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Antinociceptive and anti-inflammatory effects of limonin isolated from the dried fruits of Evodia rutaecarpa var. bodinieri were investigated. Oral administration of 30 or 100 mg/kg limonin significantly decreased the frequency of licking and biting behavior within a unit of time at the late phase without affecting that of the early phase in the formalin test. Limonin inhibited the rise of vascular permeability induced by acetic acid and the increase of paw edema induced by carrageenin. Limonin also showed inhibitory effects on bradykinin-induced paw edema and arachidonic acid-induced ear swelling. These results suggest that limonin possesses an antinociceptive effect and the effect may be accompanied by an anti-inflammatory action, and that the antinociceptive activity in Evodiae Fructus is partially attributable to limonin.

The effect of supplementation of docosahexaenoic acid (DHA) on the apoptosis of HL60 cells was examined using N-acetyl sphingosine (C2-ceramide) and sphingosine as apoptosis-inducing agents. Although C2-ceramide-induced apoptosis was not affected by DHA supplementation, sphingosine-induced apoptosis was reduced almost to the background level by preincubation with 10 microM DHA for 24 h. Among the fatty acids, only DHA appeared to be endowed with the ability to reduce sphingosine-induced apoptosis, whereas, other unsaturated fatty acids, such as arachidonic acid (AA) and eicosapentaenoic acid (EPA), did not show this activity. Incubation of HL60 with DHA within 6 h did not affect the apoptosis, suggesting that DHA probably expressed the inhibitory activity after modulation of the membrane fatty acid composition. DHA also attenuated the apoptosis induced by dimethylsphingosine and H-7, but not by calphostin C, indicating that enrichment of DHA in membranous phospholipid does not necessarily prevent all of the apoptosis associated with the inhibition of protein kinase C. The mechanism of the inhibition against sphingosine-induced apoptosis by DHA remains to be further explored. However, the inhibition of cytosolic phospholipase A2 (cPLA2) may be involved in the mechanism, because distinctive inhibitory activity of DHA against cPLA2 has been demonstrated [M. Shikano, Y. Masuzawa, K. Yazawa, K. Takayama, I. Kudo, K. Inoue, Biochim. Biophys. Acta, 1212, 1994, 211-216], and arachidonyl trifluoromethylketone, a specific inhibitor of cPLA2, attenuated the apoptosis induced by sphingosine., (Copyright 1998 Elsevier Science B.V.)

In order to examine the role of protein kinase A (PKA) in the regulation of arachidonic acid availability, the interaction between cAMP agonists and the G protein activator AIF4- in their effects on phospholipid metabolism were measured in MC3T3-E1 osteoblasts. We show that forskolin and 8-brcAMP, activators of PKA, amplify the AIF4(-)-induced stimulation of phosphatidylinositol-specific phospholipase C (phosphatidylinositol inositolphosphohydrolase; EC 3.1.4.3), measured by the formation of [3H]inositol phosphates in prelabeled cells. However, the AIF4(-)-stimulated production of 1,2-diacylglycerols and the release of [3H]arachidonic acid ([3H]AA) were inhibited 50-75% by forskolin and 8-bromocAMP. Furthermore, pretreatment with PKA activators prevented much of the AIF4(-)-induced loss of [3H]AA from phosphatidylcholine and phosphatidylethanolamine in prelabeled osteoblasts. In addition, in the absence of AIF4-, forskolin was found to stimulate the incorporation of [3H]AA and [32P]orthophosphoric acid selectively into these two major phospholipids and selectively increased their mass. The effects of forskolin and 8-BrcAMP on the levels of free [3H]AA were completely reversed by pretreatment with the PKA inhibitor H-89. Therefore, our findings suggest that the activation of cAMP-dependent protein kinase can reduce the availability of free arachidonic acid for prostaglandin synthesis in osteoblast cells by stimulating its reesterification via phospholipid resynthesis.

We have reported that arachidonic acid and the metabotropic glutamate receptor agonist, trans-1-amino-cyclopentyl-1,3-dicarboxylate (ACPD), act in synergy to increase release of glutamate from synaptosomes prepared from rat dentate gyrus. The observation that prior induction of LTP in perforant path-granule cell synapses occluded this synergism suggested that the interaction between arachidonic acid and ACPD might trigger the increase in glutamate release that accompanies LTP in dentate gyrus. Our objective was to identify the mechanism underlying the synergism between arachidonic acid and ACPD in LTP. The data indicate that both agents activate phospholipase C(PLC); the arachidonic acid-induced increase in phospholipase C activation was inhibited by the tyrosine kinase inhibitor, genistein, suggesting that PLCgamma, which is stimulated by tyrosine phosphorylation may be activated by arachidonic acid. The ACPD-induced increase was inhibited by neomycin, indicating the involvement of a G-protein and suggesting that PLCbeta may be activated by ACPD. We report that arachidonic acid stimulated phosphorylation of the specific tyrosine kinase substrate, poly(Glu80,Tyr20) and direct analysis indicated that arachidonic acid increased phosphorylation of PLCgamma. PLCgamma phosphorylation was assessed in control dentate gyrus and dentate gyrus in which LTP was induced in vivo. We report that the tyrosine kinase inhibitor, genistein, blocked expression of LTP and also blocked the associated increase in phosphorylation of PLCgamma. The data presented here indicate that tyrosine phosphorylation of PLCgamma was significantly enhanced following induction of LTP, but in separate experiments, in which LTP was inhibited by intraventricular injection of genistein, phosphorylation of PLCgamma was inhibited. The evidence presented is consistent with the hypothesis that PLC acts as a coincidence detector in LTP. The data indicate that PLCbeta is activated by ACPD, PLCgamma is activated by arachidonic acid, and coincident activation of both isoforms is necessary to stimulate an increase in glutamate release.

Oligosaccharide moieties of cell-surface glycoproteins are thought to be involved in recognition events during cancer metastasis and invasion. Swainsonine, an inhibitor of the Golgi alpha-mannosidase II, has been shown to block pulmonary colonization by tumor cells and stimulate components of the immune system. Swainsonine also abrogates much of the toxicity of chemotherapeutic agents and stimulates bone marrow hematopoietic progenitor cells, suggesting additional therapeutic applications. We are currently characterizing the ability of swainsonine to modify cell growth in human and murine bone marrow progenitor cells. Furthermore, we are examining crucial steps in metastasis that depend upon cell surface molecules that play a role in cell-matrix interactions. Our work shows that tumor cell adhesion to collagen IV in vitro is rapidly stimulated by cis-polyunsaturated fatty acids and is dependent on protein kinase C activity. We are investigating the hypothesis that integrins are critical components of this adhesion and are examining potential signal transduction pathways that lead to the modulation of cell adhesion.

Gravidin is a potent phospholipase A2 inhibitor that may contribute to the maintenance of human pregnancy. The aims of this study were to determine firstly, the site of gravidin synthesis within placental membranes and secondly, whether the antiprogestin compound, RU486, regulates synthesis. Membrane explants were taken from placentae, dissected and cultured in the presence of RU486, progesterone or inhibitors of DNA or protein synthesis and gravidin production was measured by a specific enzyme-linked immunosorbent assay. Production of gravidin was consistently greatest from choriodecidua compared with amnion and decidua. The antibiotics, cycloheximide and actinomycin D inhibited production of gravidin. The progesterone receptor antagonist RU486 at 10(-8) and 10(-7) M reduced gravidin production to 55 and 39% of the control value in membranes cultured after and before the onset of labour respectively. Progesterone at 10(-6) M stimulated gravidin production by 47% after the onset of labour. The increase in gravidin production was correlated with progesterone concentration (r = 0.47, F = 6.38, P = 0.019) in labour tissue. We conclude that the data are consistent with the hypothesis that gravidin production may be partially regulated by progesterone.

