Role of sodium in intracellular calcium elevation and leukotriene B4 formation by receptor-mediated activation of human neutrophils

The role of Na(+) and Na(+) exchangers in intracellular Ca(2+) elevation and leukotriene B(4) (LTBs) formation was investigated in granulocyte macrophage colony-stimulating factor (GM-CSF)-primed, fMLP-stimulated human neutrophils. Isotonic substitution of extracellular Na(+) with N-methyl-D-glucamine(+) (NMDG(+)) resulted in over 85% inhibition of the LTBs generation observed (from 14.1+/-0.9pmol/10(6) neutrophils to 1.7+/-1.0pmol/10(6) neutrophils at 0.3 microM fMLP). Isotonic substitution of Na(+) with NMDG(+) also induced a significant inhibition of fMLP-induced rise in cytosolic Ca(2+) concentration ([Ca(2+)](i)) (from 2.17- to 0.78-fold increase over basal levels). Pretreatment with an inhibitor of the Na(+)/Ca(2+) exchanger (benzamil) did not inhibit either [Ca(2+)](i) rise or LTBs production, indicating that the observed effects of extracellular Na(+)-deprivation were unrelated to the Na(+)/Ca(2+) exchanger in receptor-mediated Ca(2+) influx, as previously hypothesized. LTBs production by thapsigargin-activated neutrophils was not affected by Na(+) depletion, but was totally abolished in the presence of EGTA, suggesting that store depletion-driven extracellular Ca(2+) influx is required for leukotriene synthesis and that this process is independent of Na(+)-deprivation. Exposure to Na(+)-free medium for the time of GM-CSF priming led to a significant decrease of intracellular pH values, suggesting a role of the Na(+)/H(+) exchanger in intracellular Na(+) depletion. Reducing the time of Na(+)-deprivation totally reversed the observed effect on LTBs production, resulting in enhanced, rather than inhibited, formation of LTBs. These results indicate that LTBs generation and [Ca(2+)](i) rise in human neutrophils primed by GM-CSF and stimulated with fMLP is dependent on intracellular Na(+) concentration, and, at variance with previously published results, unrelated to the Ca(2+) influx through the Na(+)/Ca(2+) exchanger.     

Inhibition by adenine dinucleotides of ATP-induced prostacyclin release by bovine aortic endothelial cells

Adenine dinucleotides are a group of extracellular modulators involved in maintaining blood vessel tone. We have demonstrated previously that Ap2A and Ap4A induce the synthesis of both nitric oxide (NO) and prostacyclin (PGI2) in bovine aortic endothelial cells (BAEC), whereas Ap3A, Ap5A, and Ap6A do not. In this paper, we report that Ap2A and Ap4A are partial agonists for ATP in terms of Ca2+ mobilization and PGI2 synthesis. The Ap(4)A EC50 values for Ca2+ mobilization and PGI2 synthesis were significantly higher than the corresponding values for ATP, while the Ap4A B(max) values for Ca2+ mobilization and PGI2 synthesis were significantly lower than those for ATP. Ap2A and Ap4A concentration-effect curves for Ca2+ mobilization and PGI2 synthesis demonstrated that Ap2A and Ap4A have antagonistic effects at ATP concentrations that induce responses above the maximal amount of Ca2+ mobilized or PGI2 synthesized by these two dinucleotides. On the other hand, Ap2A and Ap4A have agonistic effects at ATP concentrations that induce PGI2 synthesis below the maximal amount of PGI2 synthesized by these two dinucleotides. We also present evidence that suggests Ap3A, Ap5A, and Ap6A are antagonists for ATP in terms of PGI2 synthesis. All these data are consistent with the adenine dinucleotides being negative modulators for ATP-induced PGI2 synthesis.     

The 21-aminosteroid U74389G prevents the down-regulation and decrease in activity of CYP1A1, 1A2 and 3A6 induced by an inflammatory reaction

