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1.
The lipids of isolated Krebs perfused rabbit kidneys and hearts were labeled with [14C]arachidonic acid. Subsequent hormonal stimulation (e.g. bradykinin, ATP) of the pre-labelled tissue resulted in dose-dependent release of [14C]prostaglandins; little or no release of the precursor [14]arachidonic acid was observed. When fatty acid-free bovine serum albumin was added to the perfusion medium as a trap for fatty acids substantial release of [14C]arachidonic acid was detected following hormonal stimulation. The release of [14C]arachidonic acid was dose-dependent and >;3 fold that of [14C]prostaglandin release. Indomethacin by inhibiting the cyclo-oxygenase, completely inhibited release of [14C]prostaglandins and only slightly inhibited release of [14C]arachidonic acid. These results demonstrate that in both rabbit kidney and heart much more substrate is released by hormonal stimulation than is converted to prostaglandins. This suggests that either the deacylation reaction is not tightly coupled to the prostaglandin synthetase system or that there are two deacrylation mechanisms, one which is coupled to prostaglandin synthesis while the other is non-specific. It has previously been shown that prostaglandin release due to hormones such as bradykinin is transient despite continued presence of the hormone (tachyphylaxis). By utilizing albumin to trap released fatty acid, it was found that hormone-stimulated release of arachidonic acid is also transient. This directly demonstrates that tachyphylaxis occurs at a step prior to the cyclo-oxygenase.  相似文献   

2.
The importance of phospholipase(s) activation in the IgE-mediated and ionophoreinduced histamine release from the rat basophilic leukemia cell line has been examined. The activation of phospholipase(s) as measured by [14C]arachidonic acid release and the release of histamine both required Ca2+ and were temporally parallel. Inhibition of phospholipase(s) activity by the inhibitors mepacrine and α-parabromoacetophenone also correlated with the inhibition of histamine release. [14C]Arachidonic acid released by the phospholipase(s) was mainly metabolized to prostaglandin D2. The inhibition of the cyclooxygenase pathway by indomethacin did not affect histamine release. 5,8,11,14-Eicosatetraynoic acid inhibited both histamine and [14C]arachidonic acid release suggesting an effect not only on the cyclooxygenase and lipoxygenase pathways but also on the phospholipase(s). These results suggest that activation of phospholipase appears to be necessary for histamine release in the rat bosophilic leukemia cells.  相似文献   

3.
The relationship between the increase of intracellular Ca2+ and the release of arachidonic acid by bradykinin and pyrophosphonucleotides was studied in cultured mammary tumour cells, MMT060562. Bradykinin, ATP, UTP and UDP induced an increase of intracellular Ca2+ and the release of arachidonic acid from phospholipids into the extracellular fluid. Release of arachidonic acid was also induced by the application of the Ca2+ ionophore, A23187. Liberation of arachidonic acid by bradykinin and ATP was reduced by mepacrine, a blocker of phospholipase A2 and W-7, a calmodulin antagonist. It is suggested that the increase in cytosolic Ca2+-induced release of arachidonic acid occurs through activation of calmodulin-dependent phospholipase A2.  相似文献   

4.
Human synovial fibroblasts in culture respond to bradykinin with a 20-fold increment in intracellular cyclic AMP concentrations, however bradykinin does not directly activate adenylate cyclase activity in a particulate fraction derived from these cells. Bradykinin evokes a release of labeled arachidonic acid and prostaglandins E and F from synovial fibroblasts pre-labeled with 3H-arachidonic acid. Hydrocortisone inhibits the bradykinin induced increment in cyclic AMP and the release of arachidonic acid and prostaglandins E and F from synovial fibroblasts. Indomethacin, which also inhibits the cyclic AMP response to bradykinin, has no effect on the release of arachidonic acid from synovial fibroblasts. Indomethacin does, however, inhibit the quantity of prostaglandins released into the medium. These studies support the hypothesis that bradykinin does not activate human synovial fibroblast adenylate cyclase, but presumably activates a phospholipase whose products in turn result in the synthesis of prostaglandins. These and other investigations also suggest that a product(s) of the prostaglandin pathway causes the increment in cyclic AMP.  相似文献   

