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PubMed1993

Effects of tetrandrine on production of leukotriene B4 and thromboxane B2 in rabbit blood.

The effects of tetrandrine (Tet) on the production of 2 major metabolites of arachidonic acid (AA), the leukotriene B4 (LTB4) and the thromboxane B2 (TXB2) in rabbit whole blood were investigated by reversed phase high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA), respectively. After incubation with different doses of Tet for 15 min in vitro, the production of LTB4 and TXB2 by rabbit whole blood stimulated with calcimycin (20 mumol.L-1) was inhibited in a dose-dependent manner, with IC50 value of 17.8 +/- 8.6 and 17.7 +/- 9.2 mumol.L-1, respectively. In the presence of exogenous AA, the inhibitory effects of Tet were markedly lessened. The effects of Tet were much like those of calmodulin (CaM) antagonist fluphenazine (Flu). Dexamethasone (Dex) also inhibited the production of LTB4 and TXB2 when incubated with rabbit whole blood for 60 min. Tet iv 10 mg.kg-1 also inhibited the production of LTB4 and TXB2 in rabbit whole blood stimulated with calcimycin. These results suggest that Tet may be an antagonist of CaM, thus suppressing the release of AA which was catalyzed by CaM dependent phospholipase A2 (PLA2) from the membrane phospholipids of blood cells.

Alkaloids↗
PubMed1993

Inhibition of spermine on calcium influx during capacitation of guinea pig spermatozoa in vitro.

To investigate the mechanism of action of spermine, we measured the intracellular calcium ([Ca]i) of guinea pig spermatozoa using a probe of fluorescence, Quin 2. Results showed that spermine (0.25-2.0 mmol.L-1) suppressed the membrane permeability to Ca2+ during capacitation, which was similar to that of verapamil (a Ca2+ channel blocker). Furthermore, the rapid increase of [Ca]i induced by calcimycin (A-23187) was inhibited by spermine and verapamil, whereas trifluoperazine (an inhibitor of calmodulin) had no effect on it. The inhibition of the acrosome reaction caused by verapamil (5-100 mumol.L-1) or trifluoperazine (1-60 mumol.L-1) was reversed by calcimycin and cAMP, respectively. In addition, there was no effect on the initiation of the acrosome reaction when verapamil was added to capacitated spermatozoa. This result was in agreement with that of spermine. When addition of spermine (0.5 mmol.L-1) was combined with trifluoperazine (5 mumol.L-1), the acrosome reaction decline almost to zero, indicating that spermine might inhibit Ca2+ sensitive channel during sperm capacitation.

Acrosome↗
PubMed1998

Cysteine string protein is required for calcium secretion coupling of evoked neurotransmission in drosophila but not for vesicle recycling.

The entire deletion of the cysteine string protein (CSP) gene causes a temperature-sensitive (ts) block of evoked neurotransmission in Drosophila. CSP has been found to interact in vitro with the clathrin-uncoating ATPase HSC70, suggesting a potential role of CSP in vesicle recycling. Using FM1-43 imaging, we analyzed whether the ts block of neurotransmission in csp mutants is caused by a defect in vesicle exocytosis or vesicle recycling. We determined that FM1-43-labeled synaptic boutons of csp mutant neuromuscular junctions fail to destain at 32 degrees C after K+ depolarization, and that FM1-43 dye uptake cannot be evoked by K+ stimulation at 32 degrees C. However, when we stimulated dye uptake independent of depolarization by using black widow spider venom (BWSV), we observed endocytotic uptake of FM1-43. This suggests that endocytosis exhibits no primary ts defect. In addition, we found no ts defect of vesicle recycling at 32 degrees C that would correlate with the ts block of neurotransmission. We also discovered that BWSV and the calcium ionophore calcimycin stimulate FM1-43 destaining and quantal release in csp mutants at 32 degrees C when depolarization fails to evoke any response. The wild-type-like, calcimycin-induced response in csp null mutants indicates that some aspect of the depolarization-dependent calcium signaling pathway must be impaired, either calcium entry, calcium action, or both. Collectively, our results indicate that the csp mutation affects calcium secretion coupling of evoked exocytosis but not vesicle recycling. This supports the hypothesis that CSP links synaptic vesicles to calcium secretion coupling.

