Phospholipase A2 Inhibitor Quinacrine Research Articles

Increased coronary arteriolar contraction to serotonin in juvenile pigs with metabolic syndrome

Metabolic syndrome (MetS) is associated with alterations in coronary vascular smooth muscle and endothelial function. The current study examined the contractile response of the isolated coronary arterioles to serotonin in pigs with and without MetS and investigated the signaling pathways responsible for serotonin-induced vasomotor tone. The MetS pigs (8-weeks old) were fed with a hyper-caloric, fat/cholesterol diet and the control animals (lean) were fed with a regular diet for 12 weeks (n = 6/group). The coronary arterioles (90–180 μm in diameter) were dissected from the harvested pig myocardial tissues and the in vitro coronary arteriolar response to serotonin was measured in the presence of pharmacological inhibitors. The protein expressions of phospholipase A2 (PLA2), TXA2 synthase, and the thromboxane-prostanoid (TP) receptor in the pigs’ left ventricular tissue samples were measured using Western blotting. Serotonin (10−9–10−5 M) induced dose-dependent contractions of coronary-resistant arterioles in both non-MetS control (lean) and MetS pigs. This effect was more pronounced in the MetS vessels compared with those of non-MetS controls (lean, P < 0.05]. Serotonin-induced contraction of the MetS vessels was significantly inhibited in the presence of the selective PLA2 inhibitor quinacrine (10−6 M), the COX inhibitor indomethacin (10−5 M), and the TP receptor antagonist SQ29548 (10−6 M), respectively (P < 0.05). MetS exhibited significant increases in tissue levels of TXA2 synthase and TP receptors (P < 0.05 vs. lean), respectively. MetS is associated with increased contractile response of porcine coronary arterioles to serotonin, which is in part via upregulation/activation of PLA2, COX, and subsequent TXA2, suggesting that alteration of vasomotor function may occur at an early stage of MetS and juvenile obesity.

5-hydroxytryptamine has an endothelium-derived hyperpolarizing factor-like effect on coronary flow in isolated rat hearts

Background5-hydroxytryptamine (5-HT)-induced coronary artery responses have both vasoconstriction and vasorelaxation components. The vasoconstrictive effects of 5-HT have been well studied while the mechanism(s) of how 5-HT causes relaxation of coronary arteries has been less investigated. In isolated rat hearts, 5-HT-induced coronary flow increases are partially resistant to the nitric oxide synthase inhibitor Nω-Nitro-L-arginine methyl ester (L-NAME) and are blocked by 5-HT7 receptor antagonists. In the present study, we investigated the role of 5-HT7 receptor in 5-HT-induced coronary flow increases in isolated rat hearts in the absence of L-NAME, and we also evaluated the involvement of endothelium-derived hyperpolarizing factor (EDHF) in 5-HT-induced coronary flow increases in L-NAME-treated hearts with the inhibitors of arachidonic acid metabolism and the blockers of Ca2+-activated K+ channels.ResultsIn isolated rat hearts, 5-HT and the 5-HT7 receptor agonist 5-carboxamidotryptamine induced coronary flow increases, and both of these effects were blocked by the selective 5-HT7 receptor antagonist SB269970; in SB269970-treated hearts, 5-HT induced coronary flow decreases, which effect was blocked by the 5-HT2A receptor blocker R96544. In L-NAME-treated hearts, 5-HT-induced coronary flow increases were blocked by the phospholipase A2 inhibitor quinacrine and the cytochrome P450 inhibitor SKF525A, but were not inhibited by the cyclooxygenase inhibitor indomethacin. As to the effects of the Ca2+-activated K+ channel blockers, 5-HT-induced coronary flow increases in L-NAME-treated hearts were inhibited by TRAM-34 (intermediate-conductance Ca2+-activated K+ channel blocker) and UCL1684 (small-conductance Ca2+-activated K+ channel blocker), but effects of the large-conductance Ca2+-activated K+ channel blockers on 5-HT-induced coronary flow increases were various: penitrem A and paxilline did not significantly affect 5-HT-induced coronary flow responses while tetraethylammonium suppressed the coronary flow increases elicited by 5-HT.ConclusionIn the present study, we found that 5-HT-induced coronary flow increases are mediated by the activation of 5-HT7 receptor in rat hearts in the absence of L-NAME. Metabolites of cytochrome P450s, small-conductance Ca2+-activated K+ channel, and intermediate-conductance Ca2+-activated K+ channel are involved in 5-HT-induced coronary flow increases in L-NAME-treated hearts, which resemble the mechanisms of EDHF-induced vasorelaxation. The role of large-conductance Ca2+-activated K+ channel in 5-HT-induced coronary flow increases in L-NAME-treated hearts needs further investigation.

