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PubMed2002

Studies on the role of calcium in the 5-HT-stimulated release of glutamate from C6 glioma cells.

We recently reported that 5-hydroxytryptamine(2A) (5-HT(2A)) receptor activation on cultured glial cells induces glutamate release [J. Neurosci. Res. 67 (2002) 399]. Here we use C6 glioma cells to examine the role of calcium in this response. 5-Hydroxytryptamine (5-HT) increases glutamate release from C6 glioma cells, an effect blocked by low calcium conditions. The calcium ionophores ionomycin and calcimycin also released glutamate from C6 glioma cells in a Ca(2+)-dependent manner. The effect of 5-HT was reduced by the phospholipase C inhibitor U73122 (1-[6[[(17 beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), but not its inactive enantomer U73343(1-[6[[(17 beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione). The protein kinase C inhibitors staurosporine and calphostin C had no effect on the response to 5-HT, whereas the response was blocked by thapsigargin and caffeine. Neither the L-type calcium channel blockers, nifedipine and verapamil, nor the N-type calcium channel blocker omega-conotoxin GVIA inhibited the effect of 5-HT, whereas NiCl(2) and KCl blocked the response to 5-HT. We conclude that the 5-HT-induced efflux of glutamate from C6 glioma cells is Ca(2+)-dependent and involves, at least in part, the mobilisation of Ca(2+) from inositol (1,4,5) tris phosphate (IP(3)) sensitive intracellular stores.

Animals↗
PubMed1996

Effect of haloperidol on the sarcoplasmic reticulum Ca-dependent adenosine triphosphatase.

Several effects of the neuroleptic agent haloperidol on the sarcoplasmic reticulum (SR) Ca-dependent adenosine triphosphatase (Ca-ATPase) and Ca transport are described. Haloperidol inhibits the Ca-ATPase activity in the presence of calcimycin. The effect depends on the conditions of preexposure of the membranes to the drug: the inhibition increases with the preincubation time; Ca and Mg protect the enzyme against the effect of the drug. The inhibitory effect of haloperidol decreases upon increasing [Ca2+], at constant [Mg], and disappears at 20 mM [Mg] for any [Ca2+], and at 0.5 mM [Ca2+] for any [Mg2+]. Haloperidol also inhibits phosphorylation of the enzyme by Pi, and ATP-dependent Ca2+ uptake, in both cases with apparent Ki = 0.10-0.15 mM, and increases the rate of Ca efflux from preloaded vesicles in this concentration range. The results suggest that haloperidol interacts with the catalytic site, interfering with the effect of the divalent catalytic cation, but not at other steps of the enzymatic cycle, where Mg2+ and Ca2+ are also activators. They are consistent with a reaction model where haloperidol interacts with the E2 conformers of the enzyme, with lower affinity for the phosphoenzyme than for the dephospho species. The inhibition of Ca uptake by SR vesicles is ascribed to an increased Ca2+ permeability rather than to the inhibition of the Ca-ATPase, which requires higher concentrations of the drug.

Animals↗
PubMed1999

Regulation of calcium-dependent [3H]noradrenaline release from rat cerebrocortical synaptosomes by protein kinase C and modulation of the actin cytoskeleton.

The effects that active phorbol esters, staurosporine, and changes in actin dynamics, might have on Ca2+ -dependent exocytosis of [3H]-labelled noradrenaline, induced by either membrane-depolarizing agents or a Ca2+ ionophore, have been examined in isolated nerve terminals in vitro. Depolarization-induced openings of voltage-dependent Ca2+ channels with 30 mM KCl or 1 mM 4-aminopyridine induced limited exocytosis of [3H]noradrenaline, presumably from a readily releasable vesicle pool. Application of the Ca2+ ionophore calcimycin (10 microM) induced more extensive [3H]noradrenaline release, presumably from intracellular reserve vesicles. Stimulation of protein kinase C with phorbol 12-myristate,13-acetate increased release evoked by all secretagogues. Staurosporine (1 microM) had no effect on depolarization-induced release, but decreased ionophore-induced release and reversed all effects of the phorbol ester. When release was induced by depolarization, internalization of the actin-destabilizing agent DNAase I into the synaptosomes gave a slight increase in [3H]NA release and strongly increased the potentiating effect of the phorbol ester. In contrast, when release was induced by the Ca2+ ionophore, DNAase I had no effect, either in the absence or presence of phorbol ester. The results indicate that depolarization of noradrenergic rat synaptosomes induces Ca2+ -dependent release from a releasable pool of staurosporine-insensitive vesicles. Activation of protein kinase C increases this release by staurosporine-sensitive mechanisms, and destabilization of the actin cytoskeleton further increases this effect of protein kinase C. In contrast, ionophore-induced noradrenaline release originates from a pool of staurosporine-sensitive vesicles, and although activation of protein kinase C increases release from this pool, DNAase I has no effect and also does not change the effect of protein kinase C. The results support the existence of two functionally distinct pools of secretory vesicles in noradrenergic CNS nerve terminals, which are regulated in distinct ways by protein kinase C and the actin cytoskeleton.