Effects of epidermal growth factor (EGF) and possible mechanisms of EGF-mediated signal transduction were investigated in isolated cells of the digestive gland of the mussel (Mytilus galloprovincialis Lam. ). EGF induced a cytosolic Ca2+ transient and subsequently stimulated DNA synthesis; both effects were dose-dependent in the nanomolar range and inhibited by pretreatment with an inhibitor of tyrosine kinase activity, suggesting specific EGFR-like receptors. The EGF-induced cytosolic Ca2+ transient was mainly due to a Ca2+ influx through the plasma membrane, possibly involving voltage-insensitive Ca2+ channels. Such a Ca2+ response was abolished by pretreatment with indomethacin and NDGA, inhibitors of arachidonic acid metabolism; similarly, the EGF-stimulated increase in DNA synthesis was significantly reduced. Indomethacin, a cyclooxygenase inhibitor, had the greatest effect on both EGF-induced responses. Results suggest the presence of EGF-responsive cells in the mussel digestive gland. A possible role for arachidonic acid and its metabolites in mediating the effects of EGF is also indicated.

In recent years, our knowledge concerning pathophysiological changes in brain metabolism after traumatic brain injury (TBI) has greatly expanded. This, in turn, has enabled the development of specific pharmacological strategies for the supplementary treatment of brain-injured patients with the aim of reducing secondary brain damage. The present article focuses on the pathophysiology of TBI and the possibilities for pharmacological intervention. While some of the substances reviewed and presently used in the treatment of TBI, others are under experimental and clinical evaluation at different stages.

Unlike gastric mucosa, it has been considered that lipoxygenase metabolites protect the esophageal mucosa and that prostaglandins are only secreted in the presence of esophageal inflammation. The aim of this study was to determine the profile of arachidonic acid metabolites and their response to regulatory compounds in rabbit esophageal mucosal cells in culture. Eicosanoids secreted into the medium were extracted and identified by HPLC and RIA. Esophageal mucosal cells in culture metabolized arachidonic acid mainly through the cycloxygenase pathway and PGE2 was the major arachidonic acid metabolite secreted. The addition of IL-1 beta and A23187 (calcium ionophore) stimulated PGE2 synthesis. In basal conditions neither leukotrienes nor HETEs were detected. However, the addition of the NDGA induced the secretion of lipoxygenase metabolites identified as 12-15 HETEs. In conclusion, rabbit esophageal epithelial cells in culture metabolize arachidonic acid via both cycloxygenase and lipoxygenase pathways. In our system, PGE2 was the main arachidonic acid metabolite.

Aims: The aim of this study was to compare the effects of aspirin on platelet function as measured by the 'classical' template bleeding time with a new ex vivo method measuring closure times using the PFA-100 machine. Platelet aggregation in response to arachidonic acid was also measured ex vivo., Methods: The trial was a randomized, double-blind, placebo-controlled crossover design, with each volunteer taking 750 mg aspirin (BP) or placebo, three times a day for 5 days, with an 18 day wash-out period between treatments. Bleeding times and closure times were measured before the first dose on the first day and 0.5 h after the last dose on the fifth day of each treatment period. They were also measured 2 weeks after the last day of the trial., Results: Baseline bleeding times (pre-placebo) were 415 s using the Simplate, whilst baseline closure times were 115 s using the PFA-100. Aspirin treatment caused an increase of both the template bleeding time (61%) and the closure time of the PFA-100 (79%) when compared with the effects of placebo. The platelet aggregatory response to arachidonic acid was completely inhibited following aspirin treatment and was unaffected following placebo. Two weeks after the end of the trial, all values had returned to pre-treatment levels. The template bleeding time was unaltered in 1 of the 12 volunteers during aspirin treatment and was significantly prolonged in 3 of the 12 volunteers during placebo treatment. The PFA-100 closure time was unaltered in 1 of the 12 volunteers during aspirin treatment and was prolonged in 1 subject during placebo treatment., Conclusions: The change in closure time using the PFA-100 is as sensitive and reproducible to the effects of aspirin on platelet function as is the template bleeding time test. However, the PFA-100 produced less variable effects with fewer false positive results.

We have established an assay based on gas chromatography-mass spectrometry to profile and quantitate endogenous cytochrome P450 monooxygenase (P450)-hydroxyeicosatetraenoic acids (HETEs) exiting the isolated perfused rabbit kidney in response to hormonal stimulation. In response to angiotensin II (Ang II) P450-derived HETEs (16-, 17-, 18-, 19- and 20-) are released from the isolated Kreb's perfused rabbit kidney. Ang II produced a several-fold increase in the levels of P450-HETEs above basal levels in both urinary (such as for 20-HETE from 0.93 +/- 0.7 to 2.31 +/- 0.9 ng/min) and venous (from 0.1 +/- 0.05 to 0.3 +/- 0.05 ng/min) effluents. However, inhibition of P450, which reduced basal release, did not prevent Ang II-induced release of P450-AA products from the rabbit kidney; for example, urinary 20-HETE in the presence of 17-ODYA (1 microM) was undetectable and increased to 0.93 +/- 0.4 ng/min with Ang II and venous 20-HETE increased from 0.06 +/- 0.03 to 0.24 +/- 0.07 ng/min. Similar results were obtained with clotrimazole (1 microM). As 16-, 18-, 19- and 20-HETEs are vasodilators in the rabbit kidney and 16- and 17-HETEs inhibit proximal tubular ATPase activity, we investigated their possible sites of esterification. Cortical and medullary lipids were extracted, separated by HPLC and P450-HETEs quantitated following alkaline hydrolysis. The P450-HETEs were incorporated into both neutral lipids (NL) and phospholipids [phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and phosphatidylcholine (PC)]. However, the assignment of a HETE to a specific phospholipid pool must be regarded as tentative as the appropriate standards containing P450-HETEs in the Sn-2 position (such as 20-HETE-PF., 20-HETE-PC, etc.) were not available. Esterified HETEs were found in larger quantities in the cortex as compared to the medulla (34.40 +/- 1.12 versus 22.76 +/- 0.53 ng/g). The PI fraction in the cortex yielded the largest quantity of HETEs and the PC fraction the lowest. In the medulla, the largest quantities of esterified HETEs were found in neutral lipids and only slightly lesser amounts in PE and PI. Esterified 18-HETE was localized only to the NI fraction. This fraction also contained the other HETEs, 19- and 20-HETE being the most abundant. Notably only 16- and 17-HETE were present in PF, whereas, 19- and 20-HETE were also present in PI, PS and PC. Thus, P450-HETEs, like EETs are stored in the kidney and are, presumably, subject to release by peptide activation of acylhydrolases.

Leukotrienes (LT) are potent vasoconstrictors in the pulmonary circulation. We have investigated the synthesis of LTC4 by intrapulmonary arteries and veins of perinatal lambs. Paired vessels of near-term fetal 146 +/- 2- and 2- to 7-day-old newborn lambs (each n = 7), were incubated for 10 min at 37 degrees C in baseline, with 1 mumol/L A32187 or 0.1 mmol/L arachidonic acid. Produced leukotriene C4 was assayed from media by ELISA. Baseline production of leukotriene (ng/mg tissue, means +/- SEM) by vessels for arteries was 0.006 +/- 0.001 and 0.059 +/- 0.009 for fetus and newborn, respectively. In veins, the values were 0.013 +/- 0.003 and 0.073 +/- 0.007 for fetus and newborn, respectively. On stimulation with the calcium ionophore A23187, production by arteries increased 25-fold in the fetus, but 4-fold in the newborn. The corresponding values for stimulated veins were 37-fold and 9-fold in fetus and newborn, respectively. Generally, production by veins was greater than production by the matching arteries. In all instances, the fetal vessels produced less leukotrienes than the newborn vessels. Western analysis of stimulated and unstimulated vessel membrane protein showed greater expression of 5-lipoxygenase in veins than in arteries (P < 0.05). Our data show that veins produce more LTC4 due to greater expression of 5-lipoxygenase in the vessels and thus suggest that veins of perinatal lamb lungs may be more susceptible to LT-induced vasoreactivity in the perinatal pulmonary circulation. We speculate that a higher production of LTC4 by fetal veins may be necessary to maintain a high venous tone in fetal lungs.