Inhibition of spawning in zebrafish (Danio rerio): Adverse outcome pathways of quinacrine and ethinylestradiol

This study determined the effects of the estrogen receptor agonist ethinylestradiol (EE2) and the phospholipase A2 inhibitor quinacrine (QUIN) on the pathways controlling follicular development, steroidogenesis, oocyte maturation, ovulation and spawning success in adult zebrafish. Both EE2 and QUIN inhibited spawning but did so through different mechanisms. EE2 affected follicular development (reduced ovarian size and reduction in the proportion of cortical alveolus, vitellogenic and mature follicle stages), steroidogenesis (reduced expression of aromatase), maturation (reduced luteinizing hormone receptor expression) and ovulation (reduced expression of cytosolic phospholipase A2 and the nuclear progesterone receptor). Although EE2 alters the proportion of follicle stages within the ovary, the downregulation of gene expression as a consequence of EE2 exposure was primarily due to a decline in expression of the genes of interest in vitellogenic and mature ovarian follicles. QUIN targeted ovulation via a reduction of the steroid 17α,20β dihydroxy-4-prenen-3-one (17α,20β-P) and decreased expression of the prostaglandin metabolizing enzyme cyclooxygenase 2. This study demonstrates the usefulness in defining the impacts of toxicants at the molecular and cellular, organ and whole organism level and how connections between these impacts can be used to describe the adverse outcome pathways (AOPs) that mediate toxicant action. Histological analysis and gene expression were effective tools in defining the AOPs of QUIN and EE2 while the measurement of reproductive hormones level did not provide much valuable information regarding the toxicant’s mode of action.

Dexmedetomidine-induced contraction involves c-Jun NH2 -terminal kinase phosphorylation through activation of the 5-lipoxygenase pathway in the isolated endothelium-denuded rat aorta.

Vasoconstriction induced by dexmedetomidine, a highly selective alpha-2 adrenoceptor agonist, mainly involves c-Jun NH2 -terminal kinase (JNK) phosphorylation in the isolated endothelium-denuded aorta. We carried out an in vitro study to determine the main arachidonic acid metabolic pathway that is involved in dexmedetomidine-induced JNK activation. Cumulative dexmedetomidine concentration-contractile response curves were generated in the endothelium-denuded rat aorta in the presence or absence of the following inhibitors: the JNK inhibitor SP600125, the phospholipase A2 inhibitor quinacrine dihydrochloride, the non-specific lipoxygenase (LOX) inhibitor nordihydroguaiaretic acid, the 5-LOX inhibitor AA-861, the dual 5-LOX and cyclooxygenase (COX) inhibitor phenidone, the non-specific COX inhibitor indomethacin, the cytochrome p450 epoxygenase inhibitor fluconazole, the COX-1 inhibitor SC-560, and the COX-2 inhibitor NS-398. The effect of the alpha-2 adrenoceptor inhibitor rauwolscine and other inhibitors, such as quinacrine dihydrochloride, nordihydroguaiaretic acid, AA-861, phenidone, indomethacin and the protein kinase C inhibitor GF 109203X, on dexmedetomidine-induced JNK phosphorylation was investigated in rat aortic vascular smooth muscle cells with western blotting. The effect of dexmedetomidine on 5-LOX and COX-2 expression was investigated in vascular smooth muscle cells. SP600125, quinacrine dihydrochloride, nordihydroguaiaretic acid, AA-861, phenidone, rauwolscine and chelerythrine attenuated dexmedetomidine-induced contraction. Indomethacin slightly attenuated dexmedetomidine-induced contraction. Fluconazole and SC-560 had no effect on dexmedetomidine-induced contraction, whereas NS-398 attenuated contraction. SP600125, rauwolscine, quinacrine dihydrochloride, nordihydroguaiaretic acid, AA-861, phenidone and GF 109203X attenuated dexmedetomidine-induced JNK phosphorylation. 5-LOX and COX-2 were upregulated by dexmedetomidine. Thus, dexmedetomidine-induced alpha-2 adrenoceptor-mediated contraction is mediated mainly by 5-LOX and partially by COX-2, which leads to JNK phosphorylation.