Actins↗
PubMedDate not supplied

A new method for in vitro detection of microbially produced mitochondrial toxins.

Sperm motility inhibition assay, earlier shown valuable for the detection of food poisoning non-protein toxins of Bacillus species was developed into an assay useful for specific detection of mitochondria damaging toxins. This was done by assessing the dissipation of the mitochondrial inner membrane transmembrane potential, Deltapsim under conditions where the plasma membrane permeability barrier remained intact. The Deltapsim was estimated as the intensity of orange JC-1 fluorescence in the mitochondrial sheath of the exposed spermatozoa. The plasma membrane integrity of the same cells was assessed by observing the exclusion of propidium iodide from the cytoplasm. Three types of mitochondrial toxic responses to microbially made bioactive substances were recognised. Mitochondrial toxicity by gramicidin (A, B, C, D), nigericin, salinomycin, narasin, monensin, calcimycin and antimycin A was characterised by gradual fading of the JC-1 fluorescence in the mitochondria. Dissipation of the Deltapsim by cereulide, valinomycin and enniatin (A, A1, B, B1) was visible as spotwise quenching of the mitochondrial JC-1 fluorescence. In addition these substances caused hyperpolarisation of the plasma membrane. Oligomycin (A, B, C), ionomycin and staurosporine inhibited the spermatozoan motility, but Deltapsim was fully preserved. Surfactin and lichenysin A caused mitochondrial damage at concentrations where the plasma membrane was also damaged.

Animals↗
PubMed1998

Protein kinase C induced changes in erythrocyte Na+/H+ exchange and cytosolic free calcium in humans.

To investigate the interrelationship between erythrocyte Na+/H+ exchange rate and free cytosolic Ca2+ concentration, the effect of the (non)selective protein kinase C inhibitors staurosporine, Ro 31-8220 and CGP 41251 (1 micromol/L) and of the protein kinase C activator phorbol-12-myristate-13-acetate (PMA, 1 micromol/L) was studied in vitro on these variables. PKC depleted erythrocytes were obtained after 24 h PMA down-regulation of the cells and intracellular Ca2+ clamping was obtained using quin-2 AM and fluo-3 AM. PMA increased (P < .05) the erythrocyte Na+/H+ exchange activity and this rise was accompanied by an increase in the free cytosolic Ca2+ concentration. When staurosporine and Ro 31-8220 were added to erythrocytes in suspension, a decrease in free cytosolic Ca2+ concentration was also found, whereas no significant change was observed after CGP 41251 administration. The Na+/H+ exchange rate was decreased in the 24 h PMA down-regulated erythrocytes as well as in Ca2+-clamped cells. Addition of Ca2+ in a concentration range of 0 to 1 mmol/L in the presence of calcimycin resulted (P < .001) in a stimulation of Na+/H+ exchange by 74%. Calcium increased the Vmax for cellular pHi or external Na+ activation of Na+/H+ exchange, whereas it did not affect the Km for H+(i) or external Na+ activation. However, in PKC down-regulated cells, calcium did not activate the Na+/H+ exchange in erythrocytes and the calpain inhibitor E-64d did not prevent this inactivation. Our data show a concomitant increase in free cytosolic Ca2+ concentration and Na+/H+-exchange rate upon protein kinase C activation and a corresponding decrease in both variables upon PKC inhibition, indicating a Ca2+ requirement for protein kinase C activation of Na+/H+ exchange.

Calcium↗
PubMed2002

Evaluation of the separate equilibrium processes that dictate the upper detection limit of neutral ionophore-based potentiometric sensors.