In vivo, the 21-aminosteroid U74389G prevents the decrease in cytochrome P450 (P450) activity produced by a turpentine-induced inflammatory reaction (TIIR). To investigate the underlying mechanism of action, four groups of rabbits were used, controls receiving or not U74389G, and rabbits with the inflammatory reaction receiving or not U74389G. Hepatocytes were isolated 48h later and incubated for 4 and 24h with the serum of the rabbits. In vivo, the TIIR diminished CYP1A1/2 and 3A6 expression, and enhanced hepatic malondialdehyde (MDA) and nitric oxide (NO*) concentrations (p<0.05). U74389G prevented the increase in MDA, as well as the decrease in CYP1A1/2 amounts and activity, but increased CYP3A6 expression by 40% (p<0.05). In vitro, compared with serum from control rabbits (S(CONT)), incubation of serum from rabbits with TIIR (S(TIIR)) for 4 and 24h with hepatocytes from rabbits with TIIR (H(TIIR)) reduced CYP1A2 and CYP3A6 activity (p<0.05) and increased the formation of NO* and MDA. In rabbits with TIIR pretreated with U74389G, the S(TIIR+U) failed to reduce CYP1A2 activity or to increase MDA, although increased NO* and further reduced CYP3A6 activity. On the other hand, in hepatocytes harvested from rabbits with TIIR pretreated with U74389G, S(TIIR) did not decrease CYP1A2 activity and did not enhance MDA, but still increased NO*. In vitro, the reduction of CYP1A2 and CYP3A6 activity by S(TIIR) is not associated to NF-kappaB activation. In conclusion, U74389G prevents CYP1A1/2 down-regulation and decrease in activity by a double mechanism: hindering the release of serum mediators and by averting intracellular events, effect possibly associated with its antioxidant activity. On the other hand, U74389G up-regulates CYP3A6 but inhibits its catalytic activity.     

Signal transduction pathways implicated in the decrease in CYP1A1, 1A2 and 3A6 activity produced by serum from rabbits and humans with an inflammatory reaction

Incubation of serum from rabbits with a turpentine-induced inflammatory reaction and from humans with an upper respiratory viral infection with hepatocytes from rabbits with a turpentine-induced inflammatory reaction for 4h reduces total cytochrome P450 content and activity of cytochrome P450 isoforms CYP1A1/1A2 and 3A6 without affecting the expression of these proteins. To document the signal transduction pathways implicated in the decrease in CYP1A1/1A2 and 3A6 activity, hepatocytes from rabbits with a turpentine-induced inflammatory reaction were incubated with serum from rabbits with a turpentine-induced inflammatory reaction, serum from individuals with a viral infection and interleukin-6 for 4h in presence of inhibitors of protein kinases. The sera-induced decrease in CYP1A1/1A2 and 3A6 activity was partially prevented by the inhibition of Janus-associated protein tyrosine kinase, double-stranded RNA-dependent protein kinase, protein kinase C, and p42/44 mitogen-activated protein kinase. The serum from rabbits with a turpentine-induced inflammatory reaction increased the phosphorylation of Erk1/2, effect prevented by PD98059 but not by bis-indolylmaleimide, a specific inhibitor of protein kinase C. The results demonstrated that the decrease in total cytochrome P450 content and in CYP1A1/1A2 and 3A6 activity by sera and interleukin-6 involves the activation of protein tyrosine kinases, p42/44 mitogen-activated protein kinase and protein kinase C. Indirect evidence supported that nitric oxide is implicated in the decrease in activity of these enzymes.     

Reduction of high glucose and phorbol-myristate-acetate-induced endothelial cell permeability by protein kinase C inhibitors LY379196 and hypocrellin A

Endothelial barrier dysfunction plays a pivotal role in the pathogenesis of diabetic vascular complications. Although recent studies have established a link between protein kinase C (PKC) pathway and hyperglycaemic-induced vascular permeability, it is unclear which PKC isoforms involve increased endothelial cell permeability. In the present study, we investigated whether high glucose induced endothelial hyperpermeability via distinct PKC isoforms in human umbilical vein endothelial cells (HUVECs) and whether increased endothelial permeability could be substantially reversed by PKC inhibitors LY379196 and hypocrellin A (HA). High glucose (20 mM) and phorbol-myristate-acetate (PMA)-induced endothelial hyperpermeability was almost abolished by 150 nM HA and partially reduced by 30 nM PKC beta inhibitor (LY379196). LY379196 and HA inhibited the membrane fraction of PKC activity in a dose-dependent manner. Western blot analysis revealed high-glucose-induced overexpression of PKC alpha and PKC beta2 in the membrane fraction of HUVECs. LY379196 (30 and 150 nM) selectively inhibited PKC beta2 with no significant effect on PKC alpha expression. HA (150 nM) significantly reduced PKC alpha expression with no inhibitory effect on PKC beta2. At higher concentrations (300 nM), both LY379196 and HA were no longer selective for PKC beta or alpha, respectively. This study showed that both PKC alpha and beta2 contributed to endothelial hyperpermeability. Since reduction of endothelial hyperpermeability was greater with inhibition of PKC alpha rather than PKC beta2, we conclude that PKC alpha may be a major isoform involved in endothelial permeability in HUVECs, and that PKC alpha-mediated endothelial permeability was significantly reversed by the PKC inhibitor HA.     