Prostaglandins induce vasodilatation of the microvasculature during muscle contraction and induce vasodilatation independent of adenosine

Blood flow data from contracting muscle in humans indicates that adenosine (ADO) stimulates the production of nitric oxide (NO) and vasodilating prostaglandins (PG) to produce arteriolar vasodilatation in a redundant fashion such that when one is inhibited the other can compensate. We sought to determine whether these redundant mechanisms are employed at the microvascular level. First, we determined whether PGs were involved in active hyperaemia at the microvascular level. We stimulated four to five skeletal muscle fibres in the anaesthetized hamster cremaster preparation in situ and measured the change in diameter of 2A arterioles (maximum diameter 40 μm, third arteriolar level up from the capillaries) at a site of overlap with the stimulated muscle fibres before and after 2 min of contraction [stimulus frequencies: 4, 20 and 60 Hz at 15 contractions per minute (CPM) or contraction frequencies of 6, 15 or 60 CPM at 20 Hz; 250 ms train duration]. Muscle fibres were stimulated in the absence and presence of the phospholipase A2 inhibitor quinacrine. Further, we applied a range of concentrations of ADO (10(-7)-10(-5) M) extraluminally, (to mimic muscle contraction) in the absence and presence of L-NAME (NO synthase inhibitor), indomethacin (INDO, cyclooxygenase inhibitor) and L-NAME + INDO and observed the response of 2A arterioles. We repeated the latter experiment on a different level of the cremaster microvasculature (1A arterioles) and on the microvasculature of a different skeletal muscle (gluteus maximus, 2A arterioles). We observed that quinacrine inhibited vasodilatation during muscle contraction at intermediate and high contraction frequencies (15 and 60 CPM). L-NAME, INDO and L-NAME + INDO were not effective at inhibiting vasodilatation induced by any concentration of ADO tested in 2A and 1A arterioles in the cremaster muscle or 2A arterioles in the gluteus maximus muscle. Our data show that PGs are involved in the vasodilatation of the microvasculature in response to muscle contraction but did not obtain evidence that extraluminal ADO causes vasodilatation through NO or PG or both. Thus, we propose that PG-induced microvascular vasodilation during exercise is independent of ADO.

Impaired contractile response of human peripheral arterioles to thromboxane A-2 after cardiopulmonary bypass

We studied the contractile response of human peripheral microvasculature to thromboxane A-2 (TXA-2) before and after cardiopulmonary bypass (CPB), with and without the blockade of TXA-2 receptors, or the inhibition of phospholipase C (PLC), phospholipase A-2 (PLA-2) or protein kinase C (PKC)-α. We also examined the protein/gene expression and localization of TXA-2 receptors, TXA-2 synthase, PLC, and other TXA-2-related proteins. Skeletal muscle arterioles (90-180 μm in diameter) were harvested pre- and post-CPB from patients (n = 28) undergoing cardiac surgery. The post-CPB contractile response of skeletal muscle arterioles to TXA-2 analog U-46619 was impaired compared with pre-CPB (P < .05). The presence of TXA-2 receptor antagonist SQ-29548 (10(-6)mol/L) prevented the contractile response to U-46619 (P < .05). Pretreatment with the PLC inhibitor U-73122 (10(-6)mol/L) significantly inhibited the U-46619-induced contractile response (P < .01). Administration of the PLA-2 inhibitor quinacrine (10(-6)mol/L) or PKC-α inhibitor safingol (2 × 10(-5)mol/L), however, failed to affect U-46619-induced contraction. Total protein levels and gene expression of TXA-2 receptors, and TXA-2 synthase of skeletal muscle, were not altered post-CPB. Confocal microscopy showed no differences in the expression of PLCβ-3 in the microcirculation. PLCβ-3 was localized to both smooth muscle and endothelium. CPB decreases the contractile response of human peripheral arterioles to TXA-2 soon after cardiac surgery. This response may be in part responsible for the decrease in vascular tone, and accompanying hypotension sometimes observed after cardiac operations utilizing CPB.

Trans-Serosal Leakage of Proinflammatory Mediators during Abdominal Aortic Aneurysm Repair: Role of Phospholipase A2 in Activating Leukocytes.

Gut barrier failure and the resultant translocation of luminal bacteria and bacterial products into the systemic circulation have been proposed as pathogenic mechanisms of multiorgan dysfunction syndrome (MODS) in open repair of abdominal aortic aneurysm (AAA). Our study aimed to demonstrate the direct release of gut-derived inflammatory mediators via the trans-serosal route in humans. Fifteen patients who underwent elective infrarenal open repair of AAA were randomized into two groups. In Group I patients (n = 10), the small intestine was exteriorized into a bowel bag. In Group II patients (n = 5), the small intestine was packed within the peritoneal cavity using large gauzes. We collected the bowel bag fluid in Group I and the ascites fluid, squeezed out from the gauzes at the end of surgery, in Group II. Leukocytes were collected from patients' blood samples. Incubation with the bowel bag fluid and ascites fluid caused a significant increase in both granulocyte pseudopod formation and CD11b expression compared to that with control fluid (p < 0.01). The addition of phospholipase A2 (PLA2) inhibitor quinacrine abolished leukocyte activation by the bowel bag fluid. Based on these results, we consider that trasns-serosal leakage of gut-derived mediators occurred during the open repair of AAA; further, sPLA2 may be the most potent mediator in the activation of leukocytes among such gut-derived mediators in AAA surgery.