The upper detection limit of polar ionophore-based ion-selective electrode membranes is predicted by utilizing the coextraction constant of dissociated electrolyte, the stability constant of the ionophore, and the membrane composition. The coextraction constant of dissociated electrolytes into the polar poly(vinyl chloride) membrane plasticized with o-nitrophenyl octyl ether (PVC-NPOE) is here measured by a novel approach. The sandwich membrane technique is utilized, with one membrane segment containing a lipophilic cation exchanger and the other containing an anion exchanger. This yields information about the coextraction constant and the free ion concentrations of the electrolyte in the two segments. Predictions correlate quantitatively with the upper detection limit observed for ion-selective electrodes based on the ionophores valinomycin, tert-butylcalix[4]arene tetraethyl ester, and calcimycin. The difficulties of the prediction of the upper detection limit for nonpolar poly(vinyl chloride) membranes plasticized with bis(2-ethylhexyl sebacate) (PVC-DOS) due to ion association are discussed in detail. A thermodynamic cycle experiment with a series of sandwich membranes shows that the principal processes governing the upper detection limit of PVC-DOS membranes are identical to those for the PVC-NPOE membranes. However, the stability of the ion pairs between the ionophore-metal ion complexes and the extracted anion are different from that of ion pairs formed between the same anion and the lipophilic anion exchanger. This makes it difficult to quantitatively predict the upper detection limit on the basis of simple apparent coextraction and complexation data alone. The approach reported herein is useful not only for mechanistic purposes but also to shed light onto the many cases where coextraction effects need to be understood but are not directly experimentally accessible.

Algorithms↗
PubMed1998

Differentiation-specific increase in ALA-induced protoporphyrin IX accumulation in primary mouse keratinocytes.

A treatment regimen that takes advantage of the induction of intracellular porphyrins such as protoporphyrin IX (PPIX) by exposure to exogenous 5-amino-laevulinic acid (ALA) followed by localized exposure to visible light represents a promising new approach to photodynamic therapy (PDT). Acting upon the suggestion that the effectiveness of ALA-dependent PDT may depend upon the state of cellular differentiation, we investigated the effect of terminal differentiation upon ALA-induced synthesis of and the subsequent phototoxicity attributable to PPIX in primary mouse keratinocytes. Induction of keratinocyte differentiation augmented intracellular PPIX accumulation in cells treated with ALA. These elevated PPIX levels resulted in an enhanced lethal photodynamic sensitization of differentiated cells. The differentiation-dependent increase in cellular PPIX levels resulted from several factors including: (a) increased ALA uptake, (b) enhanced PPIX production and (c) decreased PPIX export into the culture media. Simultaneously, steady-state levels of coproporphyrinogen oxidase mRNA increased but aminolaevulinic acid dehydratase mRNA levels remained unchanged. From experiments using 12-o-tetradecanoylphorbol-13-acetate, transforming growth factor beta 1 and calcimycin we demonstrated that the increase in PPIX concentration in terminally differentiating keratinocytes is calcium- and differentiation specific. Stimulation of the haem synthetic capacity is seen in primary keratinocytes, but not in PAM 212 cells that fail to undergo differentiation. Interestingly, increased PPIX formation and elevated coproporphyrinogen oxidase mRNA levels are not limited to differentiating keratinocytes; these were also elevated in the C2C12 myoblast and the PC12 adrenal cell lines upon induction of differentiation. Overall, the therapeutic implications of these results are that the effectiveness of ALA-dependent PDT depends on the differentiation status of the cell and that this may enable selective targeting of several tissue types.

Aminolevulinic Acid↗
PubMed1997

Characterization of endothelium-derived relaxing factors released by bradykinin in human resistance arteries.

