Cysteinyl leukotrienes are involved in angiotensin II-induced contraction of aorta from spontaneously hypertensive rats

OBJECTIVE: Non specific lipoxygenase inhibitors have been reported to reduce the in vitro constrictor response and the in vivo pressor effect of angiotensin II in rats. The aim of this study was to assess the role of cysteinyl leukotrienes, in the vascular response to angiotensin II in spontaneously hypertensive rats (SHR). METHODS: Rings of thoracic aorta from SHR and normotensive Wistar-Kyoto rats (WKY) were compared in terms of contractile responses and release of cysteinyl leukotrienes in response to angiotensin II. RESULTS: Pretreatment with the specific 5-lipoxygenase inhibitor AA861 10 microM reduced the efficacy of angiotensin II in intact and endothelium-denuded aorta from SHR (% inhibition vs. control: 65+/-12.6% with endothelium (n=6), P<0.05; 43+/-7.2% without endothelium (n=6), P<0.05) but not in aorta from WKY. In addition, in aorta from SHR, the CysLT(1) receptor antagonist MK571 1 microM reduced by 55+/-6.1% (n=6, P<0.05) the contractile effects of angiotensin II in rings with endothelium but not in endothelium-denuded rings. Angiotensin II induced a 8.6+/-2.1-fold increase in cysteinyl leukotriene production in aorta rings from SHR with endothelium which was prevented by the AT(1) receptor antagonist losartan 1 microM but not by the AT(2) receptor antagonist PD123319 0.1 microM. In aorta rings from WKY, cysteinyl leukotriene production remained unchanged after exposition to angiotensin II. The cysteinyl leukotrienes (up to 0.1 microM) induced contractions in aorta rings from SHR but not from WKY. CONCLUSIONS: These data suggest that cysteinyl leukotrienes, acting at least in part on endothelial CysLT(1) receptors, are involved in the contractile response to angiotensin II in isolated aorta from SHR but not from WKY.     

Involvement of cysteinyl leukotrienes in angiotensin II-induced contraction in isolated aortas from transgenic (mRen-2)27 rats

OBJECTIVES : We have previously reported that 5-lipoxygenase-derived products, and particularly the cysteinyl leukotrienes (CysLTs), were involved in angiotensin II (Ang II)-induced contractions in isolated aortas from spontaneously hypertensive rats. DESIGN : The aim of this study was to assess the role of CysLTs in the vascular response to Ang II in an Ang II-dependent model of hypertension, the (mRen-2)27 transgenic rats (TGs). METHODS : Intact aortic rings from TG and normotensive Sprague-Dawley rats (SDs) were suspended in organ chambers for isometric tension development in response to Ang II. In addition, the release of CysLTs in response to Ang II (0.3 micromol/l) was measured by enzyme immunoassay. RESULTS : In isolated aortas from TG rats, pretreatment with the 5-lipoxygenase inhibitor (AA861, 10 micromol/l) or the CysLT1 receptor antagonist (MK571, 1 micromol/l) significantly (P < 0.05) reduced Ang II-induced contractions by 52 and 42%, respectively. In addition, Ang II induced a 2.6-fold increase in CysLT release (pg/mg dry weight tissue: 58.3 +/- 17.9 (Ang II, n = 7) versus 22.5 +/- 5.9 (basal, n = 7) P < 0.05), which was inhibited by the AT1 receptor antagonist losartan (1 micromol/l). In contrast, in aortas from SD rats, pretreatment with AA861 or MK571 did not alter Ang II-induced contraction and CysLT production remained unchanged after exposure to Ang II. CONCLUSION : These data suggest that CysLTs are involved in the contractile responses to Ang II in isolated aortas from TG but not from SD rats.     

Production of eicosanoids by the killifish gills and opercular epithelia and their effect on active transport of ions

Gills and opercular epithelia of the killifish (Fundulus heteroclitus) homogenized and incubated with radiolabeled arachidonic acid were found to produce prostaglandins, leukotrienes, and hydroxyeicosatetraenoic acids. These metabolites were identified using thin-layer chromatography, autoradiography, reverse-phase high-performance liquid chromatography, and ultraviolet spectroscopy. Addition of glutathione and epinephrine to the incubation mixture caused a diminution in the production of most eicosanoids (cyclooxygenase and lipoxygenase products) whereas indomethacin decreased only the cyclooxygenase metabolites. The effects of eicosanoids on short-circuit and potential difference across opercular epithelia mounted in a Ussing-type chamber were examined. Prostaglandin E2 had an inhibitory effect on ion transport whereas the sulfidopeptide leukotrienes (LTC4, LTD4, and LTE4) had a stimulatory effect. These results indicate that gills and opercular epithelia have the capacity to synthesize eicosanoids and that some of these metabolites may play a role in the regulation of ion transport in the kill fish.     