Animals↗
PubMed1996

Intracellular Ca2+ during fertilization and artificial activation in mouse oocytes.

AIM: To study the mechanism of oocyte activation in mammals. METHODS: Mouse oocytes arrested at metaphase of the second meiotic division were loaded with Fura 2-AM and then activated with ethanol, calcimycin, electric pulse or fertilization. Intracellular free Ca2+ during activation were measured by Spex AR-CM-MIC cation system. Cortical granule exocytosis after activation was detected under electron microscopy. RESULTS: Sperm penetration initiated a long lasting Ca2+ oscillation in Ca2+ containing IVF medium in mouse ova. The Ca2+ oscillation lasted for over 3-4 h until the ova developed to pronuclear stage. The Ca2+ oscillated faster as extracellular Ca2+ concentration was increased from normal 1.7 mmol.L-1 to 5.0 mmol.L-1 and ceased to oscillate when extracellular Ca2+ was removed. The ova resumed Ca2+ oscillation after transferred back to IVF (Ca2+ 1.7 mmol.L-1). The ova which exhibited Ca2+ oscillation all extruded second polar body and formed pronuclei. Suppression the Ca2+ oscillation by intracellular injection of egtazic acid (2-10 pL, 200 mumol.L-1) blocked the activation of oocytes. Heparin (100 mumol.L-1) injection failed to prevent the Ca2+ oscillation. In artificial activation, ethanol, calcimycin, and a single electric pulse usually induced a monotonic Ca2+ rise and resulted in the activation only in older oocytes (> 18 h after CG injection). Activation of freshly ovulated oocytes required multiple intracellular Ca2+ increases induced by repetitive electric pulses. Artificial activation elicited the similar cortical granule exocytosis in oocytes as occurring at fertilization. Suppression of the intracellular Ca2+ elevation by introduction of egtazic acid into the oocytes blocked the activation process. CONCLUSIONS: The increase of intracellular free Ca2+ is the primary signal responsible for the initiation of oocyte activation but there are distinct differences between fertilization and artificial activation both in Ca2+ change patterns and Ca2+ sources. Young oocytes require oscillatory Ca2+ signals for activation.

Animals↗
PubMed2003

Effects of water-soluble cigarette smoke extracts upon the release of beta-hexosaminidase from RBL-2H3 basophilic leukaemia cells in response to substance P, compound 48/80, concanavalin A and antigen stimulation.

OBJECTIVE AND DESIGN: To determine whether water-soluble constituents of cigarette smoke affect mast cell function using an in vitro model, RBL-2H3 basophilic leukaemia cells. MATERIALS AND METHODS: RBL-2H3 cells were induced to degranulate in response to compound 48/80 and substance P, as assessed by monitoring the release of the granular enzyme beta-hexosaminidase, by treatment for 7 days with 20 microM quercetin. Responses to concanavalin A and antigen were determined by measuring the beta-hexosaminidase release from cells cultured on fibronectin-coated plates. RESULTS: The beta-hexosaminidase release response to compound 48/80 induced by quercetin treatment was accompanied by a release of lactate dehydrogenase, suggesting that degranulation is not the only process triggered by compound 48/80 under these conditions. Quercetin treatment reduced the beta-hexosaminidase release response to concanavalin A. Precoating of the culture wells with rat fibronectin enhanced the beta-hexosaminidase response to calcimycin, but not to concanavalin A. Under these conditions, concanavalin A did not induce a release of lactate dehydrogenase. The responses to c48/80, substance P, calcimycin, concanavalin A and antigen (after IgE pretreatment) were reduced by treatment with cigarette smoke solution obtained from standard and low-tar cigarettes (IR3 and IR5F). The effect of cigarette smoke solution from IR5F cigarettes upon the beta-hexosaminidase release elicited by compound 48/80 (in quercetin-treated cells) and by concanavalin A (in cells cultured on fibronectin-coated wells) could be prevented by N-acetyl-L-cysteine, but not with either hemoglobin, alpha-tocopherol, catalase or palmitoylethanolamide. N-acetyl-L-cysteine also reduced the effect of cigarette smoke solution upon the degranulation response to antigen. CONCLUSIONS: Under the conditions used, oxidants present in cigarette smoke solution from IR5F cigarettes reduce the ability of RBL-2H3 cells to degranulate in response to both immunological and non-immunological stimuli.