1. Relaxing factors released by the endothelium and their relative contribution to the endothelium-dependent relaxation produced by bradykinin (BK) in comparison with different vasodilator agents were investigated in human omental resistance arteries. 2. BK produced an endothelium-dependent relaxation of arteries pre-contracted with the thromboxane A2 agonist, U46619. The B2 receptor antagonist, Hoe 140 (0.1, 1 and 10 microM), produced a parallel shift to the right of the concentration-response curve to BK with a pA2 of 7.75. 3. Neither the cyclo-oxygenase inhibitor, indomethacin (10 microM) alone, the nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME, 300 microM) alone, the nitric oxide scavenger, oxyhaemoglobin (Hb, 10 microM) alone, nor the combination of L-NAME plus Hb affected the concentration-response curve to BK. Conversely, the combination of indomethacin with either L-NAME or Hb attenuated but did not abolish the BK-induced relaxation. By contrast, the relaxations produced by the Ca2+ ionophore, calcimycin (A23187), and by the inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase, thapsigargin (THAPS), were abolished in the presence of indomethacin plus L-NAME. Also, the presence of indomethacin plus L-NAME produced contraction of arteries with functional endothelium. 4. The indomethacin plus L-NAME resistant component of BK relaxation was abolished in physiological solution (PSS) containing 40 mM KCl and vice versa. However, in the presence of KCl 40 mM, indomethacin plus L-NAME did not affect the nitric oxide donor, S-N-acetylpenicillamine-induced relaxation. 5. The indomethacin plus L-NAME resistant component of the relaxation to BK was significantly attenuated by the K+ channel blocker tetrabutylammonium (TBA, 1 mM). However, it was not affected by other K+ channel blockers such as apamin (10 microM), 4-aminopyridine (100 microM), glibenclamide (10 microM), tetraethylammonium (10 mM) and charybdotoxin (50 nM). 6. In the presence of indomethacin plus L-NAME, the relaxation produced by BK was not affected by the phospholipase A2 inhibitor, quinacrine (10 microM) or by the inhibitor of cytochrome P450, SKF 525a (10 microM). Another cytochrome P450 inhibitor, clotrimazole (10 microM) which also inhibits K+ channels, inhibited the relaxation to BK. 7. These results show that BK induces endothelium-dependent relaxation in human small omental arteries via multiple mechanisms involving nitric oxide, cyclo-oxygenase derived prostanoid(s) and another factor (probably an endothelium-derived hyperpolarizing factor). They indicate that nitric oxide and cyclo-oxygenase derivative(s) can substitute for each other in producing relaxation and that the third component is not a metabolite of arachidonic acid, formed through the cytochrome P-450 pathway, in these arteries.

Arteries↗
PubMed1998

Direct dopamine D2-receptor-mediated modulation of arachidonic acid release in transfected CHO cells without the concomitant administration of a Ca2+-mobilizing agent.

In CHO cells transfected with the rat dopamine D2 receptor (long isoform), administration of dopamine per se elicited a concentration-dependent increase in arachidonic acid (AA) release. The maximal effect was 197% of controls (EC50=25 nM). The partial D2 receptor agonist, (-)-(3-hydroxyphenyl)-N-n-propylpiperidine [(-)-3-PPP], also induced AA release, but with somewhat lower efficacy (maximal effect: 165%; EC50=91 nM). The AA-releasing effect of dopamine was counteracted by pertussis toxin, by the inhibitor of intracellular Ca2+ release, 8-(N N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), by excluding calcium from the medium, by the phospholipase A2 (PLA2) inhibitor, quinacrine, and by long-term pretreatment with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). In addition, it was antagonized by the D2 antagonists, raclopride and (-)-sulpiride--but not by (+)-sulpiride--and absent in sham-transfected CHO cells devoid of D2 receptors. The results obtained contrast to the previous notion that dopamine and other D2 receptor agonists require the concomitant administration of calcium-mobilizing agents such as ATP, ionophore A-23187 (calcimycin), thrombin, and TRH, to influence AA release from various cell lines.

Adenosine Triphosphate↗
PubMed1998

Activation by calcium of erythrocyte Na+/H+ exchange in men.

OBJECTIVE: To determine whether protein kinase C is necessary for the calcium activation of the Na+/H+ exchange in human erythrocytes by studying activation by calcium of erythrocyte Na+/H+ exchange in control cells, in protein kinase C-depleted cells after downregulation of protein kinase C with phorbol-12-myristate-13-acetate and in cells that had been treated beforehand with phorbol-12-myristate-13-acetate with and without the calpain inhibitor E-64d. METHODS: Erythrocyte Na+/H+ exchange was measured by determining the initial rates of the influx of Na+ into Na+-depleted, acid loaded cells. The effects of various concentrations (0-1 mmol/l) of CaCl2 and the effects of 1 mmol/l CaCl2 on activation of the intracellular pH and on the external Na+ activation of Na+/H+ exchange were studied. The effects of 1 mmol/l CaCl2 on Na+/H+ exchange in control cells and cells that had been incubated beforehand with and without 1 micromol/l phorbol-12-myristate-13-acetate and with E-64d and 1 micromol/l phorbol-12-myristate-13-acetate for 1, 2, 3 and 24 h were also investigated. RESULTS: Addition of Ca2+ to a concentration in the range 0-1 mmol/l in the presence of calcimycin resulted in stimulation of Na+/H+ exchange: 1 mmol/l CaCl2 increased (P< 0.001) the erythrocyte Na+/H+ exchange by 74%. Calcium increased the maximum rate for activations by intracellular pH and by external Na+ of Na+/H+ exchange, whereas it did not affect the Michaelis-Menten constants for activation by intracellular H+ and external Na+. However, calcium did not activate the Na+/H+ exchange in protein kinase C downregulated erythrocytes and administration of the calpain inhibitor E-64d could not prevent this inactivation. CONCLUSION: Our data indicate that protein kinase C is necessary for the activation by calcium of the erythrocyte Na+/H+ exchange.