Angiotensin II-mediated potentiation of endothelin-1-evoked bronchial contractions: a role for leukotrienes?

Angiotensin II potentiates the contractions evoked by endothelin-1. Previous studies have suggested that the mechanism underlying this effect may involve leukotrienes. The effects of the leukotriene synthesis inhibitor MK886 and of the leukotriene receptor antagonist ICI 198615 on angiotensin II-mediated potentiation of endothelin-1-induced contractions were examined. The ability of exogenously applied leukotriene D4 (3x10(-9)M) to potentiate the endothelin-1-induced contractions was also investigated. The presence of angiotensin II (3x10(-7)M) potentiated the constrictions evoked by endothelin-1 (e.g. 170% increase at 3x10(-7)M endothelin-1). In the presence of MK886 (10(-7)-10(-5)M), however, this potentiation was abolished. The presence of ICI 198615 (10(-7)-10(-5)M), likewise abolished the angiotensin II effect. The presence of leukotriene D4(3x10(-9)M), mimicked angiotensin II in significantly potentiating the endothelin-1-evoked responses. These data confirm previous work using nordihydroguaiaretic acid and suggest that leukotrienes contribute to the angiotensin II-mediated potentiation of endothelin-1-evoked contractions.     

Effect of valsartan on angiotensin II-induced plasminogen activator inhibitor-1 biosynthesis in arterial smooth muscle cells

Previous studies have shown that angiotensin II stimulates the synthesis of plasminogen activator inhibitor-1 in cultured vascular cells, which suggests that activation of the renin-angiotensin system may impair fibrinolysis. We have investigated the effects of angiotensin II and of valsartan, a recently developed angiotensin II antagonist that is highly specific and selective for the angiotensin II subtype 1 receptor, on plasminogen activator inhibitor-1 secretion by smooth muscle cells isolated from rat and human vessels. Angiotensin II induced a time- and concentration-dependent increase of plasminogen activator inhibitor activity in supernatants of rat aortic cells, which reached a plateau after 6 hours of incubation with 100 nmol/L angiotensin II (2.4+/-0.6-fold over control value; P:<0.001). The angiotensin II-induced plasminogen activator inhibitor activity was inhibited, in a concentration-dependent manner, by valsartan with an IC(50) value of 21 nmol/L. Valsartan fully prevented the angiotensin II-induced increase in plasminogen activator inhibitor-1 protein and mRNA. Furthermore, angiotensin II doubled the secretion of plasminogen activator inhibitor-1 by smooth muscle cells obtained from human umbilical and internal mammary arteries, and valsartan fully prevented it. Angiotensin II did not affect the secretion of tissue plasminogen activator antigen by any of the cell systems tested. Thus, valsartan effectively inhibits angiotensin II-induced plasminogen activator inhibitor-1 secretion without affecting that of tissue plasminogen activator in arterial rat and human smooth muscle cells.     

Angiotensin II constriction of rat vasa recta is partially thromboxane dependent

We tested the hypothesis that thromboxane generation mediates vasoconstriction of isolated outer medullary descending vasa recta (OMDVR) by angiotensin (Ang) II. The lipoxygenase and cyclooxygenase (COX) inhibitor eicosatetraynoic acid (1 micromol/L) and the COX inhibitor indomethacin (1 micromol/L) partially reversed Ang II (1 nmol/L) constriction of in vitro perfused OMDVR. To determine whether thromboxane is a mediator of Ang II-induced vasoconstriction, a thromboxane synthase inhibitor, U63577A (1 micromol/L), and thromboxane receptor antagonists, SQ-29548 or BMS-180,291 (1 micromol/L, each), were introduced into the bath of vessels that had been preconstricted by Ang II (1 nmol/L). These agents significantly inhibited vasoconstriction induced by Ang II. In contrast, SQ-29548 and U63557A did not affect vessels preconstricted by raising extracellular KCl from 5 to 100 mmol/L. The thromboxane receptor agonist U46619 (1 micromol/L) constricted OMDVR, an effect that was blocked by the antagonist BMS-180,291. In separate protocols, microperfused OMDVR were pretreated with U63577A or SQ-29548, after which they were exposed to luminal Ang II to induce vasoconstriction. Both agents inhibited vasoconstriction whether preexposure to them was via the bath or the perfusate. We conclude that Ang II-induced constriction of OMDVR is partly mediated by metabolites of arachidonic acid, including thromboxanes.     