5.
A method is described for the efficient incorporation of radioactive arachidonic acid into the lipids of rabbit hearts and kidneys. Infusion of 14C-arachidonate through perfused tissues resulted in the quantitative removel of label from the media. Analysis of the lipids from tissues labeled by this procedure revealed that the majority of the 14C-arachidonate was incorporated into phospholipids. Essentially all of the radioactivity in phosphatidylcholine was found in the 2-position. Subsequent to the 14C-arachidonate infusion, stimulation of prostaglandin biosynthesis (e.g. by bradykinin) resulted in the release of radioactive prostaglandins. This suggests that the 14C-arachidonate is incorporated in a manner such that it is available for homone-stimulated prostaglandin biosynthesis. The method described allows both qualitative and quantitative analysis of arachidonate metabolism in intact tissues and offers significant advantages over other presently used methods.  相似文献   

6.
We provided evidence that calcium-calmodulin plays a major role in bradykinin-induced arachidonic acid release by bovine aortic endothelial cells. In cells labeled for 16 hr with 3H-arachidonic acid, ionomycin and Ca2+-mobilizing hormones such as bradykinin, thrombin and platelet activating factor induced arachidonic acid release. However, arachidonic acid release was not induced by agents known to increase cyclic AMP (forskolin, isoproterenol) or cyclic GMP (sodium nitroprusside). Bradykinin induced the release of arachidonic acid in a dose-dependent manner (EC50 = 1.6 ± 0.7 nM). This increase was rapid, reaching a maximal value of fourfold above basal level in 15 min. In a Ca2+-free medium, bradykinin was still able to release arachidonic acid but with a lower efficiency. Quinacrine (300 μM), a blocker of PLA2, completely inhibited bradykinin-induced arachidonic acid release. The B2 bradykinin receptor antagonist HOE-140 completely inhibited bradykinin-induced arachidonic acid release. The B1-selective agonist DesArg9-bradykinin was inactive and the B1-selective antagonist [Leu8]DesArg9-bradykinin had no significant effect on bradykinin-induced arachidonic acid release. The phospholipase C inhibitor U-73122 (100 μM) decreased bradykinin-induced arachidonic acid release. The calmodulin inhibitor W-7 (50 μM) drastically reduced the bradykinin- and ionomycin-induced arachidonic acid release. Also, forskolin decreased bradykinin-induced arachidonic acid release. These results suggest that the activation of PLA2 by bradykinin in BAEC is a direct consequence of phospholipase C activation. Ca2+-calmodulin appears to be the prominent activator of PLA2 in this system. © 1996 Wiley-Liss, Inc.  相似文献   

7.
The pattern of prostaglandins produced from arachidonic acid by two sublines of MDCK canine kidney epithelia cells was different. In one subline designated MDCK1, the most prevalent prostaglandin product was PGE2, whereas the most prevalent product in the subline designated MDCK2 was PGF. This difference was observed when cells previously labeled with [1?14C]arachidonic acid were stimulated with either bradykinin or the calcium ionophore A23187, or when prostaglandins were produced from labeled arachidonic acid added directly to the assay medium. In the latter case, the difference was maintained over a 38-fold range of extracellular arachidoante concentrations. These findings indicate the there is a persistent difference in the distribution of prostaglandins produced by the two commonly used sublines of MDCK cells.  相似文献   

8.
The lipids of isolated Krebs perfused rabbit kidneys and hearts were labelled with [14C]arachidonic acid. Subsequent hormonal stimulation (e.g. bradykinin, ATP) of the pre-labelled tissue resulted in dose-dependent release of [14C]prostaglandins; little or no release of the precursor [14C]arachidonic acid was observed. When fatty acid-free bovine serum albumin was added to the perfusion medium as a trap for fatty acids substantial release of [14C]arachidonic acid was detected following hormonal stimulation. The release of [14C]arachidonic acid was dose-dependent and greater than 3 fold that of [14C]prostaglandin release. Indomethacin by inhibiting the cyclo-oxygenase, completely inhibited release of [14C]prostaglandins and only slightly inhibited release of [14C]arachidonic acid. These results demonstrate that in both rabbit kidney and heart much more substrate is released by hormonal stimulation than is converted to prostaglandins. This suggests that either the deacylation reaction is not tightly coupled to the prostaglandin synthetase system or that there are two deacylation mechanisms, one which is coupled to prostaglandin synthesis while the other is non-specific. It has previously been shown that prostaglandin release due to hormones such as bradykinin is transient despite continued presence of the hormone (tachyphylaxis). By utilizing albumin to trap released fatty acid, it was found that hormone-stimulated release of arachidonic acid is also transient. This directly demonstrates that tachyphylaxis occurs at a step prior to the cyclo-oxygenase.  相似文献   