The mechanism(s) of chronic airway inflammation in cystic fibrosis (CF) remains poorly understood. We studied Ca2+-induced release of arachidonic acid (AA), a precursor of proinflammatory lipid mediators, in epithelial cell lines with the deltaF508 mutation in CF transmembrane conductance regulator (CFTR) gene and in those lacking this mutation or cells in which this mutation was corrected by a functional CFTR gene transfer. We found that: (i) the mutant cells manifested an abnormally high Ca2+-induced AA release as compared to controls, (ii) AA release appeared to be catalyzed by a phospholipase A2 (PLA2) but not by phospholipase C followed by diacylglycerol lipase, and (iii) either correction of the CFTR-mutation or inhibition of PLA2 activity rectified this AA release abnormality. Taken together, our results suggest that CFTR mutation is associated with an intrinsic abnormality in AA release by epithelial cells carrying the deltaF508 mutation and suggest that the mechanism of chronic airway inflammation in CF, at least in part, involves this abnormality. These results also partly explain the effectiveness of high-dose ibuprofen therapy in arresting the progression of destructive lung disease in CF. Furthermore, they raise the possibility that correction of abnormal AA release by inhibiting PLA2 activity may improve the therapeutic benefits of ibuprofen.

The mechanism by which bradykinin (BK) decreases vascular resistance was investigated in the hindquarters vascular bed of the rat. Under conditions of controlled blood flow, BK produced dose-related decreases in hindquarters perfusion pressure when injected into the perfusion circuit in doses of 0.1-1.0 pg. Responses to BK were reproducible with respect to time, and HOE-140 (D-Arg, [Hyp3,Thi5,D-Tic7,Oic8] -BK), a kinin B2 receptor antagonist, decreased hindquarters vasodilator responses to the peptide. HOE-140 had no significant effect on responses to vasodilator agents, which act by a variety of pharmacologically distinct mechanisms. Nitric oxide synthase inhibitors N(omega)-nitro-L-arginine methyl ester or N(omega)-nitro-L-arginine benzyl ester did not decrease responses to BK in doses that decreased hindquarters vasodilator responses to acetylcholine. The cyclooxygenase inhibitors meclofenamate and indomethacin had no effect on responses to BK in doses that attenuated vasodilator responses to the prostaglandin precursor, arachidonic acid. Clotrimazole or 5,8,11,14-eicosatetraynoic acid (ETYA), cytochrome P-450 arachidonic acid metabolism inhibitors, in doses that attenuated vasodilator responses to arachidonic acid, had no effect on responses to BK. Glybenclamide or U-37883A (4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexyl), K+-ATP channel antagonists, in doses that attenuated responses to lemakalim, had no significant effect on responses to BK. Finally, des-Arg9-BK, a reported kinin B1 receptor agonist, decreased hindquarters perfusion pressure when injected in doses of 3-30 microg, and responses to the B1 agonist were attenuated by HOE-140. The observation that des-Arg9-BK, in high doses, induces modest HOE-140-sensitive responses suggests that kinin B1 receptors are not normally expressed in the hindquarters vascular bed of the rat. The present results indicate that BK dilates the hindquarters vascular bed by a kinin B2 receptor mechanism that is independent of the release of nitric oxide, cyclooxygenase, or P-450 metabolites of arachidonic acid or the activation of K+-ATP channels.

Dexamethasone has been shown to inhibit opiate withdrawal, in an in vitro model. In this respect, we suggested that dexamethasone could reduce opiate withdrawal by blocking the release of prostaglandins' precursor, arachidonic acid through protein synthesis dependent-mechanisms (1). Since arachidonic acid is released by the enzyme phospholipase A2 (PLA2) in the present paper we evaluate whether dexamethasone effect may by related to inhibition of PLA2 activity. Therefore, the effect of a neutralizing anti-lipocortin-1 antibody and a polyclonal anti-type II extracellular phospholipase A2 antibody on the opiate withdrawal in vitro was considered. Following a 4 min in vitro exposure to morphine a strong contracture of guinea-pig isolated ileum was observed after the addition of naloxone. Dexamethasone at concentration of 5x10(-5) M reduces of 50% morphine withdrawal and the polyclonal anti-type II extracellular PLA2 antibody (in a dilution 1:1000) mimicked dexamethasone inhibitory effect. Incubation of the ileum preparation with a neutralizing anti-lipocortin-1 antibody (at a dilution of 1:10.000) 30 min before dexamethasone reverted the steroid effects. These results suggest that dexamethasone inhibition of opiate withdrawal is due to extracellular type II PLA2 inhibition through lipocortin-1.

Using Fura-2 microfluorimetry, phenylarsine oxide (PAO) (10-50 microM), a potent tyrosine phosphatase inhibitor, was shown to induce a dose-dependent increase in the free Ca2+ intracellular concentration in rat peritoneal macrophages and human foreskin fibroblasts. The PAO-induced increase in [Ca2+]i is not due presumably to depletion of intracellular Ca2+ stores but to mainly a stimulation of Ca2+ entry from the extracellular medium. This PAO-activated Ca2+ entry is attenuated by the following pharmacological agents. Organic and inorganic Ca2+ channel blockers: (nifedipine, verapamil and Ni2+); nonselective cation channel blocker niflumic acid; tyrosine kinase inhibitors genistein and methyl-2,5-dihydroxycinnamate; SH-reagents dithiothreitol parachloromercuribenzoate and N-ethylmaleimide; arachidonic acid metabolism inhibitors 4-bromophenacyl bromide, indomethacin and caffeic acid; microtubule disrupters vinblastine, colchicine and colcemide. On the contrary, microfilament disrupters, cytochalasin B and phalloidin, enhance PAO-activated Ca2+ entry. Our data suggest that the dynamic balance between tyrosine kinase and phosphatase activity may play a central role in the maintenance of homeostatic levels of [Ca2+]i both in unstimulated cells and after agonist application.

Purpose: A murine model of squamous cell carcinoma (SCC) was used to determine the role of arachidonic acid (AA) metabolites in the growth of SCC of the head and neck., Materials and Methods: C3H/HeJ mice bearing SCC (SCC VII) were treated with cyclooxygenase inhibitors (piroxicam and nabumetone) or a 5-lipoxygenase inhibitor (ketoconazole). Growth curves were established, and final tumor weights were measured. Following sacrifice, tumor tissue homogenates were assayed for prostaglandin E2 (PGE2) and 12-hydroxyeicosatetraenoic acid (12-HETE) by enzyme-linked immunosorbent assay (ELISA), and leukotriene B4 (LTB4) by radioimmunoassay (RIA). Inflammatory cell infiltrate was assessed histologically., Results: A significant inhibition of tumor growth (P = .001) and final tumor weight (P = .002) was noted in mice treated with piroxicam and nabumetone. Inhibition of tumor growth was associated with increased tumor tissue levels of PGE2 (P = .04) and lymphocytic infiltration (P = .07). Significant inhibition of tumor growth (P = .002) and final tumor weight (P = .05) was also noted in mice treated with ketoconazole., Conclusion: These data suggest that both cyclooxygenase and lipoxygenase metabolites of AA affect tumor growth in this model and that inhibition of tumor growth by inhibitors of AA metabolism may be caused by an enhanced inflammatory cell response at the tumor site.

The synthesis of title enantiomers was accomplished and their biological behaviour as inhibitors of rabbit platelet aggregation process induced by ADP and arachidonic acid was determined. Structure-activity comparison with that of SM-12502 [(2R,5S)-(+) 3,5-dimethyl-2-(3-pyridyl)-thiazolidin-4-one hydrochloride] and Dazoxiben [4-[2-(1H-imidazol-1-yl)-ethoxy]-benzoic acid] allowed us to formulate the possible capability for the synthesized compounds to interact with the biological targets of the model molecules.

