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British Journal of Pharmacology Nov 19971. Glutamate receptor activation has been previously shown to result in mitochondrial depolarization and activation of the mitochondrial permeability transition pore in...
1. Glutamate receptor activation has been previously shown to result in mitochondrial depolarization and activation of the mitochondrial permeability transition pore in cultured neurones. In this study, we characterized the effects of two putative permeability transition inhibitors, namely trifluoperazine and dibucaine, on mitochondrial depolarization in rat intact, cultured forebrain neurones. 2. Permeability transition was monitored by following mitochondrial depolarization in neurones loaded with the mitochondrial membrane potential-sensitive fluorescent indicator, JC-1. Trifluoperazine (10 20 microM) and dibucaine (50-100 microM) inhibited or delayed the onset of glutamate-induced permeability transition. 3. We also investigated the effects of trifluoperazine and dibucaine on neuronal recovery from glutamate-induced Ca2+ loads. Trifluoperazine affected Ca2+ recovery in a manner similar to the mitochondrial Na+/Ca2+ exchange inhibitor, CGP-37157, while dibucaine had no apparent effect on Ca2+ recovery. Therefore, inhibition of permeability transition does not appear to be involved in Ca2+ recovery from glutamate-induced Ca2+ loads. 4. Trifluoperazine and dibucaine did not inhibit [3H]-dizocilpine binding at the concentrations that prevented mitochondrial depolarization. 5. These studies suggest that trifluoperazine and dibucaine inhibit permeability transition in intact neurones. Trifluoperazine also appears to inhibit mitochondrial Na+/Ca2+ exchange. These drugs should prove to be valuable tools in the further study of the role of mitochondrial permeability transition in glutamate-induced neuronal death.
Topics: Animals; Benzimidazoles; Calcium; Carbocyanines; Cells, Cultured; Clonazepam; Dibucaine; Dizocilpine Maleate; Fluorescent Dyes; Fluorometry; Glutamic Acid; Membrane Potentials; Mitochondria; Neurons; Prosencephalon; Rats; Rats, Sprague-Dawley; Sodium-Calcium Exchanger; Thiazepines; Trifluoperazine
PubMed: 9384493
DOI: 10.1038/sj.bjp.0701442 -
Antimicrobial Agents and Chemotherapy Mar 2017The efficacy of antimicrobial drugs against , an intracellular bacterial pathogen, is generally first established by testing compounds against bacteria in axenic...
The efficacy of antimicrobial drugs against , an intracellular bacterial pathogen, is generally first established by testing compounds against bacteria in axenic culture. However, inside infected macrophages, bacteria encounter an environment which differs substantially from broth culture and are subject to important host-dependent pharmacokinetic phenomena which modulate drug activity. Here, we describe how pH-dependent partitioning drives asymmetric antimicrobial drug distribution in infected macrophages. Specifically, weak bases with moderate activity against (fluoxetine, sertraline, and dibucaine) were shown to accumulate intracellularly due to differential permeability and relative abundance of their ionized and nonionized forms. Nonprotonatable analogs of the test compounds did not show this effect. Neutralization of acidic organelles directly with ammonium chloride or indirectly with bafilomycin A1 partially abrogated the growth restriction of these drugs. Using high-performance liquid chromatography, we quantified the degree of accumulation and reversibility upon acidic compartment neutralization in macrophages and observed that accumulation was greater in infected than in uninfected macrophages. We further demonstrate that the efficacy of a clinically used compound, clofazimine, is augmented by pH-based partitioning in a macrophage infection model. Because the parameters which govern this effect are well understood and are amenable to chemical modification, this knowledge may enable the rational development of more effective antibiotics against tuberculosis.
Topics: Ammonium Chloride; Anesthetics, Local; Antitubercular Agents; Biological Transport; Clofazimine; Dibucaine; Fluoxetine; Humans; Hydrogen-Ion Concentration; Macrolides; Macrophages; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Protons; Selective Serotonin Reuptake Inhibitors; Sertraline
PubMed: 28052847
DOI: 10.1128/AAC.01639-16 -
Anesthesiology Dec 1999An elevation of the intracellular calcium level, which is mediated by N-methyl-D-aspartate receptors and L-type Ca2+ channels both, activates the mitogen-activated...