9.
The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3′5′-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of 14C-arachidonic acid by washed platelets. Prostaglandin E1 (PGE1), PGE1 with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B2. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE1 with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of 14C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.  相似文献   

10.
Methylcholanthrene-transformed mouse fibroblasts synthesize prostaglandins in response to bradykinin, thrombin, serum, and the ionophore A23187. These agents activate phospholipases, thereby releasing fatty acids from phospholipids. To examine the phospholipid specificity of the phospholipases activated by bradykinin, thrombin, serum, and A23187, cells were labeled with [14C]arachidonic acid and stimulated with these agents in the presence of delipidated bovine serum albumin. Phospholipid classes were resolved by two-dimensional chromatography on silica gel-coated paper. Only phosphatidylinositol and phosphatidylcholine lost radioactivity upon stimulation. To characterize the fatty acid specificity of the phospholipases, cells were incubated with 14C-labeled stearic, oleic, linoleic, eicosatrienoic, or arachidonic acid and then exposed to the stimuli. Bradykinin, thrombin, and serum caused specific release of radioactivity into the medium only from cells labeled with arachidonic acid or eicosatrienoic acid, whereas A23187 caused release from cells labeled with any one of the five fatty acids. We conclude that bradykinin, thrombin, and serum activate phospholipases that specifically hydrolyze arachidonyl and eicosatrienoyl phosphatidylinositol and phosphatidylcholine, whereas A23187 is less specific activator of phospholipases.  相似文献   

11.
Stimulation of IgE receptors on rat basophilic leukemia cells causes a transient rise and fall of methylated phopholipids, Ca2+ influx, and release of arachidonic acid previously incorporated into phosphatidylcholine and liberation of histamine. Inhibition of phospholipid methylation by methyltransferase inhibitors, 3-deazaadenosine and homocysteine thiolactone, almost completely blocks the influx of Ca2+, and release of arachidonic acid and histamine. Stimulation of immunoglobulin E receptors by antigen releases only [14C]arachidonic acid but not [14C]linoleic acid, [14C]oleic acid and [14C]stearic acid, all of which were previously incorporated into phospholipids. [14C]Arachidonate was found to be incorporated mainly into phosphatidylcholine. The phosphatidycholine rich in arachidonate appeared to be synthesized to a considerable extent by the transmethylation pathway. These findings suggest that in rat basophilic leukemia cells, immunoglobulin E receptors, phospholipid methyltransferases, Ca2+ ion channel, and phospholipase(s) that cause release of arachidonic acid and the discharge of histamine are associated.  相似文献   

12.
Effect of various prostaglandins on the release of arachidonic acid from [14C]arachidonic acid labeled fibroblasts was studied. Prostaglandin(PG) F was found to enhance the release of radioactive arachidonic acid from the cells. The stimulatory effect was dose dependent, and was greater than that of bradykinin. The active compounds can be ranked in potency for the release of arachidonic acid from the pre-labeled cells per cent of control: PGF(200.1%)>PGF (141.8%)>PGD2 (137.1%)>thromboxane B2 (113.7%)>PGE2 (109.4%). On the other hand, PGI2 showed a strong inhibitory effect on the arachidonic acid release from the pre-labeled cells (the value was only 69% of the control), while 6-ketoPGF, an end metabolite of PGI2, had no effect.  相似文献   