Amyloid beta-peptide (A beta) deposition and loss of cholinergic neurons are characteristics of Alzheimer's disease. There is evidence that A beta is neurotoxic. The role of signal transduction pathways on A beta-induced toxicity in PC12 cells was investigated. Our results revealed that A beta-induced arachidonic acid was released in a time-dependent manner. Inhibitors of cyclooxygenase (1 microM indomethacin) and lipooxygenase (100 microM nordihydroguairetic acid) protected PC12 cells against A beta-induced toxicity. These data suggest that A beta toxicity is mediated by activation of the arachidonic acid cascade. Furthermore, protein kinase C activators (phorbol ester and 1-oleyl-2-acetyl-glycerol) and tacrine reversed A beta-induced toxicity. These results suggest that A beta toxicity can be modulated by manipulating signal transduction pathways and may provide the basis for novel therapeutic interventions.

The effects of sphingoid bases, sphingosine and dihydrosphingosine, which are protein kinase C (PKC) inhibitors, on NADPH oxidase were examined in a cell-free system. The bases inhibited cell-free activation of NADPH oxidase by arachidonic acid at lower concentration than N-acetylsphingosine. Thus, positive charge in the molecules may play a critical role in inhibition of the oxidase. Sphingosine did not change the Km value for NADPH, but shifted the optimum concentration of arachidonic acid for activation of the oxidase. Moreover, sphingosine suppressed the translocation of p47-phox, one of the cytosolic components of the oxidase, to the membrane fraction, suggesting that the base inhibits the assembly of the components.

Arachidonic acid (AA) is metabolized by the cytochrome P-450 (P-450) epoxygenase pathway to epoxyeicosatrienoic acids (EETs) in the brain parenchymal tissue and perivascular astrocytes. EETs dilate cerebral microvessels and enhance K+ current in cerebrovascular smooth muscle cells. In the current study, the effect of a subdural administration of miconazole, an inhibitor of P-450 epoxygenase, on microvascular perfusion of rat cerebral cortex was evaluated using laser-Doppler flowmetry (LDF) Baseline cerebral blood flow (CBF) decreased by 29.7 +/- 7.3% (n = 5) after administration of 20 microM miconazole into the subdural space for 30 min. Responses of CBF to sodium nitroprusside and 5-hydroxytryptamine were unaltered by miconazole treatment. Administration of vehicle alone in time-control experiments had no effect on CBF. In other experiments, the effects of miconazole on the metabolism of [14C]AA by cultured rat astrocytes and on nitric oxide synthase activity in homogenates of rat brain were examined. Miconazole inhibited conversion of AA to EETs by cultured astrocytes but had no effect on the conversion of L-arginine to L-citrulline by homogenates of rat brain. These results implicate endogenous P-450 epoxides of AA in the regulation of basal blood flow in cerebral microcirculation.

Arachidonic acid (AA) has been shown to inhibit the activity of the low-conductance ATP-sensitive K+ channel in the apical membrane of the cortical collecting duct [W. Wang, A. Cassola, and G. Giebisch. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F554-F559, 1992]. ROMK1, a K+ channel derived from the rat renal outer medulla, shares many biophysical properties of the native low-conductance K+ channel, which is localized to the apical membranes of the cortical collecting duct and thick ascending limb. This study was designed to determine whether the ROMK channel maintains the property of AA sensitivity of the native low-conductance K+ channel. Experiments were conducted in Xenopus oocytes injected with cRNA encoding the ROMK1 channel by use of patch-clamp techniques. We have confirmed previous reports that the cloned ROMK1 has similar channel kinetics, high open probability, and inward slope conductance as the native low-conductance K+ channel, respectively. Addition of 5 microM AA to an inside-out patch resulted in reversible inhibition of channel activity at a concentration similar to the inhibitor constant for AA on the native K+ channel. The effect of AA on channel activity was preserved in the presence of 10 microM indomethacin, a cyclooxygenase inhibitor, 4 microM cinnamyl-3,4-dihydroxycyanocinnamate, a lipoxygenase inhibitor, and 4 microM 17-octadecynoic acid, an inhibitor of cytochrome P-450 monooxygenases, thus indicating that the effect of AA was not mediated by metabolites of AA. The effect did not appear to be the result of changes in membrane fluidity, since 5 microM eicosatetraynoic acid, an AA analogue that is a potent modulator of membrane fluidity, had no effect. Furthermore, the addition of AA to the outside of the patch also had no effect on channel activity. These results indicate that, like the native low-conductance channel, AA is able to directly inhibit ROMK1 channel activity.

The effect of arachidonic acid (AA) on the delayed rectifier K+ current (IK) was evaluated in rat pulmonary myocytes by using the whole-cell patch-clamp technique. Externally applied AA (50 mumol/L) caused a membrane depolarization, averaging 16 mV in six cells. AA (1 to 50 mumol/L) caused a dual effect on IK. First, AA accelerated the rate of IK activation, increasing current amplitude at the beginning of voltage step. Second, AA caused a marked acceleration of current decay, thereby reducing IK amplitude measured toward the end of the depolarizing steps. These effects were not prevented by indomethacin or nordihydroguaiaretic acid, blockers of cyclooxygenase and lipoxygenase, respectively. AA did not affect the voltage dependence of current activation or inactivation. The magnitude of the inhibitory effect on IK was correlated with the number of double bonds but was independent of tail length in fatty acids containing between 14 and 22 carbons. Linoleic acid (18:2, cis-9,12) inhibited IK much more than did its trans-stereo-isomer, linolelaidic acid. Arachidonyl alcohol, which is uncharged, and arachidonyl coenzyme A, which does not 'flip' across the cell membrane, were less effective than AA in inhibiting IK; this effect of fatty acids may therefore require passage across the cell membrane. The enhancement of early IK was mimicked by the protein kinase C (PKC) stimulator 1-oleoyl-2-acetyl-sn-glycerol (10 mumol/L), was suppressed by ATP removal from the pipette solution, and was blocked by PKC inhibitors chelerythrine (10 mumol/L) and staurosporine (100 nmol/L). This effect may therefore require PKC-dependent phosphorylation.

Treatment of 3T3-L1 preadipocytes with arachidonic acid resulted in a dose-dependent inhibition of adipocyte differentiation. The cells failed to accumulate fat droplets and did not express stearoyl-CoA desaturase 1 mRNA, a marker for late-stage differentiation. The inhibition of differentiation was reversed by the addition of cyclooxygenase inhibitors ibuprofen or indomethacin. Inhibitors of the lipoxygenase and cytochrome P-450 epoxygenase pathways were unable to reverse the effect of arachidonic acid. Dexamethasone, one of the adipogenic agents normally used to induce differentiation, could be replaced with cyclooxygenase inhibitors in the differentiation cocktail. This implicated dexamethasone as a modulator of prostaglandin synthesis in culture. Prostaglandins F2 alpha (ED50 = 0.4 nM), E2, and D2 prevented differentiation, each with a specific, dose-dependent affinity. Prostaglandin F2 alpha was the most potent inhibitor of differentiation, suggesting that a prostanoid FP2 receptor (FP receptor) mediates the prostaglandin action. Fluprostenol (ED50 = 0.3 nM), a selective FP receptor agonist, prevented differentiation, confirming the involvement of an FP receptor in the inhibition of 3T3-L1 preadipocyte differentiation. Stimulation of the FP receptor for 1 h during the first day of differentiation was sufficient to cause substantial inhibition. Endogenous PGF2 alpha production was lower in differentiating cells compared to unstimulated preadipocytes. These data suggest that PGF2 alpha production by preadipocytes plays a role in maintaining the undifferentiated state.

