Thrombin causes pseudopod detachment via a pathway involving cytosolic phospholipase A2 and 12/15-lipoxygenase products.
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Thrombin causes rapid pseudopod detachment and shortening in Dunning rat prostatic carcinoma (MAT-Lu) cells. As seen by interference reflection microscopy and by immunofluorescence analysis with antibodies to paxillin and talin, the primary event is disassembly of adhesion sites. Biochemically, thrombin is a potent activator of cytosolic phospholipase A2 and increases eicosanoid production in these cells. The pseudopod effects are blocked by lipoxygenase (but not cyclooxygenase) inhibitors. Arachidonic acid and 12(S)-hydroxyeicosatetraenoic acid or 15(S)-hydroxyeicosatetraenoic acid mimic the thrombin effect. We conclude that in certain cancer cells, thrombin is a pseudopod repellent that exerts its effect via a cascade involving cytosolic phospholipase A2, 12/15-lipoxygenase, and 12(S)- and/or 15(S)-hydroxyeicosatetraenoic acid. (+info)
Cardiovascular effects of 2-arachidonoyl glycerol in anesthetized mice.
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Cannabinoids, including the endogenous ligand anandamide, elicit pronounced hypotension and bradycardia through the activation of CB1 cannabinoid receptors. A second endogenous cannabinoid, 2-arachidonoyl glycerol (2-AG), has been proposed to be the natural ligand of CB1 receptors. In the present study, we examined the effects of 2-AG on mean arterial pressure and heart rate in anesthetized mice and assessed the role of CB1 receptors through the use of selective cannabinoid receptor antagonists and CB1 receptor knockout (CB1(-/-)) mice. In control ICR mice, intravenous injections of 2-AG or its isomer 1-AG elicit dose-dependent hypotension and moderate tachycardia that are unaffected by the CB1 receptor antagonist SR141716A. The same dose of SR141716A (6 nmol/g IV) completely blocks the hypotensive effect and attenuates the bradycardic effect of anandamide. 2-AG elicits a similar hypotensive effect, resistant to blockade by either SR141716A or the CB2 antagonist SR144528, in both CB1(-/-) mice and their homozygous (CB1(+/+)) control littermates. In ICR mice, arachidonic acid (AA, 15 nmol/g IV) elicits hypotension and tachycardia, and indomethacin (14 nmol/g IV) inhibits the hypotensive effect of both AA and 2-AG. Synthetic 2-AG incubated with mouse blood is rapidly (<2 minutes) and completely degraded with the parallel appearance of AA, whereas anandamide is stable under the same conditions. A metabolically stable ether analogue of 2-AG causes prolonged hypotension and bradycardia in ICR mice, and both effects are completely blocked by SR141716A, whereas the same dose of 2-AG-ether does not influence blood pressure and heart rate in CB1(-/-) mice. These findings are interpreted to indicate that exogenous 2-AG is rapidly degraded in mouse blood, probably by a lipase, which masks its ability to interact with CB1 receptors. Although the observed cardiovascular effects of 2-AG probably are produced by an arachidonate metabolite through a noncannabinoid mechanism, the CB1 receptor-mediated cardiovascular effects of a stable analogue of 2-AG leaves open the possibility that endogenous 2-AG may elicit cardiovascular effects through CB1 receptors. (+info)
Apamin-sensitive, non-nitric oxide (NO) endothelium-dependent relaxations to bradykinin in the bovine isolated coronary artery: no role for cytochrome P450 and K+.