Calcium↗
PubMed1994

Mobile ionophores are a novel class of P-glycoprotein inhibitors. The effects of ionophores on 4'-O-tetrahydropyranyl-adriamycin incorporation in K562 drug-resistant cells.

The decrease of the intracellular concentration of drug in resistant cells compared to sensitive cells is, in most cases, correlated with the presence, in the membrane of resistant cells, of a 170-kDa P-glycoprotein responsible for an active efflux of the drug. In an attempt to identify mechanism(s) by which multidrug resistance can be circumvented, we have examined the cellular accumulation of 4'-O-tetrahydropyranyl-adriamycin, alone and in conjunction with various ionophores on the one hand and with cyclosporin A on the other hand. The present study was performed using a spectrofluorometric method with which it is possible to follow continuously the uptake and release of fluorescent molecules by living cells, as the incubation of the cells with the drug proceeds. Erythroleukemia K562 cell lines were used. Using experimental conditions in which these ionophores were unable to modify either the intracellular pH, or the transmembrane potential, or to induce an intracellular ATP depletion, we have shown that mobile ionophores as well as cyclosporin inhibit the P-glycoprotein-mediated efflux of 4'-O-tetrahydropyranyl-adriamycin in K562 resistant cells, whereas gramicidin, a channel-forming ionophore, does not. The concentration that must be used to inhibit 50% of the efflux was 0.7 microM for valinomycin, 0.4 microM for nonactin, 0.2 microM for nigericin, 1.1 microM for monensin, 0.4 microM for lasalocid, 1.2 microM for calcimycin and 0.4 microM for cyclosporin. Due to the high toxicity of the ionophores, the observation that they increased 4'-O-tetrahydropyranyl-adriamycin accumulation in the multidrug-resistant cells is not correlated with an effect of these compounds on drug resistance. However, the correlation exists in the case of cyclosporin. From our data showing that lipophilic neutral complexes, formed between carboxylic ionophores and metal ions, are both able to inhibit the P-glycoprotein-mediated efflux of anthracycline we can infer that the lipophilicity but not the cationic charge is an important physical property.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Evidence for differential roles of nitric oxide (NO) and hyperpolarization in endothelium-dependent relaxation of pig isolated coronary artery.

1. The possible roles of endothelial and smooth muscle cell hyperpolarization and nitric oxide (NO) in endothelium-dependent relaxation were examined in isolated rings of pig right coronary artery. 2. The effects of hyperpolarization were prevented with high K+ (30-125 mM), isotonic Krebs solutions. Functional antagonism due to high K(+)-induced smooth muscle contraction was prevented with 0.3 microM nifedipine (in all treatments, for consistency). All rings were contracted with the thromboxane-mimetic U46619, (1-100 nM) to bring them to an initial active force of within 30-50% of maximum contraction. 3. High K+ had no effects on the sensitivity (EC50) or time course of endothelium-dependent (substance P, SP; bradykinin, BK; calcimycin, A23187) and -independent (sodium nitroprusside, SNP) agents. Maximum relaxations (Rmax) to SP, BK and A23187 were reduced significantly by approximately 20% but only with 125 mM K+. 4. In normal K+ Krebs solution (5.9 mM), NG-nitro-L-arginine (L-NOARG; 100 microM) caused 40%, 20% and no reduction in Rmax for SP, BK and SNP respectively. EC50s for SP and BK were decreased significantly by approximately two fold whereas that for SNP was increased significantly by approximately ten fold. At all high K+ concentrations (30-125 mM), L-NOARG (100 microM) caused complete inhibition of relaxations to SP and BK but those to SNP were unaffected. 5. High K+ (30 mM) unmasked potent and concentration-dependent inhibition of relaxations of SP by L-NOARG. At 10 microM L-NOARG, all relaxation responses to SP were abolished and at the higher concentrations of SP (1-10 nM) small but significant contractions were observed. 6. N0-monomethyl-L-arginine (L-NMMA) had similar effects on relaxations to SP in the presence of 30 mM K+ except that maximum inhibition (40%) of Rmax was achieved at 10 MicroM L-NMMA and this was not increased with either 100 or 1000 MicroM L-NMMA. In normal K+, L-NMMA (1000 MicroM) only decreased the EC50 by approximately two fold, without affecting Rmax.7. High choline+ (25, 75 and 125 mM) isotonic Krebs also had no direct effect on the relaxations to SP,but like high K+, enabled L-NOARG (100 MicroM) to inhibit these responses completely. Neither charybdotoxin(30 nM) nor substitution of 25 mM NaCl with 50 mM sucrose had any direct effect on relaxations to SP or on the block of relaxations to SP by L-NOARG (100 MicroM).8. In conclusion, most if not all of the endothelium-dependent relaxation in the pig coronary artery in vitro is due to NO, but hyperpolarization can supplement 60% -80% of this response if NO synthesis is blocked. Multiple endothelium-derived factors could not only explain heterogeneity of the degree of block of endothelium-dependent relaxation responses by L-arginine analogues, but also constitute important 'back-up' mechanisms for control of arterial diameter.

