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British Journal of Pharmacology Jan 19751 Cells of the aortic endothelium isolated from the guinea-pig and bathed at 37 degrees C with a calcium-free superfusion fluid had membrane potentials of minus 41 plus...
1 Cells of the aortic endothelium isolated from the guinea-pig and bathed at 37 degrees C with a calcium-free superfusion fluid had membrane potentials of minus 41 plus or minus 7 mV (mean plus or minus s.e mean). 2 Depolarization was produced by addition of potassium (50-200 mM) or certain other monovalent metal cations to the superfusion fluid. Depolarization was rapidly reversed on return to the original superfusate. 3 Several divalent metal cations, notably calcium (16 mM), caused depolarization which was only slowly and incompletely reversed on return to the original calcium-free superfusate. 4 Repolarization after exposure to calcium was accelerated and made more complete by addition of indomethacin (0.25 mM) to the superfusate, 5 The trivalent cations of lanthanum, aluminium or iron (0.1 mM) inhibited the depolarizing effect of calcium (16 mM). 6 Exposure to histamine (100 mug/ml) or heating to 45 degrees C for 1 h caused depolarization in the presence but not in the absence of calcium. Subsequent removal of histamine or cooling again to 37 degrees C in the continued presence of calcium permitted only slow and partial repolarization. However, repolarization was more rapid and complete in the presence of indomethacin (0.25 mM). 7 Heating to 45 degrees C for 5 h in the presence of calcium caused progressive and almost complete depolarization. Lanthanum, cinchocaine, indomethacin, flufenamic, meclofenamic and salicylic acids, phenylbutazone and aminopyrine each reduced the depolarization, but hydrocortisone, chloroquine, benzindamine, isoprenaline and aminophylline did not.
Topics: Animals; Anti-Inflammatory Agents; Aorta, Thoracic; Calcium; Endothelium; Guinea Pigs; Histamine; In Vitro Techniques; Membrane Potentials; Mesentery; Metals; Portal Vein; Temperature; Time Factors; Venae Cavae
PubMed: 1125483
DOI: 10.1111/j.1476-5381.1975.tb07337.x -
Journal of Biochemistry Sep 1989During anoxic incubation, depletion of mitochondrial ATP was followed by release of Ca2+ with concomitant increase in the rate of state 4 respiration due to disruption...
During anoxic incubation, depletion of mitochondrial ATP was followed by release of Ca2+ with concomitant increase in the rate of state 4 respiration due to disruption of the diffusion barrier against protons. The external addition of ATP and its non-metabolizable analog, beta,gamma-methylene adenosine 5'-triphosphate, prevented both the release of Ca2+ and increase in the rate of state 4 respiration. Addition of EGTA, which did not prevent release of the ion, resulted in little increase in the respiration rate. Addition of an inhibitor of mitochondrial phospholipase A2, such as quinacrine, dibucaine, or chlorpromazine, also prevented increase in the respiration rate without affecting Ca2+ release from mitochondria during anoxic incubation. Non-esterified polyunsaturated fatty acids were also found to be liberated from anoxic mitochondria. External addition of the ATP-analog, EGTA, and inhibitors of phospholipase A2 suppressed the liberation of non-esterified polyunsaturated fatty acids. Melittin and Ca2+, which activate phospholipase A2, increased the rate of state 4 respiration and the liberation of fatty acids. These findings support the hypothesis proposed previously that the following sequence changes occurs in mitochondria during anoxia; depletion of ATP, liberation of free calcium from mitochondria, and disruption of the diffusion barrier against H+ of the inner membrane. The results also indicate another event; activation of phospholipase A2 by release Ca2+ which results in H+ leakiness of the inner membrane.
Topics: Animals; Calcium; Enzyme Activation; Hypoxia; Intracellular Membranes; Male; Mitochondria, Liver; Phospholipases; Phospholipases A; Phospholipases A2; Rats; Rats, Inbred Strains
PubMed: 2606905
DOI: 10.1093/oxfordjournals.jbchem.a122887 -
The Journal of Biological Chemistry Mar 1995Two membrane-associated phosphoinositide-specific phospholipase Cs (mPI-PLC-1 and mPI-PLC-2) and a cytosolic enzyme (cPI-PLC) that were activated by brain G-protein beta...
