An isothiourea derivative (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methane sulfonate (KB-R7943), a widely used inhibitor of the reverse Na + /Ca 2+ exchanger (NCXrev), was instrumental in establishing the role of NCXrev in glutamate-induced Ca 2+ deregulation in neurons. Here, the effects of KB-R7943 on N-methyl-D-aspartate (NMDA) receptors and mitochondrial complex I were tested. EXPERIMENTAL APPROACHFluorescence microscopy, electrophysiological patch-clamp techniques and cellular respirometry with Seahorse XF24 analyzer were used with cultured hippocampal neurons; membrane potential imaging, respirometry and Ca 2+ flux measurements were made in isolated rat brain mitochondria. KEY RESULTSKB-R7943 inhibited NCXrev with IC50 = 5.7 Ϯ 2.1 mM, blocked NMDAR-mediated ion currents, and inhibited NMDA-induced increase in cytosolic Ca 2+ with IC50 = 13.4 Ϯ 3.6 mM but accelerated calcium deregulation and mitochondrial depolarization in glutamate-treated neurons. KB-R7943 depolarized mitochondria in a Ca 2+ -independent manner. Stimulation of NMDA receptors caused NAD(P)H oxidation that was coupled or uncoupled from ATP synthesis depending on the presence of Ca 2+ in the bath solution. KB-R7943, or rotenone, increased NAD(P)H autofluorescence under resting conditions and suppressed NAD(P)H oxidation following glutamate application. KB-R7943 inhibited 2,4-dinitrophenol-stimulated respiration of cultured neurons with IC50 = 11.4 Ϯ 2.4 mM. With isolated brain mitochondria, KB-R7943 inhibited respiration, depolarized organelles and suppressed Ca 2+ uptake when mitochondria oxidized complex I substrates but was ineffective when mitochondria were supplied with succinate, a complex II substrate. CONCLUSIONS AND IMPLICATIONSKB-R7943, in addition to NCXrev, blocked NMDA receptors in cultured hippocampal neurons and inhibited complex I in the mitochondrial respiratory chain. These findings are critical for the correct interpretation of experimental results obtained with KB-R7943 and a better understanding of its neuroprotective action. AbbreviationsDy, mitochondrial membrane potential; [Ca 2+ ]c, cytosolic Ca 2+ concentration; CNQX, 6-cyano-7-nitroquinoxaline-2, 3-dione; DMSO, dimethyl sulfoxide; FCCP, carbonylcyanide-p-trifluoromethoxyphenylhydrazone; phenyl]ethyl]isothiourea methane sulfonate; MK801 (5R,10S)-(+)-5-methyl-10,11-dihydro-BJP British Journal of Pharmacology DOI:10.1111DOI:10. /j.1476DOI:10. -5381.2010 British Journal of Pharmacology (2011) IntroductionIn neurons exposed to glutamate, prolonged stimulation of glutamate receptors, particularly the N-methyl-D-aspartate (NMDA) subtype, results in massive Ca 2+ influx into the cell, disruption of calcium homeostasis and eventual cell death (Choi, 1988;Manev et al. 1989 (Blaustein and Lederer, 1999). Predictably, inhibition of NCX forward mode by removing external Na + leads to increased [Ca 2+ ]c (Mattson et al., 1989). However, under conditions of prolonged exposure to glutamate or NMDA, when cytosolic Na + is elevated and the plasma membrane is depol...
Glutamate-induced delayed calcium dysregulation (DCD) is a causal factor leading to neuronal death. The mechanism of DCD is not clear but Ca2+ influx via N-methyl-D-aspartate receptors (NMDAR) and/or the reverse plasmalemmal Na+/Ca2+ exchanger (NCXrev) could be involved in DCD. However, the extent to which NMDAR and NCXrev contribute to glutamate-induced DCD is uncertain. Here, we show that both NMDAR and NCXrev are critical for DCD in neurons exposed to excitotoxic glutamate. In rat cultured hippocampal neurons, 25μM glutamate produced DCD accompanied by sustained increase in cytosolic Na+ ([Na+]c) and plasma membrane depolarization. MK801 and memantine, noncompetitive NMDAR inhibitors, added shortly after glutamate, completely prevented DCD whereas AP-5, a competitive NMDAR inhibitor, failed to protect against DCD. None of the tested inhibitors lowered elevated [Na+]c or restored plasma membrane potential. In the experiments with NCX reversal by gramicidin, MK801 and memantine robustly inhibited NCXrev while AP-5 was much less efficacious. In electrophysiological patch-clamp experiments MK801 and memantine inhibited NCXrev-mediated ion currents whereas AP-5 failed. Thus, MK801 and memantine, in addition to NMDAR, inhibited NCXrev. Inhibition of NCXrev either with KB-R7943, or by collapsing Na+ gradient across the plasma membrane, or by inhibiting Na+/H+ exchanger with 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and thus preventing the increase in [Na+]c failed to preclude DCD. However, NCXrev inhibition combined with NMDAR blockade by AP-5 completely prevented DCD. Overall, our data suggest that both NMDAR and NCXrev are essential for DCD in glutamate-exposed neurons and inhibition of individual mechanism is not sufficient to prevent calcium dysregulation.