Animals↗
PubMed2005

Evaluation of 2 celecoxib derivatives: analgesic effect and selectivity to cyclooxygenase-2/1.

AIM: To evaluate the analgesic effects of 2 celecoxib derivatives and their inhibitory effects on cyclooxygenase (COX). METHODS: Four antinociceptive assays were used: the acetic acid-induced writhing test, hot plate test, hot tail-flick test and formalin test. Three doses were used in the analgesic assays and ED50 values were calculated. For the selectivity assay, macrophages were incubated with test compounds at various concentrations and then stimulated with calcimycin or lipopolysaccharide (LPS). The amounts of 6-keto-prostaglandin F1alpha(6-keto-PGF1alpha) and prostaglandin E2 (PGE2) in the supernatant were examined by radioimmunoassay (RIA). The selectivity of the test compounds was expressed as the IC(50,COX-1)/IC(50,COX-2) value. RESULTS: Celecoxib and its 2 derivatives had a significant analgesic effect. The ED50 values of celecoxib, PC-406 and PC-407 were 94.2, 67.9, and 63.3 mg/kg, respectively, for the acetic acid-induced writhing test; 104.7, 89.1, and 30.0 mg/kg, respectively, for the hot tail-flick response test; 60.7, 56.7, and 86.2 mg/kg, respectively, for the hot plate response test; 67.1, 55.8, and 68.8 mg/kg, respectively, for the formalin-induced response. That is, the ED50 of PC-406 was the lowest for the formalin and hot plate tests, which focus on changes above the spinal cord level; however, the ED50 of PC-407 was lowest for the tail-flick and writhing tests, which focus on changes at the spinal cord level. Celecoxib and PC-407 inhibited COX-1 with IC50 values of 39.8 and 27.5 nmol/L, respectively. PC-406 inhibited COX-1 with an IC50 value of more than 1000 nmol/L. The IC50 values for the effect of celecoxib, PC-406 and PC-407 on COX-2 were 4.8, 8.9, and 1.9 nmol/L respectively. The IC(50, COX-1)/IC(50,COX-2) ratios for celecoxib and PC-407 were 8.3 and 14.4, respec-tively. For PC-406, the ratio was greater than 112.2. CONCLUSION: Derivatives of celecoxib via substitution with an isopropyl or naphthyl group at the 5 position in the pyrazole ring still have analgesic effects and the ability to selectively inhibit COX-2. Substitution with a naphthyl group may have more effect on the peripheral pain pathway, whereas substitution with an isopropyl group may have more effect on the central pain pathway. This phenomenon occurs partly because substitution with an isopropyl group is more beneficial for COX-2 selectivity than is substitution with a naphthyl group.

6-Ketoprostaglandin F1 alpha↗
PubMed2002

Origins of prostaglandin E2: involvements of cyclooxygenase (COX)-1 and COX-2 in human and rat systems.