Two membrane-associated phosphoinositide-specific phospholipase Cs (mPI-PLC-1 and mPI-PLC-2) and a cytosolic enzyme (cPI-PLC) that were activated by brain G-protein beta gamma subunits have been isolated from human platelets. The truncation of mPI-PLC-1 that was mediated by mu-calpain induced much higher activation by beta gamma subunits (Banno, Y., Asano, T., and Nozawa, Y. (1994) FEBS Lett. 340, 185-188). On the basis of size and immunological cross-reactivity, mPI-PLC-1 (155 kDa) was PLC-beta 3, and mPI-PLC-2 (100 kDa) was its truncated form. The cPI-PLC (140 kDa) was recognized by the antibody selective for internal sequences of PLC-beta 3 but not by the antibody raised against its carboxyl terminus, indicating that it may be related to PLC-beta 3. Treatment of human platelets with A23187 and dibucaine, activators of calpain, caused cleavage of actin-binding protein and talin in a time-dependent manner. At the same time, decrease of PLC-beta 3 (155 and 140 kDa) and concomitant increase of the 100-kDa product of cleavage were observed on immunoblots with the antibody to internal sequences of PLC-beta 3. Furthermore, stimulation of platelets by natural agonists, thrombin and collagen, caused the cleavage of PLC-beta 3 (155 and 140 kDa) and an increase of 100 kDa PLC-beta 3 in a time- and dose-dependent manner. The cleavage of these PLC-beta 3 enzymes was completely blocked by calpain inhibitor, calpeptin, indicating that the PLC-beta 3 modification may be a consequence of platelet activation leading to activation of calpain. This is the first demonstration that PLC-beta 3 is indeed cleaved by calpain upon platelet activation by physiological agonists. The cleavage of PLC-beta 3 evoked by thrombin and collagen but not ADP was correlated with irreversible aggregation, suggesting that the PLC-beta 3 modification may play a role in secondary irreversible aggregation in agonist-stimulated human platelets.
Topics: Amino Acid Sequence; Blood Platelets; Blotting, Western; Calpain; Collagen; Enzyme Activation; GTP-Binding Proteins; Humans; Hydrolysis; Isoenzymes; Molecular Sequence Data; Phospholipase C beta; Thrombin; Type C Phospholipases
PubMed: 7876193
DOI: 10.1074/jbc.270.9.4318 -
Biochemical Genetics Oct 1980This investigation was prompted by the findings that (1) dibucaine-resistant homozygotes and heterozygotes for plasmacholinesterase also exhibit resistance to fluoride...
This investigation was prompted by the findings that (1) dibucaine-resistant homozygotes and heterozygotes for plasmacholinesterase also exhibit resistance to fluoride inhibition, (2) the differentiation of dibucaine-resistant from the fluoride-resistant genotypes is ambiguous with the method of Harris and Whittaker, (3) the plasmacholinesterase inhibition by Na fluoride (FN) is markedly influenced by the temperature. Therefore, we modified their method by increasing (1) the temperature of the reaction from 25C to 37C and (2) the concentration of Na fluoride from 5.0 x 10(-5) M to 2.5 x 10(-4) M. With this method, gentically normal individuals have a mean FN + or - SD equals 43.0 + or - 10.0 and atypical dibucaine-resistant heterozygotes 67.0 + or - 5.37. Since a linear correlation was observed between DN and FN by our new method, a fluoride number 2 SD lower than the predicted FN from the DN can distinctly identify the fluoride-resistant plasmacholinesterase genotype Ef.
Topics: Cholinesterases; Cross Reactions; Dibucaine; Drug Resistance; Fluorides; Genes; Genotype; Head; Humans
PubMed: 7225083
DOI: 10.1007/BF00500119 -
American Journal of Human Genetics May 1978Human plasma cholinesterase from five different genotypes -- E1U E1U, E1U E1A, E1A E1A, E1U E1S, E1A E1S, and E1U E1U C5+ -- was purified 8,000 fold from serum by a...