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Since cytochrome P(450)-derived metabolites of arachidonic acid and K(+) have been implicated in endothelium-derived hyperpolarizing factor (EDHF)-dependent responses, the aim of this study was to determine whether such factors contribute to non-nitric oxide (NO), endothelium-dependent relaxation to bradykinin (BK) in bovine isolated coronary artery. In rings of artery contracted with U46619 and treated with indomethacin (3 microM) and N(G)-nitro-L-arginine (L-NOARG; 100 microM), relaxation to BK (0.01 nM-0.3 microM) was blocked by approximately 60% after inhibition of K(+) channels with either high extracellular K(+) (high [K(+)](o); 15 - 67 mM) or apamin (0.3 microM). Ouabain (1 microM), an inhibitor of Na(+)/K(+)-ATPase, decreased the sensitivity to BK without affecting the maximum response. In L-NOARG-treated rings, ouabain had no further effect on the relaxation to BK. An inhibitor of inward-rectifying K(+) channels, Ba(2+) (30 microM), had no effect on relaxations to BK in the absence or presence of either L-NOARG or ouabain. KCl (2.5 - 10 mM) elicited small relaxations ( approximately 20%) that were abolished by nifedipine (0.3 microM) and ouabain. Both the high [K(+)](o)/apamin-sensitive relaxation to BK, and the relaxation to the K(ATP) channel-opener, levcromakalim (0.6 microM), were unaffected by the cytochrome P(450) inhibitor, 7-ethoxyresorufin (10 microM), or by co-treatment with a phospholipase A(2) inhibitor, arachidonyl trifluoromethyl ketone (AACOCF(3); 3 microM) and a diacylglycerol (DAG)-lipase inhibitor, 1, 6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC 80267; 30 microM). The non-NO/high [K(+)](o)-insensitive, approximately 40% relaxation to BK was, however, abolished by these treatments. Therefore, neither cytochrome P(450)-derived metabolites of arachidonic acid nor K(+) appear to mediate the EDHF-like relaxation to BK (i.e the non-NO, high [K(+)](o)/apamin-sensitive component) in bovine coronary arteries. Cytochrome P(450)-derived metabolites may be released at higher BK concentrations to act in parallel with NO and the high [K(+)](o)/apamin-sensitive mechanism. (+info)
LTA(4)-derived 5-oxo-eicosatetraenoic acid: pH-dependent formation and interaction with the LTB(4) receptor of human polymorphonuclear leukocytes.
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5-oxo-(7E,9E,11Z,14Z)-eicosatetraenoic acid (5-oxo-ETE) has been identified as a non-enzymatic hydrolysis product of leukotriene A(4) (LTA(4)) in addition to 5,12-dihydroxy-(6E,8E,10E, 14Z)-eicosatetraenoic acids (5,12-diHETEs) and 5,6-dihydroxy-(7E,9E, 11Z,14Z)-eicosatetraenoic acids (5,6-diHETEs). The amount of 5-oxo-ETE detected in the mixture of the hydrolysis products of LTA(4) was found to be pH-dependent. After incubation of LTA(4) in aqueous medium, the ratio of 5-oxo-ETE to 5,12-diHETE was 1:6 at pH 7.5, and 1:1 at pH 9.5. 5-Oxo-ETE was isolated from the alkaline hydrolysis products of LTA(4) in order to evaluate its effects on human polymorphonuclear (PMN) leukocytes. 5-Oxo-ETE induced a rapid and dose-dependent mobilization of calcium in PMN leukocytes with an EC(50) of 250 nM, as compared to values of 3.5 nM for leukotriene B(4) (LTB(4)500 nM for 5(S)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE). Pretreatment of the cells with LTB(4) totally abolished the calcium response induced by 5-oxo-ETE. In contrast, the preincubation with 5-oxo-ETE did not affect the calcium mobilization induced by LTB(4). The calcium response induced by 5-oxo-ETE was totally inhibited by the specific LTB(4) receptor antagonist LY223982. These data demonstrate that 5-oxo-ETE can induce calcium mobilization in PMN leukocyte via the LTB(4) receptor in contrast to the closely related analog 5-oxo-(6E,8Z,11Z, 14Z)-eicosatetraenoic acid which is known to activate human neutrophils by a mechanism independent of the receptor for LTB(4). (+info)
Carrier-mediated uptake of the endogenous cannabinoid anandamide in RBL-2H3 cells.
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Anandamide (N-arachidonylethanolamide) is an endogenous cannabinoid that mimics the pharmacologic effects of Delta(9)-tetrahydrocannabinol, the major bioactive substance in marijuana. Anandamide appears to be synthesized, released, and inactivated by mechanisms similar to those for other neurotransmitters. Of interest to the present studies are reports that anandamide undergoes carrier-mediated uptake into neuronal or glial cells after release, followed by rapid intracellular degradation by the intracellular fatty acid amidohydrolase. In addition to effects in the brain, anandamide has multiple effects in the periphery, particularly on cells of the immune system that express both a peripheral cannabinoid receptor and amidohydrolase enzyme. We have performed a detailed characterization of anandamide uptake in the cognate mast cell line RBL-2H3 to test the hypothesis that the uptake system in peripheral cells is also carrier-mediated and functionally similar to that observed in the central nervous system. RBL-2H3 cells exhibited robust, saturable transport of [(3)H]anandamide that was both time- and temperature-sensitive. This transport activity was not dependent on extracellular ion gradients for uptake and was inhibited selectively by other fatty acid-derived molecules, anandamide congeners, and the psychoactive cannabinoids such as Delta(9)-tetrahydrocannabinol. We conclude that anandamide transport in the RBL-2H3 cells is carrier-mediated, and uptake in peripheral cells is functionally and pharmacologically identical with that observed in neurons and astrocytes. (+info)
Correlation of increased concentration of ovine endometrial cyclooxygenase 2 with the increase in PGE2 and PGD2 in the late luteal phase.