Inflammation and inducible nitric oxide synthase have no effect on monoamine oxidase activity in glioma cells

Heightened monoamine oxidase (MAO) and inducible nitric oxide synthase (iNOS) activity can contribute to oxidative stress, the formation of active neurotoxins, and associated neurodegenerative diseases of the brain. Although these enzymes co-exist within astrocytes, there has been little research examining the correlation between the two during inflammation. In this study, C6 glioma cells were stimulated with lipopolysaccharide (LPS):Escherichia coli 0111:B4 (6 micro g/mL):rat interferon-gamma (IFN-gamma) (100U/mL). In LPS/IFN-gamma-treated cells, the MAO substrates dopamine (DA) and tyramine caused a concentration-dependent attenuation of NO(2)(-) and NO(3)(-). In contrast, treatment with an MAO-A inhibitor (clorgyline) or an MAO-B inhibitor ((-)-deprenyl) did not reverse these effects. MAO activity was inhibited effectively by clorgyline and deprenyl; however, neither MAO inhibitor had an effect on NO(2)(-) in stimulated cells. Inversely, increasing concentrations of LPS/IFN-gamma resulted in heightened iNOS protein expression and NO(2)(-); however, these events did not correlate with any distinctive change in MAO enzyme activity. Moreover, a selective iNOS inhibitor, N(6)-(1-iminoethyl)-L-lysine, in LPS/IFN-gamma-stimulated cells caused a concentration-dependent attenuation of NO(2)(-) with no effects on MAO activity or iNOS protein expression. The attenuating effects of DA on iNOS were blocked completely by ICI 118-551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride], indicating a role for the beta(2)-adrenergic receptor. In conclusion, these data indicate that activity or expression of iNOS does not influence MAO activity in activated rat glioma cells. Moreover, DA exerts an inhibitory effect on glial iNOS through a receptor-mediated cascade.     

N6-Substituted adenosine derivatives: selectivity,efficacy, and species differences at A3 adenosine receptors

The activation of the human A(3) adenosine receptor (AR) by a wide range of N(6)-substituted adenosine derivatives was studied in intact CHO cells stably expressing this receptor. Selectivity of binding at rat and human ARs was also determined. Among N(6)-alkyl substitutions, small N(6)-alkyl groups were associated with selectivity for human A(3)ARs vs. rat A(3)ARs, and multiple points of branching were associated with decreased hA(3)AR efficacy. N(6)-Cycloalkyl-substituted adenosines were full (/=6 carbons) hA(3)AR agonists. N(6)-(endo-Norbornyl)adenosine 13 was the most selective for both rat and human A(1)ARs. Numerous N(6)-arylmethyl analogues, including substituted benzyl, tended to be more potent in binding to A(1) and A(3) vs. A(2A)ARs (with variable degrees of partial to full A(3)AR agonisms). A chloro substituent decreased the efficacy depending on its position on the benzyl ring. The A(3)AR affinity and efficacy of N(6)-arylethyl adenosines depended highly on stereochemistry, steric bulk, and ring constraints. Stereoselectivity of binding was demonstrated for N(6)-(R-1-phenylethyl)adenosine vs. N(6)-(S-1-phenylethyl)adenosine, as well as for the N(6)-(1-phenyl-2-pentyl)adenosine, at the rat, but not human A(3)AR. Interestingly, DPMA, a potent agonist for the A(2A)AR (K(i)=4nM), was demonstrated to be a moderately potent antagonist for the human A(3)AR (K(i)=106nM). N(6)-[(1S,2R)-2-Phenyl-1-cyclopropyl]adenosine 48 was 1100-fold more potent in binding to human (K(i)=0.63nM) than rat A(3)ARs. Dual acting A(1)/A(3) agonists (N(6)-3-chlorobenzyl- 29, N(6)-(S-1-phenylethyl)- 39, and 2-chloro-N(6)-(R-phenylisopropyl)adenosine 53) might be useful for cardioprotection.     

Stereoselectivity of 8-OH-DPAT toward the serotonin 5-HT1A receptor: biochemical and molecular modeling study

The great majority of pharmacological investigations of 5-HT1A receptors' reactivity has been performed using racemic 8-OH-DPAT, therefore the biochemical as well as behavioral profiles of both 8-OH-DPAT enantiomers are not circumstantiated. In the biochemical study capability of racemic 8-OH-DPAT (0.05, 0.1 mg/kg s.c.) and its counterparts R-8-OH-DPAT (0.05, 0.1 mg/kg s.c.) and S-8-OH-DPAT (0.05, 0.1 mg/kg s.c.) to influence 5-HT synthesis rate in rats' prefrontal cortex, hypothalamus, hippocampus and brainstem was evaluated by HPLC/ED technique. Biochemical results are supported by the exhaustive computational study of possible differences between R- and S-enantiomer toward the 5-HT1A receptor. A reliable 3D model of the rat 5-HT1A receptor was constructed from the amino acid sequence using the crystal structure of bovine rhodopsin as a structural template. The structure of the receptor model was validated through docking studies and molecular dynamics simulations that gave results consistent with experimental data. Docking studies and the dynamics of ligand-receptor complexes emphasized different profiles of both enantiomers at the molecular level. The results of both biochemical and computational studies confirmed that R-enantiomer in contrast to S-8-OH-DPAT acts as full and potent agonist, whilst racemic form may display similar pharmacological profile to R-8-OH-DPAT.     