Binge Ethanol-induced Neurodegeneration in Rat Organotypic Brain Slice Cultures: Effects of PLA2 Inhibitor Mepacrine and Docosahexaenoic Acid (DHA)

Using rat organotypic hippocampal-entorhinal cortical (HEC) slice cultures, we examined whether phospholipase A2 (PLA2) activity is involved in binge alcohol (ethanol)-induced neurodegeneration, and whether docosahexaenoic acid (DHA; 22:6n-3), a fish oil-enriched fatty acid that is anti-inflammatory in brain damage models, is neuroprotective. Assessed with propidium iodide and lactate dehydrogenase (LDH) leakage, neurodamage from ethanol (6 days 100 mM ethanol with four withdrawal periods) was prevented by the PLA2 pan-inhibitor, mepacrine. Also, ethanol-dependent neurodegeneration-particularly in the entorhinal region-was significantly ameliorated by DHA supplementation (25 microM); however, adrenic acid, a 22:4n-6 analog, was ineffective. Consistent with PLA2 activation, [(3)H] liberation was approximately fivefold greater in [(3)H]arachidonic acid-preloaded HEC slice cultures during ethanol withdrawal compared to controls, and DHA supplementation suppressed [(3)H] release to control levels. DHA might antagonize PLA2 activity directly or suppress upstream activators (e.g., oxidative stress); however, other DHA mechanisms could be important in subdueing ethanol-induced PLA2-dependent and independent neuroinflammatory processes.

The effects of the glucocorticoid receptor antagonist RU486 and phospholipase A2 inhibitor quinacrine on acoustic injury of the mouse cochlea

Glucocorticoids are used clinically for the treatment of acoustic injury. However, the protective mechanism of glucocorticoid in acoustic injury has not been completely clarified. Also, the effects of phospholipase A2 (PLA2) on acoustic injury have not been examined to the best of our knowledge. The purpose of the present study was to examine the effects of methylprednisolone, a glucocorticoid receptor inhibitor (RU486) and a phospholipase A2 inhibitor (quinacrine) on cochlear injury induced by acoustic overexposure. Seventy-eight mice were exposed to a 4 kHz pure tone at 128 dB SPL for 4 h. The auditory brainstem response (ABR) was used to examine the hearing thresholds. Cochlear morphology was examined to estimate the outer hair cell loss induced by acoustic overexposure. Methylprednisolone and quinacrine significantly alleviated the hearing threshold shift and hair cell loss induced by acoustic overexposure. RU486 antagonized the protective effect of methylprednisolone. The present findings suggest firstly that glucocorticoids exert protective effects against acoustic injury; secondly, that the protective effect of methylprednisolone was exerted by binding glucocorticoid receptors, and finally that activation of PLA2 may be involved in acoustic injury.

PGE2 in the regulation of programmed erythrocyte death

Hyperosmotic shock, energy depletion, or removal of extracellular Cl(-) activates Ca(2+)-permeable cation channels in erythrocyte membranes. Subsequent Ca(2+) entry induces erythrocyte shrinkage and exposure of phosphatidylserine (PS) at the erythrocyte surface. PS-exposing cells are engulfed by macrophages. The present study explored the signalling involved. Hyperosmotic shock and Cl(-) removal triggered the release of prostaglandin E(2) (PGE(2)). In whole-cell recording, activation of the cation channels by Cl(-) removal was abolished by the cyclooxygenase inhibitor diclophenac. In FACS analysis, phospholipase-A(2) inhibitors quinacrine and palmitoyltrifluoromethyl-ketone, and cyclooxygenase inhibitors acetylsalicylic acid and diclophenac, blunted the increase of PS exposure following Cl(-) removal. PGE(2) (but not thromboxane) induced cation channel activation, increase in cytosolic Ca(2+) concentration, cell shrinkage, PS exposure, calpain activation, and ankyrin-R degradation. The latter was attenuated by calpain inhibitors-I/II, while PGE(2)-induced PS exposure was not. In conclusion, hyperosmotic shock or Cl(-) removal stimulates erythrocyte PS exposure through PGE(2) formation and subsequent activation of Ca(2+)-permeable cation channels.

Phospholipase A2 activating protein (PLAA) is required for 1alpha,25(OH)2D3 signaling in growth plate chondrocytes.