Levels of cysteinyl‐leukotrienes in exhaled breath condensate are not due to saliva contamination

Background and Aims: Saliva contamination has been suggested to be a major contributor to levels of cysteinyl leukotrienes in exhaled breath condensate (EBC). The aim of this study was to compare the levels of cysteinyl‐leukotrienes (CysLT) and α‐amylase activity in EBC to induced sputum and saliva collected from the same subjects (asthmatics and control). We thereby aimed to find out whether saliva contamination could be a plausible explanation to the levels found in EBC or not. Methods: EBC, saliva and induced sputum were collected from 11 asthmatic and 19 healthy adults. These samples were analyzed for CysLT concentration and α‐amylase activity. Results: No significant correlation was found between CysLT concentration and α‐amylase activity in EBC, saliva or sputum. In addition, we show that the saliva contamination (measured as α‐amylase activity) was negligible, as the relative amount of saliva CysLT was only 0.6% of that found in EBC. The amount of CysLT correlated between all three compartments (EBC, saliva and sputum), but no similar correlation was seen for the α‐amylase activity in EBC compared to saliva and sputum. The levels of CysLT were higher in asthmatic patients compared to healthy controls in EBC, saliva and sputum. Conclusion: We conclude that the amount of CysLT in EBC cannot be explained by saliva contamination. Please cite this paper as: Tufvesson E, van Weele LJ, Ekedahl H and Bjermer L. Levels of cysteinyl‐leukotrienes in exhaled breath condensate are not due to saliva contamination. The Clinical Respiratory Journal 2010; 4: 83–88.     

Cyclooxygenase-1 is involved in endothelial dysfunction of mesenteric small arteries from angiotensin II-infused mice

Angiotensin II induces endothelial dysfunction by reducing NO availability and increasing reactive oxygen species. We assessed whether cyclooxygenase (COX)-1 or COX-2 participate in the angiotensin II-induced endothelial dysfunction in murine mesenteric small arteries and examined the role of reduced nicotinamide-adenine dinucleotide phosphate-dependent reactive oxygen species production. Mice received angiotensin II (600 ng/kg per minute, SC), saline (controls), angiotensin II + apocynin (reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor, 2.5 mg/day), or apocynin alone for 2 weeks. Endothelial function of mesenteric arteries was assessed by pressurized myograph. In controls, acetylcholine-induced relaxation was inhibited by NG-monomethyl-L-arginine and unaffected by DFU (COX-2 inhibitor), SC-560 (COX-1 inhibitor), or ascorbic acid. In angiotensin II-infused animals, the attenuated response to acetylcholine was less sensitive to NG-monomethyl-L-arginine, unaffected by DFU, and enhanced by SC-560 and, similarly, by SQ-29548, a thromboxane-prostanoid receptor antagonist. Moreover, response to acetylcholine was unchanged by ozagrel, a thromboxane synthase inhibitor, and normalized by ascorbic acid. Apocynin prevented the angiotensin II-induced vascular dysfunctions. In angiotensin II-infused mice, RT-PCR analysis showed a significant COX-2 downregulation, whereas COX-1 expression was upregulated. These changes were unaffected by apocynin. Modulation of COX isoform by angiotensin II was also documented by immunohistochemistry. In small mesenteric vessels, the reduced NO availability and oxidant excess, which characterize endothelial dysfunction secondary to angiotensin II, are associated with a reduced COX-2 and an increased COX-1 function and expression. Angiotensin II causes an oxidative stress-independent COX-1 overexpression, whereas angiotensin II-mediated oxidant excess production stimulates COX-1 activity to produce a contracting prostanoid endowed with agonist activity on thromboxane-prostanoid receptors.     

