non‐specific Cationic Channels by Phospholipase C‐linked Glutamate Receptors in Synaptoneurosomes?

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Effect of Thiol Reagents on Phosphoinositide Hydrolysis in Rat Brain Synaptoneurosomes

Cited by 16 publications

References 25 publications

Analysis of Steroid Conjugates in Sewage Influent and Effluent by Liquid Chromatography−Tandem Mass Spectrometry

Analysis of Steroid Conjugates in Sewage Influent and Effluent by Liquid Chromatography−Tandem Mass Spectrometry

Intracellular Cysteines of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulate Channel Gating

Stimulation of Ca2+‐Activated Non‐specific Cationic Channels by Phospholipase C‐linked Glutamate Receptors in Synaptoneurosomes?

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Stimulation of Ca²⁺‐Activated Non‐specific Cationic Channels by Phospholipase C‐linked Glutamate Receptors in Synaptoneurosomes?