13.
The tissue lipids of isolated, perfused rabbit hearts and hydronephrotic kidneys were labelled with [14C]-arachidonic acid by two different techniques: direct infusion of [14C]-arachidonic acid in a protein free media into the perfused organ (method A), and recirculation of [14C]-arachidonic acid in a solution containing albumin (method B). Autoradiography of the labelled organs demonstrated that method A resulted in selective labelling of arteries and arterioles in both perfused organs as well as glomeruli in the kidney. Labelling with method B resulted in a non-specific radioisotope incorporation in both the vasculature and myocardial cells in the heart; and of the vasculature and renal tubules in the perfused kidneys. Analysis of the tissue lipids shows similar patterns of incorporation of radioactivity between methods A and B.Peptide hormone stimulation (bradykinin) and non-specific noxious stimulation (with transient ischemia) were employed to elicit lipase activation (i.e., release of [14C]-arachidonate) and prostaglandin (PG) synthesis. It was found that in both hearts and hydronephrotic kidneys, the radioactive PG release in response to bradykinin and ischemia was much higher with method A (vascular labelling) than with method B (diffuse labelling) despite the appearance of comparable amounts of bioassayable PG release, thus indicating the sites of PG synthesis in these organs is predominantly localized in the vascular tissue. Furthermore, the radioactive arachidonic acid release in response to bradykinin stimulation in the hydronephrotic kidneys was 3 times higher with method A than with method B, suggesting the predominant sites of hormone specific lipase activation in the renal cortex is also in the vasculature. However, the radioactive arachidonic acid release in response to ischemia was much higher with method B than with method A in both hearts and hydronephrotic kidneys, indicating the sites of non-specific lipase activation in these organs are more diffusely distributed, and present also in the myocardial cells and renal tubules.  相似文献   

14.
Serum and/or arachidonic acid stimulated prostaglandin production by dog kidney (MDCK) cells. Epidermal growth factor (EGF) at concentrations of 10?9 to 10?10 M stimulated the biosynthesis of prostaglandins by MDCK cells but not that by human fibroblasts (D-550), mouse fibroblasts (3T3), transformed mouse fibroblasts (MC5-5), and rabbit aorta endothelial cells (CLO). EGF also stimulated the release of radioactivity from MDCK cells radioactively labelled with [3H]arachidonic acid.  相似文献   

15.
Vascular endothelial cells respond to a variety of physiological and pharmacological stimuli by releasing free arachidonic acid from membrane phospholipids, thus initiating synthesis of prostacyclin. Previous work in our laboratory has demonstrated that the thrombin-stimulated deacylation is specific for arachidonate and structurally similar polyunsaturated fatty acids that contain a delta-5 double bond. We now report that histamine, bradykinin, and the calcium ionophore A23187 exhibit the same fatty acid specificity as does thrombin. Experiments with both human umbilical vein and calf pulmonary artery endothelial cells indicate that these agonists stimulate the release of previously incorporated [14C]arachidonate but not 8,11,14-[14C]eicosatrienoate or [14C]docosatetraenoate. By contrast, melittin stimulates the release of 8,11,14-eicosatrienoate, docosatetraenoate, and oleate as well as arachidonate. These results suggest that histamine, bradykinin, and A23187 activate a common calcium-dependent phospholipase A2. Melittin appears either to alter the substrate specificity of the receptor-linked phospholipase A2 activity or to activate additional enzymes as well.  相似文献   

16.
Arachidonic acid release and the effect of phospholipase inhibitors on various types of cell injuries and death to rabbit renal proximal tubule suspensions were determined. Proximal tubules were exposed to the mitochondrial inhibitor antimycin A (0.1 μM), the protonophore carbonyl cyanide ρ-trifluoromethoxypheitylhydrazone (1 μM FCCP), the oxidant tertbutyl hydroperoxide (0.5 mM TBHP), or the calcium ionophore ionomycin (5 μM) in the absence or presence of the putative phospholipase inhibitors dibucaine, mepacrine, chlorpromazine, or U-26384. The phospholipase inhibitors had no effect on the proximal tubule lactate dehydrogenase (LDH) release (a marker of cell death) produced by FCCP, antimycin A, or ionomycin after 1,2, or 2 hours of exposure, respectively. Only dibucaine and mepacrine decreased LDH release in TBHP-treated proximal tubules without decreasing TBHP-induced lipid peroxidation. Antimycin A and ionomycin did not release arachidonic acid from proximal tubules prelabeled with [1-14C] arachidonic acid. In contrast, TBHP released arachidonic acid from proximal tubules prior to the onset of cell death, and dibucaine and mepacrine decreased the TBHP-induced release. Thus, phospholipase inhibitors were cytoprotective in those injuries that produced arachidonic acid release. These results suggest that arachidonic acid release and phospholipase A2 activation play a contributing role in oxidant-induced renal proximal tubule cell injury and death but not in mitochondrial inhibitor- or calcium ionophore-induced proximal tubule cell injury and death.  相似文献   