Animals↗
PubMed1996

Vasorelaxant and antiaggregatory properties of the endothelium: a comparative study in normocholesterolaemic and hereditary and dietary hypercholesterolaemic rabbits.

1. A comparison of the effects of dietary and genetically-induced hypercholesterolaemia on the vasodilator and antiaggregatory capacity of the endothelium was made in rabbit isolated subclavian artery rings. 2. Dietary-induced hypercholesterolaemia in NZW rabbits decreased the maximum relaxation to carbachol (0.01-10 microM) and calcimycin (0.01-0.1 microM) in vessel rings precontracted with 5-hydroxytryptamine (5-HT), 0.1 microM), when compared to responses observed in rings obtained from control normocholesterolaemic NZW rabbits. The relaxant responses to SIN-1 (3-(4-morpholinyl)-sydnonimine hydrochloride) were attenuated but were not significantly different from controls. In Froxfield genetically hypercholesterolaemic (FHH) rabbits, the maximum relaxations to carbachol, calcimycin and SIN-1 were all reduced significantly. 3. Neither genetic nor dietary-induced hypercholesterolaemia modified the contractile responses of vessel rings to either KCl (10-100 mM) or 5-HT (0.01-10 microM). 4. Endothelium-dependent inhibition of collagen-induced platelet aggregation in whole blood was demonstrated by stimulation of a vessel ring, incorporated into the blood sample, with carbachol (10 microM, final blood concentration). This effect was inhibited by NG-nitro-L-arginine (L-NOARG, 100 microM). SIN-1 (10 microM, final blood concentration) also decreased whole blood platelet aggregation, but only in the presence of an unstimulated vessel ring, and this was unaffected by L-NOARG. Superoxide dismutase (150 u ml-1) did not influence the inhibition of aggregation by either a carbachol-stimulated vessel ring or by SIN-1. 5. Carbachol-stimulated artery rings from FHH rabbits inhibited platelet aggregation to a similar extent to that seen with rings from control normocholesterolaemic rabbits. Rings from hypercholesterolaemic NZW rabbits, however, did not significantly inhibit platelet aggregation when stimulated with carbachol. SIN-1 inhibited platelet aggregation in the presence of rings from either group of hypercholesterolaemic rabbits. 6. Hypercholesterolaemia induced by dietary modification induces changes in endothelial function which are characteristically different from those seen in genetically hypercholesterolaemic rabbits. It appears that dietary-induced hypercholesterolaemia primarily decreases NO release from the endothelium, while in genetically-induced hypercholesterolaemic vessel rings NO is released but there is a decreased responsiveness of the vascular smooth muscle cells to NO. This may reflect differences in the age and severity of the atherosclerotic lesions in the two groups of rabbits.

Animals↗
PubMed2002

Inhibitory effect of 3,4-diaryl-3-pyrrolin-2-one derivatives on cyclooxygenase 1 and 2 in murine peritoneal macrophages.

AIM: To develop a whole-cell assay based on murine peritoneal macrophages and evaluate the inhibitory effect of candidate compounds on cyclooxygenase-1 (COX-1) and COX-2. METHODS: Macrophages were stimulated with calcimycin or lipopolysaccharide (LPS) for various periods. Their abilities to convert endogenous arachidonic acid to 6-keto-PGF1alpha or PGE2 were examined by radioimmunoassay (RIA). RNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and COX-1/2 was detected by reverse transcription polymerase chain reaction (RT-PCR) using specific primers. RESULTS: Rofecoxib selectively inhibited LPS-induced, COX-2-derived PGE2 synthesis with an IC50 value of (4.7+/-0.5) nmol/L compared with maximum inhibitory ratio of 17.3 % for the inhibition of calcimycin induced, COX-1-derived 6-keto-PGF1alpha synthesis. Indomethacin exhibited dual inhibitory effects on COX-1 and COX-2 with IC50 of (4.7+/-1.1) nmol/L and (7.1+/-1.2) nmol/L, respectively. Two series of 17 compounds were tested. Most of compounds in series II showed comparable inhibitory activities to rofecoxib on COX-2. The relative position of the sulfonylphenyl group to the lactam carbonyl group has important effects on COX-2 inhibitory activity. CONCLUSION: The established whole cell assay is appropriate for drug-design oriented in vitro assay. 3,4-Diaryl-3-pyrrolin-2-one derivatives were proved to be prospective new type of COX-2 selective inhibitors.