Prostaglandin (PG) E2 is a major cyclooxygenase (COX) product at inflammatory sites where it contributes to local increases in blood flow, edema formation, and pain sensitization. Using rats in vivo and rat and human blood in vitro, we have examined the roles of COX-1 and COX-2 in the production of PGE2. In anesthetized rats treated with bacterial lipopolysaccharide (LPS) to induce the expression of COX-2, the marked increase in PGE2 production that followed bolus intravenous injection of arachidonic acid (3 mg x kg(-1)) was strongly inhibited by diclofenac but largely unaffected by the COX-2-selective inhibitor DFP (5,5- dimethyl-3-(2-propoxy)-4-methanesulfonylphenyl)-2(5H)-furanone). In rat blood in vitro, aspirin strongly inhibited the production of PGE2 that followed either acute exposure to calcium ionophore, A23187 (calcimycin) (50 microM, 15 min), or incubation with LPS for 18 h. In contrast, human whole blood only produced significant levels of PGE2 when incubated with LPS. Rat leukocytes expressed COX-2 and produced PGE2 when exposed to LPS but not when acutely stimulated with A23187. Rat platelets, but not human platelets, also produced significant amounts of PGE2 when acutely stimulated with A23187. These data show that when exposed to an inflammatory stimulus, rat whole blood produces increased levels of PGE2 through induction of COX-2 in blood leukocytes. Rat blood, unlike human blood, may also produce copious amounts of PGE2 via the actions of COX-1 enzyme constitutively present in platelets. These data may well explain why in rats COX-2-selective inhibitors have been reported not to produce the full anti-inflammatory effects associated with standard nonsteroid anti-inflammatory drugs.

Adult↗
PubMed2003

Modulatory effect of bolinaquinone, a marine sesquiterpenoid, on acute and chronic inflammatory processes.

The marine metabolite bolinaquinone is a novel inhibitor of secretory phospholipase A(2) (sPLA(2)), with a potency on the human synovial enzyme (group II) higher than that of manoalide. This activity on the sPLA(2) was confirmed in vivo in the 8-h zymosan rat air pouch on the secretory enzyme accumulation in the pouch exudate. Additionally, bolinaquinone decreased potently the synthesis and release of leukotriene B(4) (LTB(4)) in calcimycin (A23187)-stimulated human neutrophils as a consequence of the inhibition of 5-lipoxygenase activity, as well as PGE(2) and NO production on zymosan-stimulated mouse peritoneal macrophages. This compound exerted anti-inflammatory effects by topical and oral routes on the mouse ear edema induced by 12-O-tetradecanoylphorbolacetate, with ID(50) values of 76.7 microg/ear and 5.6 mg/kg, respectively, with a significant decrease in PGE(2), LTB(4), and tumor necrosis factor-alpha (TNF-alpha) levels being more effective than indomethacin. This effect was confirmed in the mouse paw carrageenan edema after oral administration. Moreover, bolinaquinone was able to reduce the inflammatory response of adjuvant arthritis by inhibiting PGE(2), NO, and TNF-alpha production in paw homogenates without affecting PGE(2) levels in the stomach. Additionally, bolinaquinone inhibited inducible nitric oxide synthase expression and reduced the degree of bone resorption, soft tissue swelling, and osteophyte formation.

Animals↗
PubMed2003

Endoplasmic reticulum stress due to altered cellular redox status positively regulates murine hepatic CYP2A5 expression.

Murine hepatic cytochrome P450 2A5 (CYP2A5) is uniquely induced by a variety of agents that cause liver injury and inflammation, conditions that are typically associated with downregulation of P450s. We hypothesized that induction of CYP2A5 occurs in response to hepatocellular damage resulting in endoplasmic reticulum (ER) stress. Treatment of mice in vivo and mouse hepatocytes in primary culture with the CYP2A5 inducer pyrazole resulted in overexpression of the ER stress biomarker glucose-regulated protein (GRP) 78. Treatment of primary hepatocytes with ER stress activators thapsigargin, tunicamycin, and trans-4,5-dihydroxy-1,2-dithiane (DTT(ox)) and the calcium ionophore A23187 (calcimycin) resulted in elevated GRP78 mRNA levels; however, only the reducing agent DTT(ox) induced levels of CYP2A5 mRNA, protein, and coumarin 7-hydroxylase activity. To test the hypothesis that CYP2A5 induction is due to liver injury resulting from altered cellular redox status, we demonstrated that CYP2A5 induction, elevated serum alanine aminotransferase, and oxidative protein damage occur concurrently in pyrazole-treated mice. Pyrazole also induced the expression of cytosolic alpha and mu class glutathione S-transferase expression both in vivo and in primary mouse hepatocytes. Moreover, treatment of hepatocytes with the redox cycling quinone menadione resulted in overexpression of CYP2A5 and GSTM1 mRNA. Finally, pretreatment of hepatocytes with the antioxidants N-acetylcysteine and vitamin E attenuated pyrazole-mediated increases in CYP2A5 mRNA levels. These findings clearly indicate that induction of mouse hepatic CYP2A5 during liver injury occurs via a novel mechanism involving ER stress due to altered cellular redox status.