Mechanism of action of angiotensin II and bradykinin on prostaglandin synthesis and vascular tone in the isolated rat kidney. Effect of Ca++ antagonists and calmodulin inhibitors

We have studied the effect of angiotensin II and bradykinin on prostaglandin output and vascular tone during extracellular calcium depletion and administration of calcium antagonists and calmodulin inhibitors to elucidate the mechanism of action in the isolated rat kidney perfused with Tyrode's solution. Administration of angiotensin II (0.028-0.28 nmol) or bradykinin (0.28-2.8 nmol) enhanced the output of prostaglandin E2 and 6-keto-prostaglandin F1 alpha in a dose-dependent manner. Angiotensin II, but not bradykinin, produced renal vasoconstriction. Omission of calcium from the medium or infusion of calcium entry blockers, diltiazem (60 microM), or nimodipine (47 microM), failed to alter prostaglandin output elicited by angiotensin II or bradykinin; however, the effect of angiotensin II to produce renal vasoconstriction was inhibited. If calcium was omitted from the medium, the intracellular calcium antagonists, 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (23 microM), dantrolene sodium (31 microM), or ryanodine (2 microM), attenuated prostaglandin output caused by angiotensin II but not bradykinin. Calmodulin inhibitors, trifluoperazine (2 microM), napthalene sulfonamide hydrochloride (2 microM), or calmidazolium (2 microM), diminished prostaglandin output elicited by angiotensin II, but not that caused by bradykinin. Trifluoperazine, but not naphthalene sulfonamide or calmidazolium, attenuated the renal vasoconstrictor effect of angiotensin II. Prostaglandin output induced by angiotensin II and bradykinin were inhibited by mepacrine and indomethacin, whereas, the prostaglandin output caused by exogenous arachidonic acid (33 nmol) was abolished by indomethacin but was unaltered by mepacrine, calcium antagonists, and calmodulin inhibitors. From these data, we conclude that angiotensin II produces renal vasoconstriction by a mechanism dependent on extracellular calcium but not calmodulin, whereas angiotensin II-induced prostaglandin output depends on intracellular calcium and calmodulin. In contrast, bradykinin appears to stimulate prostaglandin synthesis by a calcium/calmodulin-independent mechanism.     

Role of arachidonic acid metabolites in hypoxic contractions of isolated porcine pulmonary artery and vein

Recently it has been proposed that hypoxic pulmonary vasoconstriction (HPV) is mediated by local release of sulfidopeptide leukotriene products of the lipoxygenase pathway of arachidonic acid metabolism. In the present study the response to reduced oxygen supply of isolated porcine lobar pulmonary artery and pulmonary vein spiral strips has been studied. Contractions of the pulmonary artery (mean maximum tension 66.9 +/- 13.0 mg, n = 10) required an increase in baseline tone of the preparation followed by exposure to anoxia (mean bath PO2 O +/- 3 mm Hg), whereas contractions of the pulmonary vein (mean maximum tension 75.2 +/- 13.3 mg, n = 10) could be elicited in response to hypoxia alone (mean bath PO2 40 +/- 4 mm Hg). Indomethacin (5.6 microM), a cyclooxygenase inhibitor, attenuated the arterial contraction, but the mechanism may have been independent of the cyclooxygenase pathway since phenidone, an inhibitor of both cyclooxygenase and lipoxygenase pathways, had no effect. Inhibition by FPL 55712, a leukotriene end-organ antagonist, was achieved only at a high concentration (20 microM). In the case of the pulmonary vein, both indomethacin and phenidone inhibited the contractile response, whereas FPL 55712 had no effect. Contractile responses to reduced oxygen supply can be induced in isolated porcine pulmonary artery and vein strips, but probably are not mediated by leukotrienes.     

2-Arachidonoyl glycerol induces contraction of isolated rat aorta: role of cyclooxygenase-derived products