Animals↗
PubMed1990

[Effect of dazoxiben on the metabolism of arachidonic acid in isolated porcine basilar arteries].

Dazoxiben, a selective TXA2 synthetase inhibitor, was studied in the incubating sections of porcine basilar arteries with arachidonic acid (AA) 50 mumols/L and calcimycin (calcium inophore A-23187) 50 mumol/L. TXB2 and 6-keto-PGF1 alpha were determined by radioimmunoassay. Leukotrienes (LT) were extracted and purified with SEP-PAK column, identified by HPLC and determined by bioassay with ileum of guinea pig. The results showed that the production of TXB2 was unaltered whether or not the incubation of arteries were induced by AA or calcimycin. Dazoxiben and indomethacin 0.05-50 mumols/L had no effects on the production of TXB2. However, dazoxiben 0.5, 5 and 50 mumols/L increased the production of 6-keto-PGF1 alpha by 16.3%, 19.0% and 30.7%, respectively. Indomethacin 0.5, 5 and 50 mumols/L decreased the production of 6-keto-PGF1 alpha by 22.3%, 24.9% and 24.0%, respectively. Meanwhile dazoxiben 1, 10 and 100 mumols/L decreased the production of LT by 33.4%, 45.6% and 66.4%, respectively. These results suggest that the protective effect of dazoxiben on the damages which resulted from brain ischemia may be related to the change of TAX2/PGI2 balance in the brain tissue as well as the inhibition of production of LT.

6-Ketoprostaglandin F1 alpha↗
PubMed1989

[The effect of the antiarrhythmic, prajmalium bitartrate, on human thrombocyte function].

Studies of the in vitro effects of the antiarrhythmic drug prajmalium bitartrate (PBT, Neo-Gilurytmal) showed inhibitory effects on platelet aggregation and thromboxane production. PBT inhibited the primary and secondary phases of aggregation induced by adrenaline (epinephrine) or adenosine diphosphate (ADP). Platelet aggregation stimulated with collagen, platelet activating factor (PAF), and the thromboxane mimetic 9,11-azo-prostaglandin H2 (U 44064) was inhibited. The secondary phase of aggregation induced by ristocetin and aggregation caused by arachidonic acid (AA) were inhibited in samples from some donors (responders) but not in others (nonresponders). Platelet aggregation by the ionophore calcimycine (A 23187) was not inhibited, but small doses of calcimycine abolished the PBT-induced inhibition of aggregation caused by ADP. Thromboxane production of platelets with collagen of ADP was inhibited by higher concentrations of PBT, whereas AA-induced thromboxane synthesis remained unchanged. The observed antiplatelet activities of PBT are thought to result from calcium and sodium channel blocking properties of the drug.

Ajmaline↗
PubMed1999

Mechanisms underlying the hemolytic and ichthyotoxic activities of maitotoxin.

Maitotoxin (MTX), a putative Ca(2+) channel activator produced by the dinoflagellate Gambierdiscus toxicus showed extremely potent hemolytic and ichthyotoxic activities. Hemolysis of 1% mouse blood cell suspension in saline occurred at 15 nM of MTX. The activity was enhanced six-fold in the presence of 10 microM of Ca(2+) and completely blocked by EDTA2Na, indicating its dependency on external Ca(2+). The MTX-induced hemolysis was little affected by L-type Ca(2+) channel blockers (diltiazem, nifedipine, verapamil) but was strongly inhibited by calmodulin blockers (prenylamine and chlorpromazine) or a phospholipase A2 inhibitor (quinacrine). MTX was mimicked by a calcium ionophore, calcimycin. Based on these results, a series of cellular events triggered by MTX were presumed to occur in the following sequence: increased Ca(2+) entry in cells, activation of calmodulin, promotion of phospholipase A2 activity, and finally destruction of cell membrane resulting from hydrolysis of membrane lipids. The sensitivity of blood cells to MTX varied significantly, dependent on the animal sources. Nucleated blood cells of carps and chickens were 100 times more resistant than those of mammals. LC(50) of MTX to freshwater fish Tanichthys albonubes in Ca(2+) free media (pH 8) was 5 nM but was markedly lowered to 3 pM by raising pH to 8 and increasing Ca(2+) concentration to 2 mM. In a marine environment MTX was 2000 times more toxic to fish than 42-di-hydrobrevetoxin-B (PbTx-3), one of the best known ichthyotoxins of red-tide origins.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1993

Effect of a potent selective protein kinase C inhibitor on histamine release from rat mast cells.