Phospholipase A2 (PLA2) is pivotal in the rapid membrane-mediated actions of 1,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. Microarray analysis indicated that PLA2 activating protein (PLAA) mRNA is upregulated 6-fold before rat growth plate cells exhibit 1alpha,25(OH)2D3-dependent protein kinase C (PKC) increases, suggesting that it plays an important role in 1alpha,25(OH)2D3's mechanism of action. PLAA mRNA was confirmed in 1alpha,25(OH)2D3-responsive growth zone (prehypertrophic and upper hypertrophic cell zones) chondrocytes by RT-PCR and Northern blot in vitro and by in situ hybridization in vivo. PLAA protein was shown by Western blot and immunohistochemistry. PLAAs role in 1alpha,25(OH)2D3 signaling was evaluated in growth zone cell cultures using PLAA peptide. Arachidonic acid release was increased as was PLA2-specific activity in plasma membranes and matrix vesicles. PKCalpha, but not PKCbeta, PKCepsilon, or PKCzeta, was increased. PLAAs effect was comparable to that of 1alpha,25(OH)2D3 and was additive with 1alpha,25(OH)2D3. PLA2 inhibitors quinacrine and AACOCF3, and cyclooxygenase inhibitor indomethacin blocked the effect of PLAA peptide on PKC, indicating arachidonic acid and its metabolites were involved. This was confirmed using exogenous arachidonic acid. Prostaglandin acted via EP1 based on inhibition by SC19220 and not via EP2 since AH6809 had no effect. Like 1alpha,25(OH)2D3, PLAA peptide also increased activity of phospholipase C-specific activity via beta-1 and beta-3 isoforms, but not delta-1 or gamma-1; the effect of PLAA was via lysophospholipid but not via arachidonic acid. PLAA peptide decreased [3H]-thymidine incorporation to 50% of the decrease caused by 1alpha,25(OH)2D3. In contrast, PLAA peptide increased alkaline phosphatase-specific activity and proteoglycan production in a manner similar to 1alpha,25(OH)2D3. This indicates that PLAA is a specific activator of PLA2 in growth plate chondrocytes, and suggests that it mediates the membrane effect of 1alpha,25(OH)2D3, thereby modulating physiological response.

Role of EETs in regulation of endothelial permeability in rat lung.

This study tested the hypothesis that epoxyeicosatrienoic acids (EETs) derived from arachidonic acid via P-450 epoxygenases are soluble factors linking depletion of endoplasmic reticulum Ca(2+) stores and store-dependent regulation of endothelial cell (EC) permeability in rat lung. EC permeability was measured via the capillary filtration coefficient (K(f,c)) in isolated, perfused rat lungs. 14,15-EET and 5,6-EET increased EC permeability, a response that was significantly different from that of 8,9-EET, 11,12-EET, and vehicle control. The permeability response to 14,15-EET was not significantly attenuated by the nonspecific Ca(2+) channel blocker Gd(3+) (P = 0.068). In lungs perfused with low [Ca(2+)], 14,15-EET tended to increase EC permeability, although a significant increase in K(f,c) was observed only following Ca(2+) add-back. As positive control, we showed that the 3.7-fold increase in K(f,c) evoked by thapsigargin (TG), a known activator of store depletion-induced Ca(2+) entry, was blocked by both Gd(3+) and low [Ca(2+)] buffer. Nonetheless, the permeability response to TG could not be blocked by the phospholipase A(2) inhibitors mepacrine or methyl arachidonyl fluorophosphonate or the P-450 epoxygenase inhibitors 17-octadecynoic acid or propargyloxyphenyl hexanoic acid. Similarly, combined pretreatment with ibuprofen and dicyclohexylurea to block EET metabolism had no effect on the permeability response to TG. We conclude that EETs have a heterogeneous impact on EC permeability. Despite a requirement for Ca(2+) entry with both TG and 14,15-EET, our data suggest that distinct signaling pathways or heterogeneity in EC responsiveness is responsible for the observed EC injury evoked by EETs and store depletion in the isolated rat lung.

11,12,15-Trihydroxyeicosatrienoic acid mediates ACh-induced relaxations in rabbit aorta.