OBJECTIVES: Endocannabinoids have been shown to play a role in the regulation of vascular tone. The effects of 2-arachidonoyl glycerol (2-AG) on induced-tone were examined in rat aortic rings in vitro. METHODS: Aortic rings from Wistar Kyoto (WKY) rats were suspended in organ chambers for recording isometric tension development in response to 2-AG. The production of TXA2 in response to 2-AG was also assessed by enzyme immunoassay. RESULTS: In endothelium-intact rings pre-contracted to PGF(2alpha), 2-AG (10 nM-30 microM) induced a biphasic effect: a weak relaxation from 10 nM to 0.1 microM, which turned into a concentration-dependent contraction from 3 to 30 microM. Endothelium-denudation did not change 2-AG-mediated vascular effects. 2-AG-induced contraction was unaffected by both the cannabinoid CB1 receptor antagonist SR141716A (3 microM) and the CB2 receptor antagonist SR144528 (1 microM). In contrast, the anandamine transport inhibitor (AM404, 100 microM) and the amino hydrolase inhibitor (PMSF, 30 microM) attenuated (P<0.05) the contractile response evoked by 2-AG in endothelium-intact and rubbed aortic rings. In addition, the cyclooxygenase inhibitor (indomethacin, 10 microM) and the thromboxane A2 (TXA2) receptor (TP receptor) antagonist GR32191 (0.3 microM) totally abolished the contraction elicited by 2-AG in endothelium-intact and rubbed aortic rings. Challenge of isolated aortic rings with 2-AG (10 microM) evoked a significant increase in TXA2 level (measured as TXB2 level) in endothelium-intact and rubbed aortic rings. CONCLUSION: These data suggested that the contraction elicited by 2-AG resulted from the vascular smooth muscle cell uptake and conversion of 2-AG to constrictor prostanoid TXA2, which in turn caused vasoconstriction through the stimulation of TP receptor.     

Angiotensin-(1-7) is a modulator of the human renin-angiotensin system.

The renin-angiotensin system is important for cardiovascular homeostasis. Currently, therapies for different cardiovascular diseases are based on inhibition of angiotensin-converting enzyme (ACE) or angiotensin II receptor blockade. Inhibition of ACE blocks metabolism of angiotensin-(1-7) to angiotensin-(1-5) and can lead to elevation of angiotensin-(1-7) levels in plasma and tissue. In animal models, angiotensin-(1-7) itself causes or enhances vasodilation and inhibits vascular contractions to angiotensin II. The function of angiotensin-(1-5) is unknown. We investigated whether angiotensin-(1-7) and angiotensin-(1-5) inhibit ACE or antagonize angiotensin-induced vasoconstrictions in humans. ACE activity in plasma and atrial tissue was inhibited by angiotensin-(1-7) up to 100%, with an IC(50) of 3.0 and 4.0 micromol/L, respectively. In human internal mammary arteries, contractions induced by angiotensin I and II and the non-ACE-specific substrate [Pro(11),D-Ala(12)]-angiotensin I were antagonized by angiotensin-(1-7) (10(-5) mol/L) in a noncompetitive way, with a 60% inhibition of the maximal response to angiotensin II. Contractions to ACE-specific substrate [Pro(10)]-angiotensin I were also inhibited, an effect only partly accounted for by antagonism of angiotensin II. Angiotensin-(1-5) inhibited plasma ACE activity with a potency equal to that of angiotensin I but had no effect on arterial contractions. In conclusion, angiotensin-(1-7) blocks angiotensin II-induced vasoconstriction and inhibits ACE in human cardiovascular tissues. Angiotensin-(1-5) only inhibits ACE. These results show that angiotensin-(1-7) may be an important modulator of the human renin-angiotensin system.     

Characterization of the vascular thromboxane A2/prostaglandin endoperoxide receptor in rabbit aorta. Regulation by dexamethasone

Recently, we have shown that dexamethasone treatment of rabbits specifically reduces vascular smooth muscle responsiveness to agonists that interact with the vascular thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. One potential site at which dexamethasone can influence prostanoid-mediated vasoconstriction may be at the level of the vascular TXA2/PGH2 receptor. Therefore, we characterized the vascular TXA2/PGH2 receptor in rabbit aortic membranes and examined the influence of dexamethasone treatment on vascular TXA2/PGH2 receptor affinity and number. The binding of [125I][1S-(1 alpha,2 beta(5Z),3 alpha(1E,3R)4 alpha)]-7-[3-(3- hydroxy-4-(p-iodophenoxy)-1-butenyl)-7-oxabicyclo[2.2.1] heptan-2-yl]-5-heptanoic acid ([125I]BOP), a potent TXA2/PGH2 receptor agonist, to rabbit aortic membranes was saturable, displaceable, and dependent on protein concentration. Scatchard analysis of equilibrium binding data disclosed one class of high affinity binding sites with a Kd of 0.44 +/- 0.13 nM and a Bmax of 114.4 +/- 5.2 fmol/mg protein (n = 7). Removal of the endothelium before membrane preparation did not significantly alter the affinity or number of binding sites for [125I]BOP. Kinetic analysis of the rates of [125I]BOP association/dissociation yielded a Kd of 0.62 nM. The ability of various agonists at the TXA2/PGH2 receptor to displace [125I]BOP from vascular membranes correlated well with their contractile potencies in rabbit aortic rings. Moreover, stereospecific displacement of [125I]BOP binding in aortic membranes and inhibition of U46619-mediated aortic contractions were obtained with the stereoisomers L657925(-) and L657926(+). Collectively, these data suggest that this binding site represents the functionally relevant vascular TXA2/PGH2 receptor. In functional experiments, [127I]BOP induced concentration-dependent contractions of the rabbit aorta, which were reduced by 52% in vessels from dexamethasone-treated rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional recognition of a distinct receptor preferential for leukotriene E4 in mice lacking the cysteinyl leukotriene 1 and 2 receptors