ATP-sensitive K-channels (K(ATP)) play a major role in controlling blood vessel tone, particularly in coronary vasculature. Arachidonic acid (AA) exhibits potent vasorelaxant effects on coronary arteries with unclear mechanisms. We examined the activity of AA on the K(ATP) channel of dog coronary artery smooth muscle cells using the whole cell voltage-clamp technique. AA was found to have dual effects or the K(ATP) current. In cells dialyzed with 1 mM ATP pipette solution, external application of AA activated the K(ATP) current with a bell-shaped concentration-response curve. K(ATP) current (-50 mV) increased from 1.0 +/- 1.1 to 4.6 +/- 3.0, 25 +/- 9, 43 +/- 13, 26 +/- 10, and 23 +/- 16 pA by 1, 2, 4, 8, and 16 microM AA, respectively. If K(ATP) current was activated by dialyzing the cell with ATP-free pipette solution, external application of AA (from 2 to 16 microM) inhibited the K(ATP) current (half-maximal inhibitory concentration = 3.8 microM). Pretreatment of cells with 10 microM indomethacin or 10 microM nordihydroguaiaretic acid, which respectively blocked the cyclooxygenase or lipoxygenase pathway of AA metabolism, did not prevent the dual effects of AA. Eicosatetraynoic acid (ETYA), the nonmetabolizable analogue of AA, also produced dual effects on the K(ATP) current. ETYA (4 microM) increased the K(ATP) current (-50 mV) from 2.9 +/- 1.2 to 37 +/- 7 pA in 1 mM intracellular ATP, whereas 8 microM ETYA inhibited the K(ATP) current in 0 mM intracellular ATP by 71 +/- 3%. These results suggest that the dual effects of AA on the K(ATP) current of dog coronary smooth muscle cells do not require AA metabolism. Activation of K(ATP) channels in smooth muscle cells may contribute to the AA-induced coronary vasodilation.

IBD is a disease of unknown cause that involves an amplification of the inflammatory response in the intestinal mucosa. Although not the only offending agents leading to the disease, eicosanoids, the collective group of AA metabolites, may a significant role in the pathogenesis of IBD. This article reviewed the biochemical pathways of eicosanoid formation and the clinical relevance of eicosanoids to IBD. Potential strategies designed to interfere with various aspects of AA metabolism were also outlined. Further clinical trials of newer compounds may soon prove them effective in the management of IBD.

Five new prenylflavones, yinyanghuo A (1), yinyanghuo B (2), yinyanghuo C (3), yinyanghuo D (4), and yinyanghuo E (5), along with six known flavonoids, chrysoeriol, quercetin, apigenin, apigenin 7,4'-dimethyl ether, kaempferol, and luteolin, were isolated from the leaves of Epimedium sagittatum. Their structures were determined from spectral analysis. Compounds 1 and 2 showed significant antiplatelet induced by arachidonic acid.

Condensed tannin, isolated from cotton bracts extract (CBE), increases arachidonic acid (AA) release from rabbit alveolar macrophages and inhibits its subsequent reacylation. We determined whether tannin from CBE had any effect upon AA release in bovine tracheal epithelial cells (BTE). [14C] AA release was measured at timed intervals after addition of various concentrations of tannin to BTE cells grown to confluence in the presence of [14C] AA. Tannin caused a time- and dose-dependent release of AA from airway cells, with a maximum release occurring at 1 min in the presence of 100 micrograms/ml of tannin, and was confirmed by high-pressure liquid chromatography. The pattern of release was similar to that observed with bradykinin (2 x 10(-6) M). AA release by tannin was partially inhibited by indomethacin (10(-5) M) but not by 5,8,11,14-eicosatetraynoic acid (ETYA; 10(-5) M. Both of these drugs were effective in inhibiting bradykinin-induced AA release. In addition, AA release was not inhibited by cycloheximide. Endotoxin at 100 pg/ml and higher also caused a time-dependent release of AA that was not inhibitable by indomethacin or ETYA. Tannin-induced AA release was inhibited by pretreatment with pertussis toxin but not by neomycin, an inhibitor of phospholipase C (PLC). Neither pertussis toxin nor neomycin had any effect upon endotoxin-induced AA release. In other experiments, neither tannin nor endotoxin had any effect on [14C]AA uptake by BTE. These data demonstrate that tannin at low concentrations and endotoxin at high concentrations increase AA release by BTE cells. The AA release by tannin is partially metabolized by the cyclooxygenase pathway. We hypothesize that tannin-induced AA release is not mediated by PLC but may be mediated by other phospholipases, including PLA2.

Platelet aggregate size was determined with a newly-developed platelet aggregometer, PA-100 (KOWA), which can quantitatively evaluate the size and number of platelet aggregates by means of the particle counting method based upon light scattering. Epinephrine-induced platelet aggregation consists of two phases, the former characterized by the formation of small-sized aggregates (less than 100 cells), which is followed by the phase of large aggregate formation with concomitant decrease in the number of small aggregates. These findings suggest that small aggregates fuse to form large aggregates. Effects of various inhibitors and antibodies directed against platelet membrane glycoproteins were evaluated on the size of platelet aggregates induced by epinephrine. Cyclooxygenase inhibitors, thromboxane A2 receptor antagonists, and a Na+/H+ exchanger inhibitor (ethylisopropylamiloride) inhibited the formation of large aggregates (more than 100 cells) but not that of small aggregates. Cytochalasin B, which interferes with microfilaments, suppressed large aggregate formation, whereas taxol, which reacts with microtubules, had no effects. Anti-GPIIb/IIIa monoclonal antibody (MoAb) inhibited both the formation of small and large platelet aggregates, while antibodies directed against GPIb, thrombospondin, P-selectin, or PECAM-1 had no effects on platelet aggregate formation. These findings, taken together, suggest that intracellular alkalinization, thromboxane A2 formation and microfilament rearrangement are prerequisites for large platelet aggregate formation. GPIIb/IIIa is involved in the formation of small as well as large aggregates, but a membrane glycoprotein(s) responsible for the transition of small aggregates into large aggregates awaits to be determined.

General anesthetics inhibit Ca(2+)-activated potassium (BK) channels at clinically relevant concentrations. This study examined the possibility that general anesthetics produce their effect on BK channels by disrupting the phospholipase A2 (PLA2)-arachidonic acid signal transduction pathway. Treatment of excised patches with exogenous arachidonic acid (2.5 microM) resulted in a 3.6 +/- 1.3-fold increase in BK channel activity. Subsequent exposure of these patches to concentrations of halothane (0.6 mM), ketamine (100 microM), or etomidate (10 microM) that would normally block the channel by approximately 60-80% in the absence of arachidonic acid did not reduce the channel activity. Arachidonic acid resulted in a significant increase in the 50% effective concentration for the ketamine dose-response curve from 3.4 +/- 0.4 to 693 +/- 379 microM (P < 0.001) as well as a significant decrease in slope from 1.40 +/- 0.21 to 0.59 +/- 0.05 (P < 0.001). The PLA2 inhibitors quinacrine (1 microM), aristolochic acid (250 microM), and octadecylbenzoylacrylic acid (7 microM) inhibited BK channels by 61 +/- 6, 47 +/- 2, and 30 +/- 9%, respectively, and in a manner indistinguishable from general anesthetics inhibition. Aristolochic acid and ketamine significantly inhibit the PLA2-mediated production of arachidonic acid in GH3 cells.

Head and neck tumors are known to synthesize arachidonic acid metabolites. The authors have postulated that these substances may confer growth advantage to cancer cells, and by interfering with arachidonic acid metabolism squamous cancer growth may be altered. The tongue derived squamous carcinoma cell line, SCC-25, was treated with three leukotriene synthesis inhibitors and indomethacin. A dose-dependent decrease in DNA synthesis occurred with leukotriene inhibition, but not prostaglandin inhibition. All leukotriene synthesis inhibitors produced a dramatic and immediate effect ( > 70% inhibition by 4 hours) without cytotoxicity ( > 90% trypan blue exclusion). Cell populations at 96 hours were decreased when compared to control populations. In conclusion, leukotrienes or other lipoxygenase products may play a role as growth factors for squamous cell carcinoma, and arachidonic acid inhibition may be a novel target for chemotherapeutic intervention.

Signal transduction pathways shared by different autocrine growth factors may provide an efficient approach to accomplish clinically significant control of lung cancer growth. In this study, we demonstrate that two autocrine growth factors activate 5-lipoxygenase action of the arachidonic acid metabolic pathway in lung cancer cell lines. Both growth factors increased the production of 5(S)-hydrooxyeicosa-6E,8Z,11Z,14Z-tetraeno ic acid (5-HETE), a major early 5-lipoxygenase metabolic product. Exogenously added 5-HETE stimulated lung cancer cell growth in vitro. Inhibition of 5-lipoxygenase metabolism by selective antagonists resulted in significant growth reduction for a number of lung cancer cell lines. Primary clinical specimens and lung cancer cell lines express the message for the 5-lipoxygenase enzymes responsible for the generation of active metabolites. In vivo evaluation demonstrated that interruption of 5-lipoxygenase signaling resulted in enhanced levels of programmed cell death. These findings demonstrate that 5-lipoxygenase activation is involved with growth factor-mediated growth stimulation for lung cancer cell lines. Pharmacological intervention with lipoxygenase inhibitors may be an important new clinical strategy to regulate growth factor-dependent stages of lung carcinogenesis.