17.
5,8,11,14-eicosatetraynoic acid (ETYA), a widely used inhibitor of cyclooxygenase and lipoxygenase, inhibited the incorporation of 14C-arachidonic acid into cell lipids of the murine thymoma EL4 whereas oleic acid had no effect. Inhibition appeared to result from the ability of ETYA to compete with arachidonic acid for esterification enzymes and to be itself incorporated into cell lipids. The positional specificity for ETYA incorporation was similar to that of arachidonic acid. ETYA, but not oleic acid competed with arachidonate for activation by a selective arachidonoyl CoA synthetase in lymphocytes. This may explain in part the apparent specificity of effects seen on incorporation into whole cells. In addition ETYA, unlike other arachidonate analogs tested previously, caused significant inhibition of the nonselective acyl CoA synthetase in lymphocytes. These results are discussed with respect to the use of ETYA to examine the role of intrinsic arachidonic acid metabolism in cellular processes.  相似文献   

18.
Oligomycin, antimycin, and 2,4-dinitrophenol, compounds that are known to inhibit oxidative phosphorylation by different mechanisms, inhibit the production of prostaglandins by serum-stimulated MC5-5 cells. The prostaglandin production that is stimulated by thrombin and bradykinin is inhibited by 2,4-dinitrophenol. Prostaglandin synthesis by MC5-5 cells from exogenously-supplied arachidonic acid, however, is not affected by 2,4-dinitrophenol. Antimycin and 2,4-dinitrophenol also inhibit the serum-stimulated release of arachidonic acid from the cellular lipids, suggesting that it is the expression of phospholipase activity, a prerequisite for synthesis of prostaglandins by MC5-5 cells, that is dependent on oxidative phosphorylation.  相似文献   

19.
Human endometrial cells were dispersed with collagenase and maintained in culture overnight. The synthesis of PGF by the dispersed cells incubated at 37 degrees C in serum-free medium was stimulated by estradiol (10(-7)M - 10(-5)M), histamine (5X10(-7)M - 5X10(-5)M), bradykinin (10(-6)M), phorbol myristate (PMA, 3X10(-8)M) and arachidonate (5X10(-6)M). Preincubation of the cells for 3 h with cortisol (5X10(-7)M - 5X10(-5)M), progesterone (10(-6)M) or mepacrine (10(-6)M - 2X10(-4)M) inhibited the response to histamine, bradykinin and PMA but not to arachidonate. Perfusion of the cultured cells in filtration chambers yielded similar results to those obtained in the incubation system but differences in the onset and duration of the responses to stimuli were found. In the perifusion system the responses to histamine and bradykinin were rapid and of short duration (peak response in less than 60 min) while the responses to PMA and arachidonate were of longer duration with a slower onset. We conclude that these observations using dispersed endometrial cells are consistent with previous work showing that histamine, bradykinin and PMA act by stimulating acylhydrolase activity, thereby liberating precursors such as arachidonic acid which are converted to prostaglandins by the cyclo-oxygenase complex.  相似文献   

20.
Ethanolamine plasmalogens (1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamines) of many tissues contain high levels of arachidonate at their 2-position, and in certain tissues have been implicated as possible donors of arachidonate required in the synthesis of prostaglandins and thromboxanes. In the present study, [3H]arachidonate-labeled phospholipids of HSDM1C1 cells, a cell line derived from a mouse fibrosarcoma, were examined to determine the donor of the arachidonic acid released upon bradykinin stimulation of the synthesis of PGE2. HSDM1C1 cells labeled with [3H]arachidonic acid for 24 hr in serum-free medium were used in most of the experiments and had the following distribution of label among the cellular lipids; phosphatidylcholine (33%), phosphatidylinositol (20%), diacyl-sn-glycero-3-phosphoethanolamine (15%), ethanolamine plasmalogen (15%), and less polar lipids (16%). Bradykinin treatment stimulated a rapid hydrolysis of [3H]arachidonate from the cellular lipids and conversion of the released acid to PGE2, which was secreted into the medium. The label was released predominantly from phosphatidylinositol and possibly from phosphatidylcholine with no detectable change in the labeling of diacyl- or 1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. The ethanolamine plasmalogens, therefore, do not appear to be involved in the stimulated release of arachidonate in the HSDM1C1 cells. Indomethacin blocked the bradykinin-stimulated synthesis of PGE2 and to a lesser degree inhibited the release of [3H]-arachidonate from the cellular lipids into the medium.  相似文献   

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