3,10-Dihydroxy-10-[(dimethylamino)methyl]-2,3,9,10,11,12-hexahydro-9- methyl-9,12-epoxy-1H-diindolo[1,2,3-fg-3',2',1'-k1]pyrrolo[3,4- l][1,6]benzodiazocin-1-one (UCN-01) strongly and dose-dependently inhibited histamine release from rat peritoneal mast cells that was induced by anti-immunoglobulin E (anti-IgE), calcimycin (A 23187), and 1,2-o-tetradecanoyl-13-acetate (TPA). The concentrations of UCN-01 required for 50% inhibition of histamine release induced by anti-IgE, A23187, and TPA were 1.5, 2.7, and 1.4 nM, respectively; these values are similar to those for 50% inhibition of protein kinase C. These results suggest possible participation cells induced by various secretagogues.

Alkaloids↗
PubMed1990

Cytotoxic enzyme release and oxygen centered radical formation in human neutrophils are selectively inhibited by E-type prostaglandins but not by PGI2.

The action of PGE1, PGE2, PGI2 and iloprost on superoxide anion generation, lysosomal enzyme release, and changes of Ca2+ fluxes in human polymorphonuclear leukocytes (PMN) was studied in vitro. Both PGE-type compounds were equipotent inhibitors of FMLP-and PAF-stimulated superoxide anion generation, beta-glucuronidase release (IC50 3-5 mumol/l) and Ca2+ influx while PGI2 and iloprost were ineffective at concentrations up to 10 mumol/l. These inhibitory actions of PGE1 and PGE2 were paralleled by an increase in cAMP level of the PMN while no change occurred with PGI2 and iloprost. None of the prostaglandins affected the initial intracellular Ca2+ liberation after challenge with FMLP or PAF. Preincubation of PMN with PGE1 and PGE2 but not with iloprost resulted in subsequent desensitization against a second administration of these compounds. None of the compounds affected PMN activation produced by arachidonic acid or calcimycin (A 23187). These data demonstrate that PGE-type compounds are effective inhibitors of receptor-mediated (PAF, FMLP) activation of human PMN while prostacyclins are considerably less potent. This suggests that the inhibitory prostaglandin receptor on human PMN belongs to the E-type being functionally different from the inhibitory prostaglandin receptor on human platelets. These results suggest that compounds, such as PGE1 and PGE2 might be superior to prostacyclins to prevent PMN-associated generation of reactive oxygen species and lysosomal enzyme release in situations with endogenous PMN activation, i.e. inflammatory reactions.

Arachidonic Acid↗
PubMed1984

Ionophoric activity of the antibiotic X.14547 A in the mitochondrial membrane.

Release of Ca++, Mg++ and K+ by the carboxylic ionophore X-14547 A was studied in the mitochondrial membrane. A comparison was made with A.23187 ( Calcimycin ) and X.537 A (Lasalocid A) under the same experimental conditions. It was shown that in this test system X.14547 A is primarily a K+ carrier comparable with X.537 A.

Animals↗
PubMed2000

Effect of carticaine on the sarcoplasmic reticulum Ca2+-dependent adenosine triphosphatase.