Rabbit aortic endothelium metabolizes arachidonic acid (AA) by the 15-lipoxygenase pathway to vasodilatory eicosanoids, hydroxyepoxyeicosatrienoic acids (HEETAs), and trihydroxyeicosatrienoic acids (THETAs). The present study determined the chemical identity of the vasoactive THETA and investigated its role in ACh-induced relaxation in the rabbit aorta. AA caused endothelium-dependent, concentration-related relaxations of the rabbit aorta. Increasing the extracellular KCl concentration from 4.8 to 20 mM inhibited the relaxations to AA by approximately 60%, thereby implicating K+-channel activation in the relaxations. In addition, AA caused an endothelium-dependent hyperpolarization of aortic smooth muscle from -39.6 +/- 2.7 to -56.1 +/- 3.4 mV. In rabbit aortic rings, [14C]AA was metabolized to prostaglandins, HEETAs, THETAs, and 15-hydroxyeicosatetraenoic acid. Additional purification of the THETAs by HPLC resolved the mixture into its 14C-labeled products. Gas chromatography/mass spectrometry identified the metabolites as isomers of 11,12,15-THETA and 11,14,15-THETA. The 11,12,15-THETA relaxed and hyperpolarized the rabbit aorta, whereas 11,14,15-THETA had no vasoactive effect. The relaxations to 11,12,15-THETA were blocked by 20 mM KCl. In aortic rings pretreated with inhibitors of nitric oxide and prostaglandin synthesis, ACh caused a concentration-related relaxation that was completely blocked by 20 mM KCl. Pretreatment with the phospholipase A2 inhibitors mepacrine and 7,7-dimethyl-5,8-eicosadienoic acid, the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, nordihydroguaiaretic acid, and ebselen, or the hydroperoxide isomerase inhibitors miconazole and clotrimazole also blocked ACh-induced relaxations. ACh caused a threefold increase in THETA release. These studies indicate that AA is metabolized by endothelial cells to 11,12,15-THETA, which activates K+ channels to hyperpolarize the aortic smooth muscle membrane and induce relaxation. Additionally, this lipoxygenase pathway mediates the nonnitric oxide, nonprostaglandin relaxations to ACh in the rabbit aorta by acting as a source of an endothelium-derived hyperpolarizing factor.

Searching for mechanisms of N-methyl-D-aspartate-induced glutathione efflux in organotypic hippocampal cultures.

N-Methyl-D-aspartate (NMDA)-receptor stimulation evoked a selective and partly delayed elevated efflux of glutathione, phosphoethanolamine, and taurine from organotypic rat hippocampus slice cultures. The protein kinase inhibitors H9 and staurosporine had no effect on the efflux. The phospholipase A2 inhibitors quinacrine and 4-bromophenacyl bromide, as well as arachidonic acid, a product of phospholipase A2 activity, did not affect the stimulated efflux. Polymyxin B, an antimicrobal agent that inhibits protein kinase C, and quinacrine in high concentration (500 microM), blocked efflux completely. The stimulated efflux after but not during NMDA incubation was attenuated by a calmodulin antagonist (W7) and an anion transport inhibitor (DNDS). Omission of calcium increased the spontaneous efflux with no or small additional effects by NMDA. In conclusion, NMDA receptor stimulation cause an increased selective efflux of glutathione, phosphoethanolamine and taurine in organotypic cultures of rat hippocampus. The efflux may partly be regulated by calmodulin and DNDS sensitive channels.

Progesterone regulates IL12 expression in pregnancy lymphocytes by inhibiting phospholipase A2.

Progesterone-induced blocking factor (PIBF) is one of the pathways that mediate the immunological effects of progesterone. PIBF inhibits natural killer (NK) cytotoxic activity. Recently we showed that neutralization of PIBF results in an increased interleukin (IL)-12 expression, which is corrected by cyclooxygenase inhibitors. As exogenous arachidonic acid (AA) voids the NK blocking effect of PIBF, it is likely that PIBF acts before the level of the cyclooxygenase enzyme. Therefore in this study we investigated the effect of PIBF neutralizing antibody and simultaneous phospholipase A2 inhibitor quinacrine (Q) treatment on IL-12 production. Pregnancy lymphocytes were treated with anti-PIBF antibody or lipopolysaccharide (LPS) as a positive control, in the presence or absence of Q. IL-12 expression by PBMC was detected by immunocytochemistry. Neutralization of PIBF as well as LPS treatment resulted in an increased IL-12 expression, which was corrected by simultaneous Q treatment. Pre-treatment of lymphocytes with progesterone prevented the stimulating effect of LPS on IL-12 production. Progesterone binding of the lymphocytes is followed by the release of PIBF that inhibits AA release. The subsequent block of prostaglandin synthesis reduces IL-12 production and results in a lowered cytotoxic NK activity, which may contribute to a normal pregnancy outcome.

Hypoxia alters the sensitivity of the L-type Ca(2+) channel to alpha-adrenergic receptor stimulation in the presence of beta-adrenergic receptor stimulation.