The cysteinyl leukotrienes (cys-LTs) are a family of potent lipid mediators of inflammation derived from arachidonic acid. Activation of certain cell types results in the biosynthesis and export of leukotriene (LT) C(4), which then undergoes extracellular metabolism to LTD(4) and LTE(4). LTE(4), the most stable cys-LT, is only a weak agonist for the defined type 1 and type 2 cys-LT receptors (CysLT(1)R and CysLT(2)R, respectively). We had recognized a greater potency for LTE(4) than LTC(4) or LTD(4) in constricting guinea pig trachea in vitro and comparable activity in eliciting a cutaneous wheal and flare response in humans. Thus, we hypothesized that a vascular permeability response to LTE(4) in mice lacking both the CysLT(1)R and CysLT(2)R could establish the existence of a separate LTE(4) receptor. We now report that the intradermal injection of LTE(4) into the ear of mice deficient in both CysLT(1)R and CysLT(2)R elicits a vascular leak that exceeds the response to intradermal injection of LTC(4) or LTD(4), and that this response is inhibited by pretreatment of the mice with pertussis toxin or a Rho kinase inhibitor. LTE(4) is approximately 64-fold more potent in the CysLT(1)R/CysLT(2)R double-deficient mice than in sufficient mice. The administration of a CysLT(1)R antagonist augmented the permeability response of the CysLT(1)R/CysLT(2)R double-deficient mice to LTC(4), LTD(4), and LTE(4). Our findings establish the existence of a third receptor, CysLT(E)R, that responds preferentially to LTE(4), the most abundant cys-LT in biologic fluids, and thus reveal a new target for therapeutic intervention.     

Thromboxane A2 and prostaglandin endoperoxide receptors in platelets and vascular smooth muscle

9,11-Dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyphenyl)-13, 14-dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2 (I-PTA-OH), a recently synthesized thromboxane (TX) A2/prostaglandin (PG) H2 receptor antagonist, was shown to be a competitive antagonist of human platelet aggregation induced by the stable endoperoxide analog U46619. This antagonism was due to competitive blockade of the platelet TXA2/PGH2 receptor since I-PTA-OH did not antagonize the first phase of ADP-induced aggregation which is TXA2/PGH2 independent, nor did it inhibit TXA2 synthesis. In addition, analysis of dose-response curves to U46619 (0.1 to 40 microM) in the presence of increasing concentrations of I-PTA-OH (0.5 to 10 microM) showed that I-PTA-OH produced a parallel rightward shift of the dose-response curve. Further analysis of the data in the form of a Schild plot yielded a straight line with a slope (m = 1.03) not significantly different from -1. These results are consistent with the notion that I-PTA-OH acts as a competitive antagonist of the TXA2/PGH2 receptor.     

Inhibitors of arachidonic acid metabolism have variable effects on calcium signaling pathways