The sarcoplasmic reticulum Ca2+-ATPase (calcium-dependent adenosine triphosphatase) transports Ca2+ from the myoplasm to the reticulum lumen at the expense of free energy from ATP hydrolysis. Carticaine is a local anesthetic of frequent use in dentistry which is now entering other clinical fields. We studied the action of carticaine on the sarcoplasmic reticulum (SR) skeletal muscle Ca2+-ATPase. SR vesicles from rabbit fast skeletal muscle were used. Carticaine inhibits the enzymatic activity. The inhibition of the enzymatic activity depends on pH, [Ca2+] and the presence of calcimycin. Half-maximal carticaine concentration that inhibits the ATPase activity tends to a maximal value upon increasing [Ca2+]. Carticaine concentrations required to inhibit the enzymatic activity at myoplasmic calcium concentration are lower than usual clinical doses: Ki=6.0+/-1.4 mM carticaine (n=5) for 0.1 microM [Ca2+]. ATP-dependent calcium uptake is also inhibited by the local anesthetic: Ki=30.5+/-3.4 mM (n=4). Besides, carticaine inhibits the phosphorylation of the enzyme by inorganic phosphate (Pi): Ki=20.0+/-3.4 (n=5) - 33.2+/-4.6 (n=4) mM, for [Pi] 1-4 mM. Carticaine increases the membrane permeability to Ca2+. Ca2+ efflux from preloaded vesicles is prevented by Ca2+ and Mg2+. Our results suggest that the diffusion of the local anesthetic into muscle fibers might trigger undesired effects such as sustained contraction of the masticatory muscles.

Anesthetics, Local↗
PubMed1999

Evidence for a contribution of store-operated Ca2+ channels to NO-mediated endothelium-dependent relaxation of guinea-pig aorta in response to a Ca2+ ionophore, A23187.

A23187 (6S-[6alpha,8beta,9beta,11alpha]-5-(methylamino) -2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)ethyl]-1,7- dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazolecarboxylic acid, calcimycin), an antibiotic Ca2+ ionophore, produces an endothelium-dependent vascular relaxation. In the present study, pharmacological features were functionally characterized of endothelium-dependent relaxant response of guinea-pig aorta to A23187, especially focusing on the possible Ca2+ source and Ca2+ mobilization mechanisms in endothelial cells responsible for the vasorelaxant response to the Ca2+ ionophore. A23187-induced endothelium-dependent relaxation was suppressed profoundly by N(G)-nitro-L-arginine (L-NNA; 3 x 10(-4) M) or calmidazolium (3 x 10(-5) M), suggesting that nitric oxide (NO) produced by the enhanced activation of Ca2+/calmodulin-dependent endothelial NO synthase (eNOS) is largely responsible for the relaxant response of this artery to A23187. In the Ca2+-free solution without EGTA, NO-mediated endothelium-dependent relaxation induced by A23187 was almost abolished, which suggests that Ca2+ entry from extracellular space into endothelial cells plays the key role in the A23187-induced functional vasorelaxation. On the other hand, SK&F96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole; 5 x 10(-5) M) and Ni2+ (3 x 10(-4) M), both of which inhibit capacitative Ca2+ influx through store-operated Ca2+ channels (SOCCs), attenuated significantly NO-mediated endothelium-dependent relaxation by A23187. Furthermore, A23187-induced endothelium-dependent relaxation was suppressed more strongly than endothelium-independent relaxation induced by SIN-1 (3-morpholino-sydnonimine), an NO donor, when aortic preparation was preconstricted with high KCl instead of agonistic stimulation (prostaglandin F2alpha). These findings suggest that NO-mediated endothelium-dependent relaxant response of guinea-pig aorta to A23187 is preceded by the increase in endothelial cytosolic free Ca2+ concentration ([Ca2+]cyt) due to the enhanced Ca2+ influx from extracellular space. In the enhanced Ca2+ entry leading to the stimulation of eNOS and NO-mediated functional relaxant response of guinea-pig aorta to A23187, activation of SOCCs but not the Ca2+ entry through plasma membrane Ca2+-specific routes made by A23187 seems to play the predominant role. It is most likely that A23187 acts primarily at the Ca2+ store sites in endothelial cells, which subsequently depletes stored Ca2+ to activate SOCCs via unidentified mechanisms.

Animals↗
PubMed2001

Ca++ regulation of paracellular permeability in the middle intestine of the eel, Anguilla anguilla.