1 The blocker of endoplasmic reticulum Ca(2+)-ATPase, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ) was shown to inhibit formation of prostaglandin E2 and prostacyclin in the osteoblast-like cell lines, MC3T3-E1 and ROS 17/2.8, respectively, in a dose-dependent manner with an IC50 of 0.5-1 microM. Inhibition was observed with various stimuli (arachidonic acid, bradykinin, melittin and calcium ionophore, A23187). 2 This effect was also observed in human platelets, where BHQ dose-dependently blocked thromboxane biosynthesis and formation of 12-hydroxy-heptadecatrienoic acid after stimulation with arachidonic acid, but not formation of 12-hydroxy-eicosatetraenoic acid. 3 Inhibition of prostaglandin E2 formation in MC3T3-E1 cells was not observed with thapsigargin after stimulation with arachidonic acid, A23187 or melittin, whereas bradykinin-induced prostaglandin E2 biosynthesis was blocked. 4 Taken together, the results suggest a direct inhibitory action of BHQ on the cyclo-oxygenase in these three cell systems.

We investigated the functional effects of epidermal growth factor (EGF) on guinea pig airways in vitro. EGF (3 ng/ml to 1 microgram/ml) induced a concentration-dependent contraction in epithelium-denuded strips. The average maximal contraction was 0.64 +/- 0.1 g (mean +/- S.E., for n = 27), which was 72.0 +/- 9.5% of the 100 mM KCl-induced contraction. The EC50 was 12.3 +/- 1.6 ng/ml. The presence of the epithelium significantly suppressed the EGF-induced contraction (P < 0.01). EGF-induced contraction was abolished by cyclooxygenase inhibitors (indomethacin and ibuprofen) and a 5-lipoxygenase inhibitor, 2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benz oqu inone (AA-861). It was also inhibited by a leukotriene-receptor antagonist, 8-[p-(4-phenylbutyloxy)benzoyl]amino-2-(tetrazol-5-yl)-4-oxo -4H-1-benzopyran hemihydrate (ONO-1078) but not affected by a thromboxane A2-synthetase inhibitor, (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoic acid (OKY-046) or a thromboxane A2-receptor antagonist, 9,11-epithio-11,12-methano-thromboxane A2 (ONO-3708). A phospholipase A2 inhibitor (mepacrine) inhibited the EGF-induced contraction but a diacylglycerol-lipase inhibitor, 1,6-di-(O-(carbamoyl)cyclohexanone oxime)hexane (U-57908) and a phospholipase D inhibitor (wortmannin) did not affect it. A tyrosine kinase inhibitor (genistein) abolished it. Measurement of prostanoids showed that EGF (300 ng/ml) did not increase the prostaglandin F2 alpha level in either epithelium-intact or epithelium-denuded strips. In epithelium-intact strips, EGF significantly increased the prostaglandin E2 concentration (P < 0.01). These results suggest that EGF causes contraction of guinea pig airway smooth muscle by activating tyrosine kinase followed by phospholipase A2 activation, and that arachidonic acid metabolites, especially leukotrienes, may have important roles in this contraction.

The ability of estrogens to protect against DNA damage induced by either hydrogen peroxide or arachidonic acid alone or in combination with Cu2+ was investigated. DNA strand breaks were determined by conversion of double stranded supercoiled OX-174 RFI DNA to double stranded open circular DNA and linear single stranded DNA. Estradiol-17 beta significantly decreased the formation of single and double strand breaks in DNA induced by H2O2 alone or with Cu2+. Equilin (an equine estrogen) was more effective than estradiol-17 beta at the doses tested. Arachidonic acid in the presence of Cu2+ caused the formation of high levels of linear DNA which was protected by estrogen with equilen being more effective. These studies suggest that estrogens through this protective effect on DNA damage might contribute to cardioprotection.

One major role of alveolar macrophages is the production of eicosanoids, which modulate immune and inflammatory processes in the lung. In this study, the effects were investigated of cadmium ions on the secretion of leukotriene (LT)B4 and prostaglandin (PG)E2, predominant products of lipoxygenase and cyclooxygenase, respectively. Cd2+ had an inhibitory effect on the secretion of LTB4 and PGE2 in response to A23187 stimulation at concentrations > 3 x 10(-5)M. This effect can be explained by the inhibition of arachidonic acid (20:4) liberation from membrane phospholipids by Cd2+, because Cd2+ inhibits both [3H] arachidonic acid (20:4) liberation from [3H]20:4-prelabeled macrophages and the cytosolic phospholipase A2 activity. At concentrations < 3 x 10(-5)M, Cd2+ had no effect on PGE2 secretion but showed an augmentation of LTB4 secretion. In vitro study using macrophage lysate showed enhanced LTB4 synthesis from arachidonic acid by Cd2+, which could be responsible for the augmentation of LTB4 secretion in cells. These results indicate that Cd2+ may increase inflammation by increasing LTB4 production in lung.

In rabbit platelet-rich plasma, a specific TXA2 synthase inhibitor, CV-4151 (isbogrel) alone modestly inhibited the platelet aggregation induced by arachidonic acid (AA); even at 10(-4) M the inhibition was less than 50% (IC40 value: 8.1 x 10(-5) M). Aspirin-treated rat aortic rings alone inhibited the AA-induced platelet aggregation by 7%. However, CV-4151 exhibited a potent antiplatelet action in the presence of the aortic rings and its IC40 value was 8.3 x 10(-8) M. Thus, the aortic rings caused 1000-fold enhancement of the antiplatelet action of CV-4151. Next, we investigated the effect of CV-4151 on the production of two highly biologically active prostanoids, TXA2 and PGI2, and the contribution of these prostanoids to the antiplatelet action of CV-4151. TXA2 and PGI2 were measured by a radioimmunoassay of their stable metabolites, TXB2 and 6-keto-PGF1 alpha, respectively. Under the experimental conditions, CV-4151 simultaneously inhibited TXA2 generation and enhanced PGI2 generation. There was a positive linear relationship (r = 0.975, p < 0.01) between % inhibition of platelet aggregation and PGI2 generation and a significant negative linear relationship (r = 0.994, p < 0.001) between % inhibition of platelet aggregation and TXA2 generation. CV-4151 exhibits a potent antiplatelet action in the presence of PGI2 synthase, which is the in vivo situation, by simultaneously inhibiting TXA2 generation and enhancing PGI2 generation.