The effects of alpha-adrenergic receptor (alpha-AR) stimulation alone and the effects in the presence of beta-adrenergic receptor (beta-AR) stimulation were examined on L-type Ca(2+) currents (I(Ca-L)) in the absence and presence of hypoxia. The alpha-AR agonist methoxamine either had no effect or had a slight inhibitory effect on basal I(Ca-L) in the absence and presence of hypoxia. Hypoxia significantly decreased the K(0.5) for activation of I(Ca-L) by norepinephrine from 79.8+/-6.6 to 13.3+/-0.7 nmol/L. To determine whether hypoxia specifically altered the sensitivity of the channel to alpha-AR stimulation, cells were exposed to increasing concentrations of methoxamine in the presence of 100 nmol/L isoproterenol (Iso). In the absence of hypoxia, methoxamine inhibited the Iso-activated I(Ca-L) in a concentration-dependent manner with an EC(50) of 86.9+/-9.9 micromol/L. However, in the presence of hypoxia, the EC(50) for inhibition of I(Ca-L) by methoxamine was significantly increased to 266.7+/-10.8 micromol/L. Methoxamine had little effect on I(Ca-L) activated by forskolin or histamine in the absence or presence of hypoxia. In addition, inhibition of protein kinase C by bisindolylmaleimide 1 or protein kinase C beta peptide inhibitor had no effect on the methoxamine-induced antagonism of I(Ca-L) in the absence or presence of hypoxia. The tyrosine kinase inhibitor genistein attenuated the methoxamine response in nonhypoxic cells only. However, during hypoxia it was attenuated with the phospholipase A(2) inhibitors mepacrine and indomethacin. These findings represent a novel regulation of the L-type Ca(2+) channel by the phospholipase A(2) pathway and illustrate the complexity of regulation of the channel under hypoxic conditions.

Sensitivity of Orexin-A Binding to Phospholipase C Inhibitors, Neuropeptide Y, and Secretin

The binding of [125I] orexin-A (Ox-A) to particulates from Chinese hamster ovary (CHO) cells expressing the cloned orexin-A receptor, or from rat forebrain areas, was sensitive to blockers of phosphatidylinositol-specific phospholipase C (PtdIns-PLC) U-73122 and ET-18-OCH3, little affected by phospholipase A2 inhibitor quinacrine, and not sensitive to D609, a xanthate inhibitor of phosphatidylcholine-selective PLC. Interaction of the receptor with a PtdIns-PLC was further indicated by a large sensitivity of the binding to Ca2+. Up to 50% of the binding was sensitive to the G-protein nucleotide site agonist GTP-γ-S. Ligand attachment to the orexin-A receptor thus depends on an association with both PtdIns-PLC and G-protein α-subunits. In all paradigms examined, the binding of [125I]orexin-A was competed by human/rat neuropeptide Y (hNPY) and porcine secretin with a potency similar to orexin-A (IC50 range 30–100 nM). The rank order of potency for NPY-related peptides was hNPY > porcine peptide YY (pPYY) > (Leu31, Pro34) human PYY > human PYY(3-36) > hNPY free acid > human pancreatic polypeptide. Among secretin-related peptides, the rank order of potency was porcine secretin ≥ orexin-A > human pituitary adenylate cyclase-activating peptide > orexin-B > porcine vasoactive intestinal peptide. Among opioid peptides, rat β-endorphin and camel δ-endorphin were much less active than NPY and secretin, and two enkephalins were inactive at 1 μM. In view of high abundance of NPY in forebrain, the above cross-reactivity could indicate a significant contribution of NPY to signaling via orexin-A receptors.

Effect of the inhibition of PLA2 and PAF on the neutrophilic respiratory burst and apoptosis

Background Since the exact pathogenesis of sepsis-induced ARDS has not been elucidated, the mechanisms of enhanced neutrophilic respiratory burst were probed in endotoxin primed neutrophils associated with the roles of phospholipase A2 (PLA2), platelet activating factor (PAF) and apoptosis. Methods In isolated fresh human neutrophils, effects of the inhibition of PLA2 and PAF on the apoptosis were examined by the method of Annexin-FITC/dual PI flow cytometry. The roles of PLA2 and PAF on the neutrophilic respiratory burst were also examined by measuring oxidant generation in cytochrome-c reduction assay. Activities of the PLA2 and lysoPAF acetyltransferase (lysoPAF AT) of the neutrophils were determined to understand the effect of endotoxin on these enzymatic activities which may be related to the neutrophilic respiratory burst and apoptosis. In addition, the role roles of PLA2 and PAF in neutrophilic adhesion to bovine endothelial cells were examined in vitro by neutrophil adhesion assay. To investigate the effect of oxidants on pulmonary surfactant, cytochemical ultrastructural microscopy was performed. To inhibit PLA2 and PAF, non-specific PLA2 inhibitor mepacrine (100 nM) and WEB 2086 (100 nM) or ketotifen fumarate (10 kg/ml) were used respectively in all in vitro experimental sets. WEB 2086 is PAF receptor antagonist, and ketotifen fumarate is a lyso PAF AT inhibitor. Results The mapacrine treatment, provided after the endotoxin (ETX) treatment, resulted in increased apoptosis of neutrophils (p<0.001) while treatments of WEB 2086 and ketotifen did not. The inhibition of PLA2 and PAF decreased (p<0.001) production of oxidants from PMA-stimulated neutrophils. While endotoxin increased the PLA2 activity of neutrophils (p<0.01), mepacrine supressed (p<0.001) the activity, provided after treatment of ETX. The lyso PAF actyltransferase activity (lyso PAF AT) increased(p<0.01) after treatment of ETX. In contrast, mepacrine, WEB 2086 and ketotifen showed a tendency of decreasing the activity after treatment of ETX. The Treatment of ETX increased (p<0.001) neutrophil adhesion to endothelial cells, which was reversed by inhibition of PLA2 and PAF (p<0.001). The binding of oxidants to pulmonary surfactant was identified histologically. Conclusions The enhanced neutrophilic respiratory burst by ETX plays a pivotal role in the pathogenesis of ARDS in term of oxidayive oxidative stress. Increased production of oxidants form neutrophils is mediated by the activations of PLA2 and lyso PAF AT.