Animals↗
PubMed2005

DAF-FM (4-amino-5-methylamino-2',7'-difluorofluorescein) diacetate detects impairment of agonist-stimulated nitric oxide synthesis by elevated glucose in human vascular endothelial cells: reversal by vitamin C and L-sepiapterin.

Elevated plasma glucose, as commonly seen in types I and II diabetes mellitus, is known to result in endothelial dysfunction, a condition characterized by a loss of nitric oxide (NO)-dependent regulation of vascular tone. In the present study, we have utilized a recently developed NO-sensitive fluorescent dye, DAF-FM (4-amino-5-methylamino-2',7'-difluorofluorescein) diacetate to directly examine the consequences of elevated glucose on agonist-evoked NO synthesis in cultured human vascular endothelial cells. Exposure of cells for 5 to 7 days to high (20 mM) external glucose markedly reduced NO production in response to ATP, histamine, or the calcium ionophore calcimycin A23187 compared with 5 and 10 mM glucose concentrations. However, high glucose did not affect agonist-evoked elevations in cytosolic-free calcium, as monitored by Fluo-3. The addition of vitamin C (150 microM) and L-sepiapterin (20 microM) for approximately 24 h to 20 mM glucose-treated cells improved stimulus-evoked NO synthesis but had no effect on cells exposed to either 5 or 10 mM glucose. Likewise, impaired NO production in high glucose-treated cells was largely reversed by exposure ( approximately 3 h) to superoxide dismutase. Cellular levels of endothelial nitric-oxide synthase protein were unaltered by elevated glucose treatment, and no further change was observed after the addition of vitamin C and l-sepiapterin. Taken together, the results of our study serve to directly explain at the cellular level how glucose-impaired NO production in human endothelial cells may be reversed by agents that are reported clinically to improve endothelium-dependent vasorelaxation in patients.

Adenosine Triphosphate↗
PubMed2002

Leukotriene B4 production in human mononuclear phagocytes is modulated by interleukin-4-induced 15-lipoxygenase.

The aim of this study was to evaluate the consequences of interleukin (IL)-4-induced 15-lipoxygenase (15-LO) expression on leukotriene B4 (LTB4) synthesis in human monocytes. Human monocytes incubated for 24, 48, and 72 h with IL-4 (10 ng/ml) were stimulated with Ca2+-ionophore A23187 (calcimycin; 5 microM) or opsonized zymosan. 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], LTB4, and arachidonic acid (AA) release were measured by high-performance liquid chromatography/radioimmunoassay, liquid chromatography/tandem mass spectrometry (LC/MS/MS), or gas chromatography/mass spectrometry. 15-LO activity was evaluated in AA-treated monocytes. 15-LO, 5-lipoxygenase (5-LO) and 5-LO activating protein (FLAP) expression were analyzed by reverse transcription-polymerase chain reaction. Neutrophil chemotactic activity was evaluated using a microtaxis chamber assay. A23187-induced synthesis of 15(S)-HETE was significantly increased after treatment with IL-4 (10 ng/ml) for 48 and 72 h (p < 0.001). Concomitant decrease of LTB4 release was observed after 72 h of incubation with IL-4 (p < 0.001). LC/MS/MS analysis confirmed the production of 15(S)-HETE and the significant inhibition of LTB4 synthesis in IL-4-treated monocyte after challenge with opsonized zymosan. IL-4 treatment induced 15-LO enzymatic activity as well as 15-LO mRNA, but did not affect either 5-LO or FLAP mRNA expression in monocytes. Supernatant from IL-4-treated monocytes showed significantly lower neutrophil chemotactic activity than controls. 15(S)-HETE significantly inhibited LTB4 production induced by A23187-stimulated human monocytes without affecting AA release. IL-4-induced expression of 15-LO in monocytes caused a significant reduction of LTB4 production. Whereas this effect did not reflect changes in 5-LO and FLAP mRNA expression, synthetic 15(S)-HETE was able to significantly inhibit the synthesis of LTB4, without affecting AA release.

Anti-Inflammatory Agents, Non-Steroidal↗