Study Objective The two antidotes for acute cyanide poisoning in the United States must be given by intravenous injection. In the pre-hospital setting, intravenous injection is not practical, particularly for mass casualties, and intramuscular injection would be preferred. The purpose of this study was to determine if sodium nitrite and sodium thiosulfate are effective cyanide antidotes when given by intramuscular injection. Methods We used a randomized, non-blinded, parallel group study design in three mammalian models: cyanide gas inhalation in mice, with treatment post exposure; intravenous sodium cyanide infusion in rabbits, with severe hypotension as the trigger for treatment; and intravenous potassium cyanide infusion in pigs, with apnea as the trigger for treatment. The drugs were administered by intramuscular injection, and all three models were lethal in the absence of therapy. Results We found that sodium nitrite and sodium thiosulfate individually rescued 100% of the mice, and that the combination of the two drugs rescued 73% of the rabbits and 80% of the pigs. In all three species, survival in treated animals was significantly better than in control animals (log rank test, p < 0.05). In the pigs, the drugs attenuated a rise in the plasma lactate concentration within five minutes post-antidote injection (difference: plasma lactate, saline-versus nitrite-thiosulfate-treated 1.76 [95% confidence interval 1.25-2.27]). Conclusion We conclude that sodium nitrite and sodium thiosulfate given by intramuscular injection are effective against severe cyanide poisoning in three clinically relevant animal models of prehospital emergency care.
Glutamate-induced delayed calcium dysregulation (DCD) is causally linked to excitotoxic neuronal death. The mechanisms of DCD are not completely understood, but it has been proposed that the excessive influx of external Ca2+ is essential for DCD. The NMDA-subtype of glutamate receptor (NMDAR) and the plasmalemmal Na+/Ca2+ exchanger operating in the reverse mode (NCXrev) have been implicated in DCD. In experiments with “younger” neurons, 6-8 days in vitro (6-8 DIV), in which the NR2A-containing NMDAR expression is low, ifenprodil, an inhibitor of NR2B-containing NMDAR, completely prevented DCD whereas PEAQX, another NMDAR antagonist that preferentially interacts with NR2A-NMDAR, was without effect. With “older” neurons (13-16 DIV), in which NR2A- and NR2B-NMDARs are expressed to a greater extent, both ifenprodil and PEAQX applied separately failed to prevent DCD. However, combined application of ifenprodil and PEAQX completely averted DCD. Ifenprodil and ifenprodil-like NR2B-NMDAR antagonists Ro 25-6981 and Co 101244 but not PEAQX or AP-5 inhibited gramicidin- and Na+/NMDG-replacement-induced increases in cytosolic Ca2+ mediated predominantly by NCXrev. This suggests that ifenprodil, Ro 25-6981, and Co 101244 inhibit NCXrev. The ability of ifenprodil to inhibit NCXrev correlates with its efficacy in preventing DCD and emphasizes an important role of NCXrev in DCD. Overall our data suggest that both NR2A- and NR2B-NMDARs are involved in DCD in “older” neurons, and it is necessary to inhibit both NMDARs and NCXrev to prevent glutamate-induced DCD.
QuEChERS has been widely utilized for the analysis of pesticides in produce, but it has not been as widely used in clinical test specimens, especially for smaller, sub-gram sample sizes. This study describes the application of a miniaturized QuEChERS methodology toward the analysis of two insecticides, methomyl and aldicarb, in guinea pig blood and brain tissue. Matrix effects and absolute recoveries were investigated for both analytes in the two matrices. While the matrix effects of methomyl in both matrices were minimal at most levels (i.e., from −20% to 20%), aldicarb experienced signal suppression under the described conditions (mean of −47%). However, the matrix effects were not cause for concern due to the sensitivity of the method and the use of matrix-matched standards. The precision and accuracy of the method were excellent over a range of concentrations that spanned three orders of magnitude. The limits of detection (LOD) for both carbamates were determined to be 0.1 ng mL−1 in blood and 0.2 ng g−1 in brain. Other validation parameters, such as linearity, accuracy, precision, and recovery, were also satisfactory in the blood and brain tissue. This method was demonstrated to be sensitive and reproducible, and it should be applicable to the analysis of a wide range of compounds of interest in sub-gram- and sub-milliliter-sized clinical and toxicology specimens.
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