The role of Ca++ on the regulation of the paracellular pathway permeability of the middle intestine of Anguilla anguilla was studied by measuring the transepithelial resistance and the dilution potential, generated when one half of NaCl in the mucosal solution was substituted iso-osmotically with mannitol, in various experimental conditions altering extracellular and/or intracellular calcium levels. We found that removal of Ca++ in the presence of ethylene glycol-bis(beta-aminoethyl ether) (EGTA) from both the mucosal and the serosal side, but not from one side only, reduced both the transepithelial resistance and the magnitude of the dilution potential. The irreversibility of this effect suggests a destruction of the organization of the junction in the nominal absence of Ca++. However a modulatory role of extracellular Ca++ cannot be excluded. The decrease of the intracellular Ca++ activity, produced by using verapamil to block the Ca++ entry into the cell, or by adding 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (hydrochloride) (TMB-8), an inhibitor of Ca++ release from the intracellular stores, reduced both the transepithelial resistance and the magnitude of the dilution potential, indicating a role of cytosolic Ca++ in the modulation of the paracellular permeability. However the rise of calcium activity produced by the Ca++ ionophore calcimycin (A23187) evoked an identical effect, suggesting that any change in physiological intracellular Ca++ activity alters the paracellular permeability.

Anguilla↗
PubMed2006

Increase in intracellular Ca2+ concentration is not the only cause of lidocaine-induced cell damage in the cultured neurons of Lymnaea stagnalis.

PURPOSE: To determine whether the increase in intracellular Ca2+ concentration induced by lidocaine produces neurotoxicity, we compared morphological changes and Ca2+ concentrations, using fura-2 imaging, in the cultured neurons of Lymnaea stagnalis. METHODS: We used BAPTA-AM, a Ca2+ chelator, to prevent the increase in the intracellular Ca2+ concentration, and Calcimycin A23187, a Ca2+ ionophore, to identify the relationship between increased intracellular Ca(2+) concentrations and neuronal damage without lidocaine. Morphological changes were confirmed using trypan blue to stain the cells. RESULTS: Increasing the dose of lidocaine increased the intracellular Ca2+ concentration; however, there was no morphological damage to the cells in lidocaine at 3 x 10(-3) M. Lidocaine at 3 x 10(-2) M increased the intracellular Ca2+ concentration in both saline (from 238 +/- 63 to 1038 +/- 156 nM) and Ca2+-free medium (from 211 +/- 97 to 1046 +/- 169 nM) and produced morphological damage and shrinkage, with the formation of a rugged surface. With the addition of BAPTA-AM, lidocaine at 3 x 10(-2) M moderately increased the intracellular Ca2+ concentration (from 150 +/- 97 to 428 +/- 246 nM) and produced morphological damage. These morphologically changed cells were stained dark blue with trypan blue dye. The Ca2+ ionophore increased the intracellular Ca2+ concentration (from 277 +/- 191 to 1323 +/- 67 nM) and decreased it to 186 +/- 109 nM at 60 min. Morphological damage was not observed during the 60 min, but became apparent a few hours later. CONCLUSION: These results indicated that the increase in intracellular Ca2+ concentration is not the only cause of lidocaine-induced cell damage.

Anesthetics, Local↗
PubMed1990

How to evaluate the distribution of an "invisible" amphiphile between biological membranes and water.

To evaluate the distribution of an amphiphile or its binding to membranes whose properties are affected by such binding, it is only necessary to establish to what extent the dose-response to the amphiphile depends on the membrane concentration. The measured response only needs to reflect local events. This method of evaluation does not depend on the precise shape of the dose-response curve and is particularly useful for amphiphiles devoid of properties like fluorescence or radioactivity which would allow their direct assay. In this work, we establish the validity of this approach by comparing it with direct conventional determinations. Two parameters are especially suitable for such evaluation: the perturbation of an enzyme's activity, produced by many amphiphiles, and the fluorescence quenching of membrane-embedded proteins by chromophoric amphiphiles through long-range Förster transfer. We illustrate this approach in sarcoplasmic reticulum membranes containing Ca2(+)-ATPase as the main protein constituent. The equilibrium distribution of the antioxidant 4-nonylphenol was deduced from its inhibition of ATPase activity, whereas the equilibrium distribution of the calcium ionophore calcimycin (A23187) and of its brominated analog 4-bromo-A23187 were determined from their quenching of ATPase fluorescence. Apparent partition coefficients K* in the range of 10(5) (expressed as (moles of lipid/liter)-1) were obtained for these highly hydrophobic molecules.

Calcimycin↗