Cyclooxygenase, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase MAPK, Rho kinase, and Src mediate hydrogen peroxide-induced contraction of rat thoracic aorta and vena cava

In hypertension, blood vessels exhibit increased reactive oxygen species production that may alter vascular tone. We previously observed that H2O2 contracted rat thoracic vena cava under resting tone and aorta contracted with KCl. In arteries but not veins, H2O2-induced contraction required extracellular Ca2+ influx. Because of this difference in Ca2+ utilization, we hypothesized that signaling pathways mediating H2O2-induced contraction in vena cava under resting tone differed from those mediating H2O2-induced contraction in aorta contracted with KCl. Inhibitors of cyclooxygenase (COX) 1 and 2 (indomethacin, 10 microM), thromboxane A2 (TXA2) receptors [ICI185282 (2RS,4RS,5SR-4-o-hydroxyphenyl-2-trifluoromethyl-1,3-dioxan-5-yl heptenoic acid), 10 microM], p38 mitogen-activated protein kinase (MAPK) [SB203580 (4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine), 10 microM], extracellular signal-regulated kinase (Erk) [PD98059 (2'-amino-3'-methoxyflavone), 10 microM], src [PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, 10 microM], and rho kinase [Y27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride), 10 microM], significantly reduced H2O2-induced contraction in vena cava under resting tone and aorta after KCl (30 mM) contraction. In contrast, the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, 20 microM] did not reduce aortic or venous H2O2-induced contraction. p38 MAPK, Erk MAPK, and src inhibition did not reduce aortic or venous contraction to the TXA2 receptor agonist U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy PGF(2alpha), 1 microM), whereas rho kinase inhibition significantly reduced aortic and venous contraction to U46619, and PI3-K inhibition reduced venous contraction to U46619. Our data suggest that, in rat thoracic aorta and vena cava, a COX-derived metabolite is one important mediator of H2O2 contraction, possibly via rho kinase activation, and that H2O2-induced contraction via p38 and Erk MAPK probably occurs independently of TXA2 receptor activation.     

Dexamethasone decreases contraction to electrical field stimulation in mesenteric arteries from spontaneously hypertensive rats through decreases in thromboxane A2 release

Glucocorticoids play a role in the control of vascular smooth muscle tone through the alteration of vasoconstrictor and vasodilator factor production. We studied the effect of dexamethasone on vasoconstriction induced by electrical field stimulation (EFS) in rat mesenteric arteries (MAs) and the role of hypertension in this effect. Endothelium-denuded MAs were obtained from Wistar-Kyoto rats and spontaneously hypertensive rats (SHRs). EFS response was analyzed by isometric tension recordings and cyclooxygenase (COX-1 and COX-2) expression by Western blot. Noradrenaline (NA) release was evaluated in segments incubated with [(3)H]NA. Dexamethasone (0.1 and 1 microM; 2-8 h) reduced vasoconstriction to EFS (200 mA, 0.3 ms, 1-16 Hz), in a dose- and time-dependent manner only in SHRs. However, the EFS-induced release of [(3)H]NA was increased in SHR arteries preincubated with dexamethasone (1 microM; 6 h). The thromboxane A(2) (TxA(2)) synthase inhibitor furegrelate (10 microM), the selective COX-2 inhibitor NS-398 (N-[2-cyclohexyloxy-4-nitrophenyl] methanesulfonamide; 10 microM), or the TxA(2) receptor antagonist SQ 29548 (1 microM), reduced EFS and NA induced vasoconstrictor responses. However, the effect of these drugs was abolished in arteries preincubated with dexamethasone. Both dexamethasone and phentolamine (1 microM) inhibited the increased thromboxane B(2) levels observed after EFS. COX-2 protein expression was reduced by dexamethasone in SHR arteries. Results suggest that dexamethasone reduces vasoconstriction to EFS in MAs from SHRs by decreasing COX-2 expression, thereby decreasing the smooth muscle TXA(2) release induced by alpha-adrenoceptor activation. The undetectable COX-2 expression in MAs from normotensive animals explains the noneffect of dexamethasone in their arteries.     