Elevation in intracellular Ca2+ acting via protein kinase C (PKC) is shown to regulate tight junction resistance in T84 cells, a human colon cancer line and a model Cl- secretory epithelial cell. The Ca2+ ionophore A23187, which was used to increase the intracellular Ca2+ concentration, caused a decrease in tight junction resistance in a concentration- and time-dependent manner. Dual Na+/mannitol serosal-to-mucosal flux analysis performed across the T84 monolayers treated with 2 microM A23187 revealed that A23187 increased both fluxes and that in the presence of ionophore there was a linear relationship between the Na+ and mannitol fluxes with a slope of 56.4, indicating that the decrease in transepithelial resistance was due to a decrease in tight junction resistance. Whereas there was no effect of 0.1 microM A23187, 1 or 2 microM produced a 55% decrease in baseline resistance in 1 hr and 10 microM decreased resistance more than 80%. The A23187-induced decrease in tight junction resistance was partially reversible by washing 3 times with a Ringer's-HCO3 solution containing 1% BSA. The A23187 effect on resistance was dependent on intracellular Ca2+; loading the T84 cells with the intracellular Ca2+ chelator BAPTA significantly reduced the decrease in tight junction resistance caused by A23187. This intracellular Ca2+ effect was mediated by protein kinase C and not calmodulin. While the protein kinase C antagonist H-7 totally prevented the action of A23187 on tight junction resistance, the Ca2+/calmodulin inhibitor W13 did not have any effect. Sphingosine, another inhibitor of PKC, partially reduced the A23187-induced decline in tight junction resistance. The PKC agonist PMA mimicked the A23187 effect on resistance, although the effect was delayed up to 1 hr after exposure. In addition, however, PMA also caused an earlier increase in resistance, indicating it had an additional effect in addition to mimicking the effect of elevating Ca2+. The effects of a phospholipase inhibitor (mepacrine) and of inhibitors of arachidonic acid metabolism (indomethacin for the cyclooxygenase pathway, NDGA for the lipoxygenase pathway, and SKF 525A for the epoxygenase pathway) on the A23187 action were also examined. None of these agents altered the A23187-induced decrease in resistance. Monolayers exposed to 2 microM A23187 for 1 hr were stained with fluorescein conjugated phalloidin, revealing that neighboring cells did not part one from another and that A23187 did not have a detectable effect on distribution of F-actin in the perijunctional actomyosin ring. The results indicate that elevation in intracellular Ca2+ decreases tight junction resistance in the T84 monolayer, acting through protein kinase C by a mechanism which does not involve visible changes in the perijunctional actomyosin ring.

The present study examined the effects of 20-hydroxyeicosatetraenoic acid (20-HETE) and 17-octadecynoic acid (17-ODYA), an inhibitor of the metabolism of arachidonic acid by P-450, on K(+)-channel activity in vascular smooth muscle cells (VSM) isolated from renal arterioles of the rat. Two types of K+ channels were characterized using inside-out excised membrane patches. One channel exhibited a large conductance (250.3 +/- 5 pS), was activated by membrane depolarization and elevations in cytoplasmic Ca2+ concentration, and was blocked by low concentrations (< 1 mM) of tetraethylammonium (TEA). The other K+ channel exhibited an intermediate conductance (46.3 +/- pS), was activated by membrane depolarization but not by changes in intracellular Ca2+ concentration, and was blocked by 4-aminopyridine (5 mM). Addition of 20-HETE to the bath (1-100 nM), reduced the frequency of opening of the large-conductance Ca(2+)-activated K+ channel recorded using cell-attached patches on VSM. It had no effect on the intermediate-conductance K+ channel: 17-ODYA (1 microM) increased the activity of the large-conductance Ca(2+)-activated K+ channel, and this effect was reversed by 20-HETE (10 nM). 20-HETE (1-1000 nM) reduced the diameter of isolated perfused small renal arteries of the rat by approximately 15% TEA (1 mM) blocked the vasoconstrictor response to 20-HETE (100 nM). These studies suggest that 20-HETE is an endogenously formed vasoconstrictor that acts in part by inhibiting the opening of the large-conductance Ca(2+)-activated K+ channel in renal arteriolar VSM.

We studied in vitro the effects on platelet aggregation and vascular tone of a new nitrocompound (nitroxy-butyl-acetylsalicylate: NO-ASA). In order to elucidate any possible activity due to the release of nitric oxide or the inhibition of platelet cyclo-oxygenase we compared NO-ASA to acetylsalicylic acid. NO-ASA 1 mM inhibited arachidonic acid-induced platelet aggregation (basal 75.4 +/- 2.35%; NO-ASA 22 +/- 3.46%; M +/- SEM; P < 0.001; n = 6), but proved less active than acetylsalicylic acid (complete inhibition at 2 x 10(-5) M). NO-ASA also significantly reduced thrombin-induced (0.04-0.08 U/ml) platelet aggregation in acetylsalicylic acid-treated platelets (basal 70.5 +/- 1.7%; NO-ASA 35.4 +/- 2.2%; P < 0.001; n = 10; IC50 7 x 10(-5) M). Methylene blue reduced the effects of NO-ASA on thrombin-induced (NO-ASA 46.7 +/- 5.25%; NO-ASA+MB 59.1 +/- 4.3%; P < 0.01; n = 8), but not arachidonic acid-induced platelet aggregation. The inhibitory effects of NO-ASA on platelet aggregation were partially removed by oxyhaemoglobin. Platelet thromboxane A2 production (TXB2 concentration in the supernatant of the aggregate 35.38 +/- 7.81 ng/ml; n = 8), was totally abolished by acetylsalicylic acid (0.17 +/- 0.04 ng/ml; P < 0.001; n = 8) and reduced by NO-ASA (8.3 +/- 4.05 ng/ml; P < 0.01; n = 8). In vitro studies on isolated rat aortic rings showed NO-ASA 10(-3) M, but not ASA up to 10(-3) M, induce a dose dependent vasorelaxation (100% of epinephrine-induced contraction) both in intact and endothelium denuded arteries (IC50 5 x 10(-5) M). Addition of methylene blue reversed this relaxation. In conclusion these data demonstrate that NO-ASA acts through a double mechanism: a) by inhibiting cyclo-oxygenase and b) by releasing NO active on guanylyn cyclase both in platelets and in vascular smooth muscle cells.

Protein kinase C (PKC) regulates a variety of signal transduction events implicated in the pathogenesis of inflammation, including the biosynthesis of inflammatory cytokines and superoxide and the activation of phospholipase A2. Because of the significant role of PKC in these inflammatory processes, we evaluated a specific and potent inhibitor of C kinase for efficacy in several in vitro and in vivo murine models of inflammation. Unlike the relatively nonspecific kinase inhibitor staurosporine, the bisindolylmaleimide 3-[1-[-3-(dimethylaminopropyl]-1H-indol-3-yl]- 4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (Gö 6850) demonstrated increased selectivity for C kinase in purified enzyme assays (respective IC50 values (microM) for Gö 6850 and staurosporine: protein kinase C (0.032, 0.009); myosin light-chain kinase (0.6, 0.01); protein kinase G (4.6, 0.018); protein kinase A (33, 0.04); tyrosine kinase1 (94, 0.4); tyrosine kinase2 (> 100, > 1)). Topically applied Gö 6850 inhibited phorbol myristate acetate-induced edema, neutrophil influx and vascular permeability in murine epidermis in a dose- and time-dependent manner at levels comparable to indomethacin. In a murine model of delayed type hypersensitivity, Gö 6850 inhibited dinitrofluorobenzene-induced contact dermatitis with and ID50 value of 150 micrograms/ear. Cellular studies in mouse peritoneal macrophages demonstrated that Gö 6850 was a potent inhibitor of phorbol myristate acetate-induced prostaglandin E2 production. Superoxide production in phorbol myristate acetate-stimulated murine neutrophils was also inhibited by Gö 6850 (IC50 = 88 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

A novel pyridobenzazepinone derivative (Z)-11-(5-carboxypentylidene)-6-methyl-5, 11-dihydropyrido +ad4,3-C] [1] benzazepin-5 (6H)-one (CAS 127654-03-9, KF 13218), inhibited human and bovine platelet thromboxane synthase with IC50 values of 27 +/- 5.8 nmol/l (mean +/- S.E.M.) and 36 +/- 6.9 nmol/l, respectively. The compound did not inhibit cyclooxygenase or 5-lipoxygenase up to a dose of 100 mumol/l and did not antagonize thromboxane A2/prostaglandin H2 receptors. KF13218 inhibited arachidonic acid-induced thromboxane B2 production by human intact platelets with an IC50 value of 5.3 +/- 1.3 nmol/l. The IC50 value of KF13218 for the intact platelets was about 5 times lower than that for the microsomal enzyme. The inhibition of thromboxane synthase in platelets by KF13218 was sustained after removal of the extracellular compound. After oral dosing in rat from 0.03 mg/kg to 3 mg/kg, KF13218 dose-dependently inhibited the thromboxane B2 production in serum, and the inhibition was retained for 72 h. KF13218, at a dose of 0.1 mg/kg p.o. prevented mortality induced by sodium arachidonate in rabbit. It is concluded that KF13218 is a potent, selective and long lasting thromboxane synthase inhibitor.