Dopamine D(2) receptor-induced COX-2-mediated production of prostaglandin E(2) in D(2)-transfected Chinese hamster ovary cells without simultaneous administration of a Ca(2+)-mobilizing agent

We have earlier demonstrated that dopamine stimulates the liberation of the prostaglandin E(2) (PGE(2)) precursor, arachidonic acid, in Chinese hamster ovary cells transfected with the rat dopamine D(2) receptor (long isoform), also without concomitant administration of a Ca(2+)-releasing agent [Nilsson et al., Br J Pharmacol 1998;124:1651-8]. In the present report, we show that dopamine, under the same conditions, also induces a concentration-dependent increase in the production of PGE(2), with a maximal effect of 235% at approximately 100 microM, and with an EC(50) of 794 nM. The effect was counteracted by the D(2) antagonist eticlopride, pertussis toxin, the inhibitor of intracellular Ca(2+) release TMB-8, incubation in Ca(2+)-free experimental medium, and PKC desensitization obtained by chronic pretreatment with the phorbol ester TPA. It was also antagonized by the non-specific cyclooxygenase (COX) inhibitor, indomethacin, and by the selective COX-2 inhibitor, NS-398, but not by the specific COX-1 inhibitor, valeryl salicylate. Both the non-specific phospholipase A(2) inhibitor, quinacrine, and an inhibitor of cPLA(2) and iPLA(2), AACOF3, counteracted the effect; in contrast, a selective iPLA(2) inhibitor, BEL, and a selective sPLA(2) inhibitor, TAPC, were ineffective. No effects of dopamine were obtained in control cells mock-transfected with the p3C vector only. The results reinforce previous assumptions that dopamine may interact with eicosanoid metabolism by means of D(2) receptor activation, and implicate an involvement of cPLA(2) and COX-2 in this effect. It is suggested that measurement of dopamine-induced PGE(2) production may serve as a convenient way to study D(2) receptor function in vitro.     

Synergistic effects of chemotherapeutic drugs in lymphoma cells are associated with down-regulation of inhibitor of apoptosis proteins (IAPs), prostate-apoptosis-response-gene 4 (Par-4), death-associated protein (Daxx) and with enforced caspase activation

Cytotoxic drugs mediate apoptotic tumor cell death by influencing key regulator proteins of programmed cell death. In clinical practice cytotoxic drug combinations are desired to potentiate tumor cell kill and to minimize side effects. Nevertheless, the molecular mechanisms underlying synergistic and antagonistic effects on tumor cells are still poorly understood. In order to elucidate these molecular mechanisms we established models of synergistic and antagonistic drug combinations within the same lymphoma cell lines. By combination index method we demonstrated that bendamustine in combination with either doxorubicin or mitoxantrone caused antagonistic effects on disruption of mitochondrial membrane potential as well as on the rate of apoptosis. In contrast the combination of bendamustine with cladribine acted synergistically on these parameters. By using the IC(50) (dosages causing 50% rate of apoptosis) the synergistic effect of the combination of bendamustine and cladribine was associated with an enhanced mitochondrial release of cytochrome c and Smac/DIABLO, by down-regulation of x-linked inhibitor of apoptosis (XIAP), cIAP1, Par-4 and Daxx as well as by a significantly increased activation of caspases-3, -6, -7, -8 and -9. At the same rate of apoptosis (IC(50)), the antagonistic combinations did not increase the release of cytochrome c or Smac/DIABLO, nor down-regulate the expression of XIAP, cIAP1, Par-4 and Daxx, nor increase the activation of caspases. The role of down-regulation of IAPs and of enforced caspase activation for synergism in this model was supported by the observation, that broad spectrum inhibition of caspases re-established expression of XIAP. Our study is the first to outline the molecular alterations caused by synergistic and antagonistic drug combinations within the same lymphoma cell model. The above described mechanisms were already assessable at a point where the effects of synergistic or antagonistic combinations could not yet be discriminated quantitatively by the level of apoptosis rate of the lymphoma cells.