Human plasma cholinesterase from five different genotypes -- E1U E1U, E1U E1A, E1A E1A, E1U E1S, E1A E1S, and E1U E1U C5+ -- was purified 8,000 fold from serum by a two-step procedure involving chromatography on DEAE-cellulose and preparative disc electrophoresis. The esterases were labeled with diisopropyl-1, 3-C14-fluorophosphate (DFP) aminoethylated, and digested by trypsin. The trytic digests were subjected to high voltage electrophoresis, and the radioactive peptides were detected by radioautography. Comparison of the peptides revealed different electrophoretic mobilities of the usual and atypical (dibucaine resistant) plasma cholinesterase peptides. The results are consistent with a structural abnormality of the active center in the variant enzyme. No difference was observed an the esteratic site of the enzyme with C5 component.
Topics: Binding Sites; Cholinesterases; Dibucaine; Drug Resistance; Electrophoresis, Paper; Genetic Variation; Humans; Protein Conformation
PubMed: 677127
DOI: No ID Found -
FEBS Letters Dec 2002The quinoline-based tertiary amine dibucaine has been shown to bind the membrane skeletal protein spectrin with a dissociation constant of 3.5x10(-5) M at 25 degrees C....
The quinoline-based tertiary amine dibucaine has been shown to bind the membrane skeletal protein spectrin with a dissociation constant of 3.5x10(-5) M at 25 degrees C. Such binding is detected by monitoring the quenching of the tryptophan fluorescence intensity with increasing concentrations of dibucaine only and not with the benzene-based local anesthetics procaine, tetracaine and lidocaine. Binding of dibucaine also indicated changes in the tertiary structure of spectrin indicated by a circular dichroism spectrum in the near-UV region due to absorption of the aromatic side chains. The thermodynamic parameters associated with the binding indicated the interaction of dibucaine and spectrin to be enthalpy-driven and insensitive to an increase in the ionic strength of the buffer.
Topics: Amines; Anesthetics, Local; Animals; Circular Dichroism; Dibucaine; Dose-Response Relationship, Drug; Erythrocyte Membrane; Goats; Hot Temperature; Ions; Kinetics; Lidocaine; Procaine; Protein Binding; Protein Structure, Tertiary; Spectrin; Spectrometry, Fluorescence; Temperature; Tetracaine; Thermodynamics; Tryptophan; Ultraviolet Rays
PubMed: 12482599
DOI: 10.1016/s0014-5793(02)03721-3 -
The Journal of Biological Chemistry Sep 1980Ca2+-activated, phospholipid-dependent protein kinase recently found in mammalian tissues (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishizuka, Y....
Ca2+-activated, phospholipid-dependent protein kinase recently found in mammalian tissues (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695) is inhibited by various phospholipid-interacting drugs such as chlorpromazine, imipramine, phentolamine, dibucaine, verapamil, and tetracaine. This effect is attributed to the inhibition of the activation process but not to the interaction with the active site of enzyme. This is supported by the fact that a catalytic fragment of this enzyme, which is obtained by limited proteolysis with Ca2+-dependent neutral protease, is fully active without without Ca2+ and phospholipid and is not susceptible to any of these drugs. Kinetic analysis suggests that these drugs cause such inhibition competitively with phospholipid. None of these drugs appears to compete with Ca2+ or to counteract the unique effect of unsaturated diacylglycerol. Unsaturated diacylglycerol has been shown previously to increase markedly the affinity of enzyme for Ca2+ as well as for phospholipid and thereby serve as an initiator for the activation of this protein kinase. Neither cyclic AMP-dependent nor cyclic GMP-dependent protein kinase is susceptible to these phospholipid-interacting drugs.
Topics: Animals; Brain; Calcium; Chlorpromazine; Cytosol; Dibucaine; Enzyme Activation; Imipramine; Kinetics; Phentolamine; Phospholipids; Protein Kinases; Rats; Tetracaine; Verapamil
PubMed: 7410368
DOI: No ID Found -
The Biochemical Journal Dec 1982The effects of calmodulin antagonists on the secretion of lysosomal enzyme and lipid metabolism in guinea-pig peritoneal macrophages were studied. Calmodulin...