Specificity of synergistic coronary flow enhancement by adenosine and pulsatile perfusion in the dog.

1. Coronary flow elevation from enhanced perfusion pulsatility is synergistically amplified by adenosine. This study determined the specificity of this interaction and its potential mechanisms. 2. Mean and phasic coronary flow responses to increasing pulsatile perfusion were assessed in anaesthetized dogs, with the anterior descending coronary artery servoperfused to regulate real-time physiological flow pulsatility at constant mean pressure. Pulsatility was varied between 40 and 100 mmHg. Hearts ejected into the native aorta whilst maintaining stable loading. 3. Increasing pulsatility elevated mean coronary flow +11.5 +/- 1.7 % under basal conditions. Co-infusion of adenosine sufficient to raise baseline flow 66 % markedly amplified this pulsatile perfusion response (+82. 6 +/- 14.3 % increase in mean flow above adenosine baseline), due to a leftward shift of the adenosine-coronary flow response curve at higher pulsatility. Flow augmentation with pulsatility was not linked to higher regional oxygen consumption, supporting direct rather than metabolically driven mechanisms. 4. Neither bradykinin, acetylcholine nor verapamil reproduced the synergistic amplification of mean flow by adenosine and higher pulsatility, despite being administered at doses matching basal flow change with adenosine. 5. ATP-sensitive potassium (KATP) activation (pinacidil) amplified the pulse-flow response 3-fold, although this remained significantly less than with adenosine. Co-administration of the phospholipase A2 inhibitor quinacrine virtually eliminated adenosine-induced vasodilatation, yet synergistic interaction between adenosine and pulse perfusion persisted, albeit at a reduced level. 6. Thus, adenosine and perfusion pulsatility specifically interact to enhance coronary flow. This synergy is partially explained by KATP agonist action and additional non-flow-dependent mechanisms, and may be important for modulating flow reserve during exercise or other high output states where increased flow demand and higher perfusion pulsatility typically co-exist.

Influence of some phospholipase A2and cytochrome P450 inhibitors on rat arterial smooth muscle K+currents

The hyperpolarizing factor that is liberated by vascular endothelial cells in response to various agonists, and known to induce relaxation by opening of smooth muscle K+ channels, has been suggested to be a product of cytochrome P450 dependent arachidonic acid metabolism. In this study, the direct influence of two phospholipase A2 inhibitors and of five structurally and mechanistically different cytochrome P450 inhibitors on K+ currents in freshly isolated vascular smooth muscle cells from the rat aorta was investigated. On stepping the cell membrane potential from -70 mV to a series of depolarized test potentials, a noisy outward current developed at test potentials > +10 mV, which showed no appreciable inactivation during the voltage pulse. It was largely abolished by 3 mM external tetraethylammonium chloride (TEA), suggesting that it predominantly consisted of current through large-conductance Ca(2+)-activated K+ channels. The phospholipase A2 inhibitor quinacrine considerably inhibited this TEA-sensitive current, while 4-bromophenacylbromide exerted no effect. The cytochrome P450 inhibitors proadifen and miconazole reversibly decreased the amplitude of I(K), while clotrimazole and 1-aminobenzotriazole had no effect. Conversely, 17-octadecynoic acid increased whole-cell I(K). These results show that some phospholipase A2 and cytochrome P450 inhibitors may interfere with K+ channel activation in the rat arterial smooth muscle cell. The relevance of these findings to studies on the involvement of cytochrome P450 dependent metabolism in the generation of the endothelium-derived hyperpolarizing factor in intact arteries is discussed.