The metabolic pathways of arachidonic acid (AA) have been shown to be important in the regulation of cellular function. Several studies have demonstrated both direct and indirect effects of products of these pathways in the regulation of vascular actions, and in particular on signaling pathways. Because intracellular calcium concentration is a significant mediator of stimulus-coupled signal transduction, we investigated the effects of AA pathway inhibitors on angiotensin II (Ang II)-induced calcium mobilization in cultured rat vascular smooth muscle cells (VSMC). Thus, specific calcium pools were examined for differential effects resulting from inhibitors of the three major pathways of AA metabolism. Inhibition of lipoxygenase (LO) with 2.5 micromol/L of 5,8,11 eicosatriynoic acid (ETI), cyclooxygenase (CO) with 2 micromol/L of ibuprofen (IBU), and cytochrome P-450 (P450) with 1 micromol/L of 7-ethoxyresorufin (7ER) all reduced total Ang II-induced intracellular calcium transients ([Ca2+]i) in fura 2-loaded cultured rat VSMC. However, the sites of action affected were unique for each inhibitor. Pretreatment of VSMC with either ETI or IBU inhibited thapsigargin (TG) (1 micromol/L)-sensitive calcium increments (control; 118.0 +/- 33.1 nmol/L, n = 9, ETI; 34.7 +/- 4.8 nmol/L, n = 9, IBU; 40.3 +/- 8.8 nmol/L, n = 8, P < .05 v control). Both caffeine (CAF) and ryanodine (RY) treatment attenuated Ang II-induced [Ca2+]i; however, pretreatment with ETI, IBU, or 7ER did not alter this effect. In other studies, the LO inhibitor ETI attenuated Ang II-induced Ca2+ influx, whereas inhibitors of CO and P450 pathways had no effect. These data show that 1) E     

Cytotoxic effects of cytokines on islet beta-cells: evidence for involvement of eicosanoids

Arachidonic acid metabolites (eicosanoids) have been implicated in mediating actions of cytokines in different tissues. In this study, we tested inhibitors of arachidonate metabolism for possible protection against the toxic effects of the cytokine combination of tumor necrosis factor (TNF, 100 U/ml) and interferon-gamma (IFN-gamma, 100 U/ml) in rat islet cell monolayer cultures, using a 51Cr release cytotoxicity assay to measure islet cell lysis (% 51Cr release). The toxic effect of TNF/IFN-gamma (26.6 +/- 3.7%) was inhibited partially by both a cyclooxygenase inhibitor, indomethacin and a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), and combination of maximally effective concentrations of Indo and NDGA (30 microM) produced further protection against TNF/IFN-gamma-induced lysis (3.5 +/- 0.9%). Also, the combined cyclo/lipoxygenase inhibitors, oxyphenbutazone and eicosa 5,8,11,14 tetrynoic acid, as well as the phospholipase A2 inhibitor, bromophenacyl bromide, significantly inhibited the cytotoxic effect of TNF/IFN-gamma. Whereas indomethacin and NDGA did not prevent TNF/IFN-gamma-induced inhibition of insulin release, this recovered after cytokine removal from cultures protected by the cyclo/lipoxygenase inhibitors. These results suggest that arachidonate metabolites may be involved in mediating the cytotoxic and not the functional inhibitory effects of TNF and IFN-gamma in islet cells.     

Role of endothelium-derived prostanoid in angiotensin-induced vasoconstriction

To test the hypothesis that prostanoids contribute to angiotensin II-induced vascular contraction, we compared the effect of angiotensin II on isometric tension development by rings of descending thoracic aorta bathed in Krebs' bicarbonate buffer with and without indomethacin (10 microM) to inhibit cyclooxygenase, CGS13080 (10 microM) to inhibit thromboxane A2 synthesis, or SQ29548 (1 microM) to block thromboxane A2/prostaglandin endoperoxide receptors. The comparisons were made in rings of aorta taken from normotensive rats and from rats with aortic coarctation-induced hypertension at 12 days and 90-113 days after coarctation. These rings released thromboxane B2, which was found to be endothelium dependent, increased in hypertensive rats, and stimulated by angiotensin II (10(-6) M) in normotensive rats and in hypertensive rats at 12 days after coarctation. The angiotensin II (10(-6) to 10(-5)M)-induced contraction of aortic rings was increased by about 30% at 12 days after coarctation and decreased at 90-113 days after coarctation. Removal of the endothelium increased the contractile effect of angiotensin II (10(-6) M) in aortic rings of normotensive rats and hypertensive rats at 90-113 days after coarctation but decreased the effect in aortic rings of hypertensive rats at 12 days after coarctation. In rats at 12 days after coarctation, the angiotensin II (10(-6) M)-induced contraction of aortic rings with endothelium was attenuated by indomethacin and SQ29548 but not by CGS13080. These data suggest that a prostanoid-mediated and endothelium-dependent mechanism of vasoconstriction contributes to the constrictor effect of angiotensin II in aortic rings of rats in the early phase of aortic coarctation-induced hypertension.     