BACKGROUND
An elevation of the intracellular calcium level, which is mediated by N-methyl-D-aspartate receptors and L-type Ca2+ channels both, activates the mitogen-activated protein (MAP) kinase signaling pathway involved in synaptic modification. It has recently been suggested that MAP kinase plays a role in coupling the synaptic excitation to gene expression in the nucleus of postsynaptic neurons. Because the effects of local anesthetics on cellular signal transduction in neuronal cells are not well-known, the authors investigated whether they affect the MAP kinase signaling pathway using PC12 cells.
METHODS
The cells were stimulated with either 50 mM KCl or 1 microM ionomycin, and activated MAP kinase was thus immunoprecipitated. The immunocomplexes were then subjected to an Elk1 phosphorylation assay. Both the phosphorylation of MAP kinase and the induction of c-Fos were detected by immunoblotting.
RESULTS
Pretreatment of the cells with 1 mM (ethylenedioxy)-diethyl-enedinitrilotetraacetic acid or 5 micron nifedipine blocked the MAP kinase activation induced by 50 mM KCl, whereas pretreatment with 2 microM omega-conotoxin GIVA did not. The expression of c-Fos induced by potassium chloride was also suppressed by dibucaine, tetracaine (concentrations that inhibited 50% of the activity of positive control [IC50s] were 16.2+/-0.2 and 73.2+/-0.7 microM, respectively), and PD 98059, a mitogen-activated/extracellular receptor-regulated kinase inhibitor. Higher concentrations of dibucaine and tetracaine were needed to suppress the activation of MAP kinase induced by ionomycin (the IC50 values of dibucaine and tetracaine were 62.5+/-2.2 and 330.5+/-32.8 microM, respectively) compared with potassium chloride (the IC50 values of dibucaine and tetracaine were 17.7+/-1.0 and 70.2+/-1.2 microM, respectively). Although probable targets of these local anesthetics might be L-type Ca2+ channels or components between Ca2+ and Ras in MAP kinase pathway, the possibility that they directly affect MAP kinase still remains.
CONCLUSIONS
Dibucaine and tetracaine at clinical concentrations were found to inhibit the activation of MAP kinase and the expression of c-Fos mediated by L-type Ca2+ channels in PC12 cells. The suppression of MAP kinase pathway may thus be a potential target site for the actions of dibucaine and tetracaine, including the modification of the synaptic functions.
Topics: Anesthetics, Local; Animals; Blotting, Western; Calcium Channels, L-Type; Dibucaine; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Ionomycin; Mitogen-Activated Protein Kinases; PC12 Cells; Phosphorylation; Potassium Chloride; Proto-Oncogene Proteins c-fos; Rats; Tetracaine
PubMed: 10598624
DOI: 10.1097/00000542-199912000-00034 -
The Journal of Biological Chemistry 2021The mitochondrial calcium uniporter (MCU) and cyclophilin D (CyD) are key players in induction of the permeability transition pore (PTP), which leads to mitochondrial...
The mitochondrial calcium uniporter (MCU) and cyclophilin D (CyD) are key players in induction of the permeability transition pore (PTP), which leads to mitochondrial depolarization and swelling, the major signs of Ca-induced mitochondrial damage. Mitochondrial depolarization inhibits ATP production, whereas swelling results in the release of mitochondrial pro-apoptotic proteins. The extent to which simultaneous deletion of MCU and CyD inhibits PTP induction and prevents damage of brain mitochondria is not clear. Here, we investigated the effects of MCU and CyD deletion on the propensity for PTP induction using mitochondria isolated from the brains of MCU-KO, CyD-KO, and newly created MCU/CyD-double knockout (DKO) mice. Neither deletion of MCU nor of CyD affected respiration or membrane potential in mitochondria isolated from the brains of these mice. Mitochondria from MCU-KO and MCU/CyD-DKO mice displayed reduced Ca uptake and diminished extent of PTP induction. The Ca uptake by mitochondria from CyD-KO mice was increased compared with mitochondria from WT mice. Deletion of CyD prevented mitochondrial swelling and resulted in transient depolarization in response to Ca, but it did not prevent Ca-induced delayed mitochondrial depolarization. Mitochondria from MCU/CyD-DKO mice did not swell in response to Ca, but they did exhibit mild sustained depolarization. Dibucaine, an inhibitor of the Ca-activated mitochondrial phospholipase A2, attenuated and bovine serum albumin completely eliminated the sustained depolarization. This suggests the involvement of phospholipase A2 and free fatty acids. Thus, in addition to induction of the classical PTP, alternative deleterious mechanisms may contribute to mitochondrial damage following exposure to elevated Ca.