The effects of calmodulin antagonists on the secretion of lysosomal enzyme and lipid metabolism in guinea-pig peritoneal macrophages were studied. Calmodulin antagonists, such as trifluoperazine, dibucaine and quinacrine, inhibited the secretion of N-acetyl-beta-d-glucosaminidase from cytochalasin B-treated macrophages when the macrophages were stimulated by the chemotactic peptide, formylmethionyl-leucyl-phenylalanine (f Met-Leu-Phe) or the Ca(2+) ionophore A23187. The effect of calmodulin antagonists on the incorporation of [(32)P]P(i) or [(3)H]glycerol into glycerolipids as well as on the redistribution of [(14)C]glycerol or [(3)H]arachidonic acid in [(14)C]glycerol- or [(3)H]arachidonic acid-prelabelled lipids were examined. Trifluoperazine, dibucaine or quinacrine stimulated [(32)P]P(i) incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) without significant effect on the labelling of phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), lysophosphatidylcholine (lyso-PtdCho) and lysophosphatidylethanolamine (lyso-PtdEtn). The incorporation of [(32)P]P(i) into phosphatidylcholine (PtdCho) was, on the contrary, inhibited. When calmodulin antagonists were added to macrophages stimulated by fMet-Leu-Phe, [(32)P]P(i) incorporation into PtdIns and PtdA was synergistically increased compared with that induced only by calmodulin antagonists. Trifluoperazine inhibited the incorporation of [(3)H]glycerol into PtdCho, triacylglycerol and PtdEtn. Also in this case, the incorporation of [(3)H]glycerol into PtdA and PtdIns was greatly enhanced. But [(3)H]glycerol incorporation into PtdSer, lyso-PtdEtn and lyso-PtdCho was not affected by the drug. On the other hand, diacylglycerol labelling with [(3)H]glycerol was maximally activated by 10mum-trifluoperazine and levelled off with the increasing concentration. When the effect of calmodulin antagonists on the redistribution of [(14)C]glycerol among lipids was examined in pulse-chase experiments, no significant effect on [(14)C]glycerol redistribution in PtdEtn, PtdCho, PtdIns, PtdSer, PtdA and tri- and di-acylglycerol could be detected. When macrophages prelabelled with [(3)H]arachidonic acid were treated with trifluoperazine, dibucaine or quinacrine, the [(3)H]arachidonic acid moiety in PtdEtn and PtdCho was decreased and that in PtdA was increased. The formation of [arachidonate-(3)H]diacylglycerol and non-esterified [(3)H]-arachidonic acid was also enhanced, but the increase in [(3)H]arachidonic acid was only observed at concentrations between 1 and 50mum. [Arachidonate-(3)H]PtdIns was not significantly affected. The activated formation of [arachidonate-(3)H]PtdA, diacylglycerol and non-esterified arachidonic acid by these drugs was synergistically enhanced in the presence of fMet-Leu-Phe.
Topics: Acetylglucosaminidase; Animals; Arachidonic Acid; Arachidonic Acids; Calcium-Binding Proteins; Calmodulin; Dibucaine; Female; Guinea Pigs; Hexosaminidases; In Vitro Techniques; Lysosomes; Macrophages; Phospholipids; Quinacrine; Trifluoperazine; Triglycerides
PubMed: 6819863
DOI: 10.1042/bj2080549 -
The Journal of Cell Biology Dec 1979Differences in cell morphology, concanavalin A-induced receptor redistributions, and the cooperativity of the inhibition of 5'-nucleotidase (AMPase) by concanavalin A...