Mitogen-activated protein kinase-activated protein kinase 2 in angiotensin II-induced inflammation and hypertension: regulation of oxidative stress

Vascular oxidative stress and inflammation play an important role in angiotensin II-induced hypertension, and mitogen-activated protein kinases participate in these processes. We questioned whether mitogen-activated protein kinase-activated protein kinase 2 (MK2), a downstream target of p38 mitogen-activated protein kinase, is involved in angiotensin II-induced vascular responses. In vivo experiments were performed in wild-type and Mk2 knockout mice infused intravenously with angiotensin II. Angiotensin II induced a 30 mm Hg increase in mean blood pressure in wild-type that was delayed in Mk2 knockout mice. Angiotensin II increased superoxide production and vascular cell adhesion molecule-1 in blood vessels of wild-type but not in Mk2 knockout mice. Mk2 knockdown by small interfering RNA in mouse mesenteric vascular smooth muscle cells caused a 42% reduction in MK2 protein and blunted the angiotensin II-induced 40% increase of MK2 expression. Mk2 knockdown blunted angiotensin II-induced doubling of intracellular adhesion molecule-1 expression, 2.4-fold increase of nuclear p65, and 1.4-fold increase in Ets-1. Mk2 knockdown abrogated the angiotensin II-induced 4.7-fold and 1.3-fold increase of monocyte chemoattractant protein-1 mRNA and protein. Angiotensin II enhanced reactive oxygen species levels (by 29%) and nicotinamide adenine dinucleotide phosphate oxidase activity (by 48%), both abolished by Mk2 knockdown. Reduction of MK2 blocked angiotensin II-induced p47phox translocation to the membrane, associated with a 53% enhanced catalase expression. Angiotensin II-induced increase of MK2 was prevented by the nicotinamide adenine dinucleotide phosphate oxidase inhibitor Nox2ds-tat. Mk2 small interfering RNA prevented the angiotensin II-induced 30% increase of proliferation. In conclusion, MK2 plays a critical role in angiotensin II signaling, leading to hypertension, oxidative stress via activation of p47phox and inhibition of antioxidants, and vascular inflammation and proliferation.     

Eicosanoids in exhaled breath condensate and BAL fluid of patients with sarcoidosis

BACKGROUND: Measurement of inflammatory mediators in exhaled breath condensate (EBC) is an easy and noninvasive diagnostic method, which has gained popularity in the past few years. However, the source of these mediators is not precisely defined. It has been only presumed that inflammatory cells present in the airway lumen are the main source. Therefore, the aim of this study was to verify the relationship between EBC and BAL fluid (BALF) eicosanoids, and the percentage, number, and activity of cells in BALF. METHODS: In 28 sarcoidosis patients and 17 healthy subjects, 8-isoprostane, cysteinyl leukotrienes (CysLTs), and leukotriene B4 (LTB4) were measured in EBC by enzyme immunoassay. Eicosanoids were also examined in BALF in the study group. Cell count, percentage, and superoxide production by BALF cells were estimated. RESULTS: The mean (+/- SEM) CysLT and 8-isoprostane concentrations were higher in the sarcoidosis group (6.5 +/- 0 vs 27.82 +/- 6.65 pg/mL, respectively; and 2.67 +/- 0.16 vs 13.95 +/- 2.59 pg/mL, respectively). There were positive correlations between EBC and BALF 8-isoprostane concentration (r = 0.68, p < 0.0001) and LTB4 concentration (r = 0.43; p = 0.026). EBC LTB4 levels correlated with the number of lymphocytes per milliliter of BALF. The percentage and number of eosinophils in BALF correlated with EBC 8-isoprostane and BALF CysLT concentrations. No positive correlation was found between concentrations of EBC eicosanoids and percentages BALF lymphocytes, BALF macrophages, or superoxide production. CONCLUSIONS: The levels of 8-isoprostane and CysLT are elevated in EBC in sarcoidosis patients; however, a lack of correlation with BALF lymphocyte percentage does not encourage us to recommend the measurement of eicosanoids as activity markers. The positive correlation of EBC 8-isoprostane and BALF CysLT concentrations with the percentage of eosinophils in BALF, and the higher percentage of eosinophils in BALF from patients with grade 3 sarcoidosis, may suggest the possible prognostic value.