In rat luteal cells labeled with [3H]oleic acid, PGF2 alpha-stimulated phospholipase D (PLD) activation was investigated. The PLD activity was detected by measuring the accumulation of [3H]phosphatidylethanol (PtdEt) in the presence of ethanol. PGF2 alpha stimulated PtdEt accumulation at concentrations of more than 100 nM in the presence of ethanol. However, PtdEt accumulation did not change in the absence of ethanol. PGF2 alpha (1 microM) increased PtdEt accumulation after 1 min, and the accumulation reached a plateau by 2-3 min. These results indicate that PGF2 alpha activates PLD in rat luteal cells. U-73122, a phospholipase C (PLC) inhibitor, and staurosporine, a protein kinase C (PKC) inhibitor, did not inhibit PGF2 alpha-stimulated [3H]PtdEt accumulation. These results suggest that PGF2 alpha-induced PLD activation is different from PLC-PKC systems. We reported previously that PGF2 alpha stimulated the release of arachidonic acid. The effects of indomethacin, nordihydroguaiaretic acid (NDGA), and 5,8,11,14-eicosatetraynoic acid (ETYA), inhibitors of arachidonic acid metabolism, on PGF2 alpha-stimulated PtdEt accumulation were examined. Pretreatment with indomethacin enhanced PGF2 alpha-induced PtdEt accumulation. In contrast, pretreatment with NDGA and ETYA inhibited PGF2 alpha-induced PtdEt accumulation. It is suggested that PGF2 alpha-stimulated PLD activation is mediated via lipoxygenase products.

Selected 21-aminosteroids (U74500A, U74006F, and U74389G) and a 2-(aminomethyl)chromans (U78517F) were tested for their efficacy in preventing arachidonate-induced lipid peroxidation and permeability alterations in brain microvessel endothelial cells (BMECs). The 21-aminosteroids and 2-(aminomethyl)chromans were effective in varying degrees in inhibiting (U74500A = U78517F > U74006F = U74389G) concentration- and time-dependent arachidonate-induced thiobarbituric acid reactive substances (TBARS) production by BMECs. Arachidonate produced a corresponding concentration-dependent increase in BMEC monolayer permeability to the membrane impermeant marker, sucrose. Pretreatment of BMEC monolayers with either the 21-aminosteroids or the 2-(aminomethyl)chromans completely blocked the arachidonate-induced increase in permeability to sucrose. Our results demonstrated that these membrane-associating antioxidants were particularly effective in preventing both arachidonic acid-induced lipid peroxidation and permeability changes in BMEC monolayers. However, concentrations of some antioxidants that only partially inhibited TBARS production, completely inhibited the arachidonic acid-induced enhancement in BMEC monolayer permeability. Therefore, arachidonic acid-induced effects on BMEC permeability were likely due in part to both lipid peroxidation and direct or indirect effects of the fatty acid on membrane integrity. This study provides further support for the application of primary cultures of BMECs as a useful in vitro system to evaluate mechanisms through which mediators of disease or injury states compromise blood-brain barrier integrity.

Exogenous arachidonic acid induces the acrosome reaction and the production of the prostaglandins PGE2 and PGF2 alpha in bovine spermatozoa. Exogenous PGE2 also induces the acrosome reaction and PGF2 alpha synthesis. To understand better the role of PGE2 in the induction of PGF2 alpha synthesis through modulation of phospholipase A2, inhibitors of this enzyme were used. The effects of PGE2 were blocked by phospholipase A2 inhibitors and this inhibition was reversed by addition of arachidonic acid. These data indicate that PGE2 activates phospholipase A2 to produce arachidonic acid. To determine whether protein kinase C modulates phospholipase A2 activity in this process, staurosporin, an inhibitor of protein kinase C, was used. The effect of PGE2 on PGF2 alpha production is inhibited by staurosporin and this inhibition was reversed by addition of arachidonic acid indicating that protein kinase C is involved in phospholipase A2 activation. The effect of exogenous arachidonic acid or PGE2 on the acrosome reaction is blocked by lipoxygenase inhibitors but not by inhibitors of cyclo-oxygenase, indicating that lipoxygenase products are involved in the mechanism of the acrosome reaction. The presented data shed light on the cross-talk between cyclo-oxygenase and lipoxygenase and their involvement in the sperm acrosome reaction. It is suggested that cyclo-oxygenase products modulate the activity of lipoxygenase which is a key enzyme in the mechanism leading to the acrosome reaction. Stimulation of cyclo-oxygenase to synthesize PGE2 activates phospholipase A2 to release arachidonic acid which is the substrate for lipoxygenase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

A series of 4,5-diphenylthiazoles containing morpholinoalkyl amino groups at the 2 position was synthesized and their anti-platelet activities were evaluated. The inhibitory effects of the compounds in which phenyl groups at the 4 position were substituted by methoxy groups were more potent than those of aspirin and ibuprofen on the collagen-induced rabbit platelet aggregation. These compounds were also shown to have potent efficacies on the arachidonic acid-induced platelet aggregation. On the other hand, these compounds did not affect the ADP-induced platelet aggregation similarly to non-steroidal anti-inflammatory agents. Moreover, their efficacies on the inhibition of prostaglandin synthesis were more potent than that of ibuprofen. Therefore, we thought that 2-(morpholinoalkyl)amino-4,5-diphenylthiazoles, in spite of their basic properties, inhibited the platelet aggregation based on cyclooxygenase.

The polyunsaturated fatty acids, arachidonic, linoleic, and linolenic acids, were potent blockers of regulatory volume decrease (RVD) and of the swelling-activated efflux of [3H]taurine, D-[3H]aspartate, [3H]inositol, and 125I (used as marker of Cl) from rat cerebellar astrocytes in culture. The monounsaturated oleic and ricinoleic acids and saturated fatty acids were ineffective. The amino acid and 125I fluxes were similarly inhibited by fatty acids, whereas inositol release was less sensitive. Polyunsaturated fatty acids appear to directly affect RVD in trypsinized astrocytes as the inhibition was immediate and fully reversible. Blockers of the arachidonic acid metabolic pathways, indomethacin (cyclooxygenase), esculetin (lipoxygenases), and metyrapone (P-450 monooxygenases), did not prevent the effect of arachidonic acid, suggesting that further metabolism is not required for displaying the effects of arachidonic acid on RVD and osmolyte fluxes. Some blockers of arachidonic acid metabolic pathways, such as nordihydroguaiaretic acid (lipoxygenases) and naphthoflavone (P-450 monooxygenases), also exhibited marked inhibitory effects on RVD and on osmolyte fluxes. The predominant arachidonic acid metabolite in astrocytes, 12-hydroxyeicosatetraenoic acid, did not affect RVD or osmolyte fluxes. These results suggest that arachidonic acid and other polyunsaturated fatty acids directly inhibit the permeability pathways correcting cell volume after swelling in cultured astrocytes.

1. Platelet behaviour in vitro in relation to cyclic AMP was studied longitudinally during pregnancy and in the same women when they were not pregnant. Subjects comprised a group of healthy primigravidae and a group of women deemed at risk of pre-eclampsia, on the basis of a previous history of the condition. 2. There was a progressive decline during pregnancy in sensitivity of platelets to inhibition of the arachidonic acid-induced release reaction by agents which act via cyclic AMP. This effect was maximum at 36 weeks' gestation. 3. Basal platelet cyclic AMP levels, and those in the presence of a phosphodiesterase inhibitor, did not change throughout the period of the study. 4. By contrast, platelet cyclic AMP accumulation in response to a variety of adenylate cyclase stimulators was reduced from early pregnancy, throughout the gestational period, compared with post-natally. This effect was noted when platelets were incubated with prostaglandins acting via different surface receptors or with forskolin and was most marked on co-incubation with a phosphodiesterase inhibitor. 5. Compared with healthy women, platelets from women with a previous history of pre-eclampsia tended to accumulate less cyclic AMP in response to adenylate cyclase stimulators. This was the case both during pregnancy and post-natally. Further investigation of adenylate cyclase activity in platelets in relation to pre-eclampsia is required.