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Ovine endometrium showed transient expression of high concentrations of the inducible isoform of cyclooxygenase, cyclooxygenase 2 (COX-2), whereas the constitutive isoform, cyclooxygenase 1 (COX-1), was expressed at much lower concentrations and did not change. In this study, the pattern of prostaglandin synthesis in endometrial luminal cells was investigated in relation to their COX-2 content. Endometrial cells from cyclic or pregnant ewes at days 9, 12, 14 and 16 were isolated and analysed for the presence of COX-1 and COX-2 proteins using western blot analysis. Freshly isolated cells were incubated with 0.5 microCi [3H]arachidonic acid ml-1. Radioactive cyclooxygenase metabolites were analysed by reverse-phase HPLC. Luminal cells produced mainly PGF2 alpha, PGE2, PGD2 and 13,14-dihydro-15-keto PGF2 alpha and to a lesser extent 6-keto PGF1 alpha, thromboxane B2 and 13,14-dihydro-15-keto PGE2. The production of PGE2 and PGD2 was proportional to the cellular concentration of COX-2. PGE2 and PGD2 release was low on day 9 when COX-2 was not expressed, whereas high concentrations of PGE2 and PGD2 were synthesized on days 12-14 when COX-2 was highly expressed, reaching 100 ng microgram-1 cellular protein. In contrast, the basal production of PGF2 alpha did not appear to be related to COX-2 concentration and was greatest on day 16. Moreover, the release of PGF2 alpha was maintained at steady state values between days 9 and 14 by the production of 13,14-dihydro-15-keto PGF2 alpha. Although PGF2 alpha output was lower at day 16 of pregnancy compared with the oestrous cycle, no difference was observed in the pattern of prostaglandin synthesis between pregnant and non-pregnant ewes. (+info)
Human mast cells take up and hydrolyze anandamide under the control of 5-lipoxygenase and do not express cannabinoid receptors.
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Human mast cells (HMC-1) take up anandamide (arachidonoyl-ethanolamide, AEA) with a saturable process (K(m)=200+/-20 nM, V(max)=25+/-3 pmol min(-1) mg protein(-1)), enhanced two-fold over control by nitric oxide-donors. Internalized AEA was hydrolyzed by a fatty acid amide hydrolase (FAAH), whose activity became measurable only in the presence of 5-lipoxygenase, but not cyclooxygenase, inhibitors. FAAH (K(m)=5.0+/-0.5 microM, V(max)=160+/-15 pmol min(-1) mg protein(-1)) was competitively inhibited by palmitoylethanolamide. HMC-1 cells did not display a functional cannabinoid receptor on their surface and neither AEA nor palmitoylethanolamide affected tryptase release from these cells. (+info)
The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1).
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The endogenous cannabinoid anandamide was identified as an agonist for the recombinant human VR1 (hVR1) by screening a large array of bioactive substances using a FLIPR-based calcium assay. Further electrophysiological studies showed that anandamide (10 or 100 microM) and capsaicin (1 microM) produced similar inward currents in hVR1 transfected, but not in parental, HEK293 cells. These currents were abolished by capsazepine (1 microM). In the FLIPR anandamide and capsaicin were full agonists at hVR1, with pEC(50) values of 5. 94+/-0.06 (n=5) and 7.13+/-0.11 (n=8) respectively. The response to anandamide was inhibited by capsazepine (pK(B) of 7.40+/-0.02, n=6), but not by the cannabinoid receptor antagonists AM630 or AM281. Furthermore, pretreatment with capsaicin desensitized the anandamide-induced calcium response and vice versa. In conclusion, this study has demonstrated for the first time that anandamide acts as a full agonist at the human VR1. (+info)