Topics: Animals; Brain; Calcium; Calcium Channels; Peptidyl-Prolyl Isomerase F; Gene Knockout Techniques; Mice; Mice, Knockout; Mitochondria; Mitochondrial Proteins
PubMed: 33864812
DOI: 10.1016/j.jbc.2021.100669 -
Aging and Disease Aug 2013Acetylcholinesterase and Butyrylcholinesterase share unravelling link with components of metabolic syndromes that's characterised by low levels of HDL cholesterol,...
Acetylcholinesterase and Butyrylcholinesterase share unravelling link with components of metabolic syndromes that's characterised by low levels of HDL cholesterol, obesity, high fast aldohexose levels, hyper-trigliceridaemia and high blood pressure, by regulation of cholinergic transmission and therefore the enzyme activity within a living system. The phosphomotifs associated with amino acid and tyrosine binding motifs in AChE and BChE were known to be common. Phylogenetic tree was constructed to these proteins usinf UPGMA and Maximum Likelihood methods in MEGA software has shown interaction of AChE and BChE with ageing diseases like Alzheimer's disease and Diabetes. AChE has shown closely related to BChE, retinol dehydrogenase and β-polypeptide. The present studies is also accomplished that AChE, BChE, COLQ, HAND1, APP, NLGN2 and NGF proteins has interactions with diseases such as Alzheimer's and D2M using Pathwaylinker and STRING. Medicinal compounds like Ortho-7, Dibucaine and HI-6 are predicted as good targets for modeled AChE and BChE proteins based on docking studies. Hence perceptive studies of cholinesterase structure and the biological mechanisms of inhibition are necessary for effective drug development.
PubMed: 23936743
DOI: No ID Found -
The Biochemical Journal Dec 1977The kinetics of uptake of Ca(2+) by rat heart mitochondria were studied by a spectrophotometric method with Arsenazo III indicator. The exponential rate coefficients...
The kinetics of uptake of Ca(2+) by rat heart mitochondria were studied by a spectrophotometric method with Arsenazo III indicator. The exponential rate coefficients measured with or without added phosphate increase with the amount of Ca(2+) added up to about 24mum. Evidence is given that the effect is attributable to a combination of formation of chelates at low concentrations to act as Ca(2+) buffers, with co-transport of substrate to provide more respiratory fuel. The inhibitory effect of Mg(2+) depends on the Ca(2+) concentration, so with a constant [Mg(2+)] the low concentrations of Ca(2+) are most inhibited, and the rate coefficients are still more Ca(2+)-dependent. Ca(2+) uptake is slowed by local anaesthetics such as butacaine and dibucaine, and also by propranolol and palmitoyl-CoA. After an uptake, the release of Ca(2+) was investigated. The spontaneous release involves an initially slow and small appearance of free Ca(2+) and is followed by an auto-accelerated phase. The release is accompanied by a gradual decrease in internal ATP; it is initiated by palmitoyl-CoA (reversed by carnitine), by lysophosphatidylcholine, by Na(+) salts (reversed by oligomycin) and by K(+) salts added to a K(+)-free medium containing valinomycin. The process is probably a response to an increased energy load imposed on the mitochondria by the various conditions, which include the spontaneous action of phospholipase activated by traces of Ca(2+). The problem of how much mitochondrial activity is participating in normal heart Ca(2+) turnover is discussed, and experiments showing only 7-14% exchange of the mitochondrial Ca(2+) occurring in vivo in 10 or 20min are reported.