Differences in cell morphology, concanavalin A-induced receptor redistributions, and the cooperativity of the inhibition of 5'-nucleotidase (AMPase) by concanavalin A (Con A) have been investigated in ascites sublines of the 13762 rat mammary adenocarcinoma cells treated with microfilament- and microtubule-perturbing drugs. By scanning electron microscopy MAT-C1 cells exhibit a highly irregular surface, covered with microvilli extending as branched structures from the cell body. MAT-A, MAT-B, and MAT-B1 cells have a more normal appearance, with unbranched microvilli, ruffles, ridges, and blebs associated closely with the cell body. MAT-C cells have an intermediate morphology. Treatment of MAT-A, MAT-B, or MAT-B1 cells with Con A causes rapid redistribution of Con A receptors. Both cytochalasins and colchicine cause alternations in the receptor redistributions. Receptors on MAT-C1 cells are highly resistant to redistribution, even in the presence of cytoskeletal perturbant drugs. The cooperativity of the inhibition of AMPase by Con A was investigated in MAT-A and MAT-C1 cells. Untreated cells exhibit no cooperativity. If either subline is treated with colchicine, cytochalasin B or D, or dibucaine, cooperativity is observed. Lumicolchicine has no effect. Theophylline or dibutyryl cyclic AMP prevents the effects of either colchicine or cytochalasin. The concentration required for half-maximal induction of cooperativity is 0.3--0.4 microM for both colchicine and cytochalasin D, which is in the appropriate range for specific microtubule and microfilament disruptions. The effectiveness of the cytochalasins (E greater than D greater than B) is consistent with their known effects on microfilaments. No direct correlation was observed between the induction of cooperativity and drug-induced changes in Con A receptor redistribution or cell morphology. The morphology of MAT-A cells is grossly altered by cytochalasins or dibucaine and somewhat less by colchicine. MAT-C1 cells exhibit more minor alterations in morphology as a result of these drug treatments. The results of this study indicate that the inhibition of AMPase, which is a Con A receptor, is a different process from the redistribution of the bulk of the Con A receptors, possibly short range membrane interactions rather than global effects on the cell.
Topics: Animals; Bucladesine; Calcium; Cell Line; Cell Membrane; Colchicine; Cytochalasins; Dibucaine; Mammary Neoplasms, Experimental; Nucleotidases; Rats; Receptors, Concanavalin A; Theophylline
PubMed: 230191
DOI: 10.1083/jcb.83.3.529 -
Anesthesiology Apr 2004Irreversible nerve injury may result from neural membrane lysis due to the detergent properties of local anesthetics. This study aimed to investigate whether local...
BACKGROUND
Irreversible nerve injury may result from neural membrane lysis due to the detergent properties of local anesthetics. This study aimed to investigate whether local anesthetics display the same properties as detergents and whether they disrupt the model membrane at high concentrations.
METHODS
Concentrations at which dodecyltrimethylammonium chloride and four local anesthetic (dibucaine, tetracaine, lidocaine, and procaine) molecules exhibit self-aggregation in aqueous solutions were measured using an anesthetic cation-sensitive electrode. Light-scattering measurements in a model membrane solution were also performed at increasing drug concentrations. The concentration at which drugs caused membrane disruption was determined as the point at which scattering intensity decreased. Osmotic pressures of anesthetic agents at these concentrations were also determined.
RESULTS
Concentrations of dodecyltrimethylammonium chloride, dibucaine, tetracaine, lidocaine, and procaine at which aggregation occurred were 0.15, 0.6, 1.1, 5.3, and 7.6%, respectively. Drug concentrations causing membrane disruption were 0.09% (dodecyltrimethylammonium chloride), 0.5% (dibucaine), 1.0% (tetracaine), 5.0% (lidocaine), 10.2% (procaine), and 20% (glucose), and osmotic pressures at these concentrations were 278, 293, 329, 581, 728, and 1,868 mOsm/kg H2O, respectively.
CONCLUSIONS
These results show that all four local anesthetics form molecular aggregations in the same manner as dodecyltrimethylammonium chloride, a common surfactant. At osmotic pressures insufficient to affect the membrane, local anesthetics caused membrane disruption at the same concentrations at which molecular aggregation occurred. This shows that disruption of the model membrane results from the detergent nature of local anesthetics. Nerve membrane solubilization by highly concentrated local anesthetics may cause irreversible neural injury.
Topics: Anesthetics, Local; Cell Membrane; Detergents; Dose-Response Relationship, Drug; Micelles; Spinal Nerves
PubMed: 15087634
DOI: 10.1097/00000542-200404000-00029