Topics: Animals; Calcium; In Vitro Techniques; Kinetics; Magnesium; Mitochondria, Heart; NAD; Palmitoyl Coenzyme A; Potassium; Rats; Temperature; Valinomycin
PubMed: 204287
DOI: 10.1042/bj1680447 -
Molecular Pharmaceutics Jun 2023In this study, we investigated the effects of drugs on membrane function in which lipid peroxidation was inhibited by the antioxidant Trolox (TRO) in liposomes...
In this study, we investigated the effects of drugs on membrane function in which lipid peroxidation was inhibited by the antioxidant Trolox (TRO) in liposomes containing egg yolk lecithin. Local anesthetics (LAs), such as lidocaine (LID) and dibucaine (DIB), were used as model drugs. The effect of LAs on the inhibitory activity of TRO was evaluated by calculating the p from the inhibition constant calculated by curve fitting. p indicates the strength of TRO membrane protective function. p indicates the strength of LA activity. LAs inhibited lipid peroxidation in a dose-dependent manner and decreased p. The effect of DIB on p was 1.9 times more than that of LID. This result indicated that LA may improve the fluidity of the membrane, which may facilitate the migration of TRO from the membrane to the liquid phase. As a result, TRO is less likely to suppress lipid peroxidation within the lipid membrane, possibly resulting in a decrease in p. The effect of TRO on p was found to be similar in both, indicating that it did not depend on the type of the model drug. These results suggest that our developed procedure successfully quantified the effects of LAs on lipid membrane functions. We were able to obtain the characteristics of model drugs independent of TRO by simultaneously measuring and analyzing the lipid peroxidation inhibitory activities of TRO and model drugs in liposomes.
Topics: Anesthetics, Local; Liposomes; Lipid Peroxidation; Antioxidants; Dibucaine; Lidocaine; Lipids
PubMed: 37104048
DOI: 10.1021/acs.molpharmaceut.2c01053 -
Journal of Pharmacy & Bioallied Sciences Jun 2021To compare and analyze the clinical adequacy of two topical anesthetic gels, Precaine (8% lidocaine + 0.8% dibucaine) and Precaine B (20% benzocaine) in children before...
AIM
To compare and analyze the clinical adequacy of two topical anesthetic gels, Precaine (8% lidocaine + 0.8% dibucaine) and Precaine B (20% benzocaine) in children before intraoral local anesthetic injections.
MATERIALS AND METHODS
This clinical study included thirty children who needed an inferior alveolar nerve block. They were divided into three groups: Group A: Precaine topical gel group, Group B: Precaine B topical gel Group, Group C: no anesthetic topical gel group (control group). These two effective topical gels were applied before giving intraoral local anesthesia, and afterward, the child's pain response was surveyed utilizing the Wong-Baker Faces Pain Rating Scale. The scores obtained were subjected to statistical analysis.
RESULTS
Intergroup comparison showed a significant mean difference between the control group and Precaine group ( > 0.05) as well as Precaine B group ( > 0.05). However, there is no significant difference obtained between Group A and Group B ( < 0.05).
CONCLUSION
It is psychologically and clinically beneficial to apply a topical anesthetic agent before injecting any intraoral anesthesia. In this study, both anesthetic gels showed a nonsignificant difference in reducing inferior alveolar injection pain, but Precaine B shows more promising results than Precaine.
PubMed: 34447172
DOI: 10.4103/jpbs.JPBS_772_20 -
Antimicrobial Agents and Chemotherapy May 2016Enteroviruses (EVs) represent many important pathogens of humans. Unfortunately, no antiviral compounds currently exist to treat infections with these viruses. We...
Enteroviruses (EVs) represent many important pathogens of humans. Unfortunately, no antiviral compounds currently exist to treat infections with these viruses. We screened the Prestwick Chemical Library, a library of approved drugs, for inhibitors of coxsackievirus B3, identified pirlindole as a potent novel inhibitor, and confirmed the inhibitory action of dibucaine, zuclopenthixol, fluoxetine, and formoterol. Upon testing of viruses of several EV species, we found that dibucaine and pirlindole inhibited EV-B and EV-D and that dibucaine also inhibited EV-A, but none of them inhibited EV-C or rhinoviruses (RVs). In contrast, formoterol inhibited all enteroviruses and rhinoviruses tested. All compounds acted through the inhibition of genome replication. Mutations in the coding sequence of the coxsackievirus B3 (CV-B3) 2C protein conferred resistance to dibucaine, pirlindole, and zuclopenthixol but not formoterol, suggesting that 2C is the target for this set of compounds. Importantly, dibucaine bound to CV-B3 protein 2C in vitro, whereas binding to a 2C protein carrying the resistance mutations was reduced, providing an explanation for how resistance is acquired.
Topics: Antiviral Agents; Carbazoles; Carrier Proteins; Clopenthixol; Dibucaine; Enterovirus; Fluoxetine; Formoterol Fumarate; HeLa Cells; Humans; Rhinovirus; Viral Nonstructural Proteins; Viral Proteins; Virus Replication
PubMed: 26856848
DOI: 10.1128/AAC.02182-15 -
Blood Mar 1990Platelet calpain has many platelet substrates, including external membrane proteins. We thus investigated whether platelet calpain II was associated with platelet...
Platelet calpain has many platelet substrates, including external membrane proteins. We thus investigated whether platelet calpain II was associated with platelet membranes in unstimulated and thrombin-activated platelets. A monospecific, goat polyclonal antibody was reared to purified platelet calpain II. Sixteen whole platelet lysates were found to contain 4.5 +/- 0.7 micrograms calpain antigen II per 10(8) platelets (mean +/- SEM) as determined by a competitive enzyme-linked immunosorbent assay. Using the dipeptide fluorogenic substrate, Suc-Leu-Tyr-MCA, 17 human platelet lysates contained 3.6 +/- 0.4 micrograms calpain activity per 10(8) platelets. Platelet calpain II was associated with the Triton X-100 insoluble platelet cytoskeletons from both unstimulated and thrombin-activated platelets. When compared with the total cell content of platelet calpain II, calpain antigen (10% to 13%) and calpain activity (24% to 28%) was associated with platelet cytoskeletons in unstimulated and thrombin-activated platelets, respectively. On immunoblot, the heavy chain (80 Kd) of calpain II was detected in platelet cytoskeletons. Subcellular fractionation studies on both unstimulated and thrombin-activated platelets, revealed that half of the total platelet calpain II antigen was associated with cytosol, and the other half was associated with the membrane fraction. Platelet calpain II was not seen on the surface of unstimulated, paraformaldehyde fixed platelets by immunofluorescence. However, on thrombin-activated platelets, rim immunofluorescence was seen, indicating that activated platelets externalize their calpain. This observation was confirmed by the finding that about 2,000 molecules per platelet of an 125I-anti-calpain II Fab' specifically bound to thrombin-activated but not unstimulated platelets. Both dibucaine (1 mmol/L) and platelet activating factor (1.86 mumol/L) in the absence of external Ca++, but not collagen (5 micrograms/mL) or ionophore A23187 (2.5 mumol/L) in the absence of external Ca++, were also able to externalize platelet calpain II antigen, as indicated by a similar level of specific 125I-anti-calpain II Fab'-platelet binding. These combined studies indicate that platelet calpain II is a major protein, comprising 2% of total platelet protein, a substantial portion of which is membrane-associated. When platelets are activated by thrombin and platelet activating factor, calpain II antigen also becomes present on the external platelet surface.
Topics: Antibodies; Blood Platelets; Calpain; Cell Fractionation; Cell Membrane; Cytoskeleton; Humans; Immunohistochemistry; Platelet Activation; Thrombin
PubMed: 2310827
DOI: No ID Found