Pharmacological characterization of 2NTX-99 [4-methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a potential antiatherothrombotic agent with antithromboxane and nitric oxide donor activity in platelet and vascular preparations

Thromboxane (TX) A(2), prostacyclin (PGI(2)), and nitric oxide (NO) regulate platelet function and interaction with the vessel wall. Inhibition of TXA(2), implemented synthesis of PGI(2), and supply of exogenous NO may afford therapeutic benefit. 2NTX-99 [4-methoxy-N(1)-(4-trans-nitrooxycyclohexyl)-N(3)-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a new chemical entity related to picotamide, showed antithromboxane activity and NO donor properties. 2NTX-99 relaxed rabbit aortic rings precontracted with norepinephrine or U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic acid; EC(50), 7.9 and 17.1 microM, respectively), an effect abolished by 10 microM 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ). 2NTX-99 inhibited arachidonic acid (AA)-induced washed platelet aggregation (EC(50), 9.8 microM) and TXB(2) formation (-71% at 10 microM), and its potency increased in the presence of aortic rings (EC(50), 1.4 microM). In whole rabbit aorta incubated with homologous platelets, AA caused contraction and TXA(2) formation, reduced by 2NTX-99 (10-40 microM): contraction, -28 and -47%, TXA(2) formation, -37 and -75.4%, respectively, with concomitant increase in PGI(2). 2NTX-99 (20-40 microM) inhibited U46619-induced aggregation in rabbit platelet-rich plasma (PRP) (-74 +/- 6.7 and -96 +/- 2.4%, respectively) and inhibited collagen-induced aggregation in human PRP (-48.2 +/- 10 and -79.2 +/- 6%), whereas ozagrel was ineffective. In human embryonic kidney 293 cells transfected with the TXA(2) receptor isophorm alpha receptor, 2NTX-99 did not compete with the ligand, [(3)H]SQ29,548 ([(3)H][1S-[1alpha,2beta(5Z),3beta,4alpha]]-7-[3-[[2-(phenylamino)-carbonyl]hydrazino]methyl]-7-oxabicyclo[2,2,1]-hept-2-yl]-5-heptanoic acid), or prevent inositol phosphate accumulation. After oral administration (50-250 mg/kg), 2NTX-99 inhibited TXA(2) production in rat clotting blood (-71 and -91%); at 250 mg/kg, an area under the curve, 0 to 16 h, of 149.5 h/microg/ml and a t(1/2) of 6 h were calculated, with a C(max) value of 31.8 +/- 8.2 microg/ml. An excellent correlation between plasma concentrations and TXA(2) inhibition occurs. 2NTX-99 controls platelet function and vessel wall interaction by multifactorial mechanisms and possesses therapeutic potential.     

Mechanism of inositol 1,4,5-trisphosphate-induced aggregation in saponin-permeabilized platelets.

The present study investigated the relationship between inositol 1,4,5-trisphosphate (IP3), thromboxane (TX)A2 and ADP in IP3-induced activation of saponin-permeabilized platelets. The time course of the different responses indicated that IP3-induced Ca++ mobilization and TXA2 production preceded both aggregation and secretion. Furthermore, platelet aggregation occurred coincident with secretion. In contrast, U46619- [15(S)-hydroxy-11,9-epoxymethano-prosta-5Z,13E-dienoic acid] and A23187-induced aggregation was commensurate with Ca++ mobilization, and preceded the secretion response. Indomethacin and SQ29,548 ([1S-[1 alpha,2 beta(5Z),3 beta,4 alpha]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]- 7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid) inhibited IP3-mediated aggregation by 100%, Ca++ mobilization by 80% and secretion by 90%. A23187 exhibited a different inhibition profile, in that only secretion was blocked (65%). U46619-induced responses were completely inhibited by SQ29,548 and unaffected by indomethacin. Pretreatment of the platelets with creatine phosphate/creatine phosphokinase (CP/CPK), which removes secreted ADP, produced 100% inhibition of IP3-induced aggregation, 90% inhibition of Ca++ mobilization and 72% inhibition of secretion. On the other hand, CP/CPK was ineffective in blocking any of the A23187-induced responses. Concerning U46619, CP/CPK produced a 30% attenuation of maximal aggregation and 78% inhibition of both Ca++ mobilization and secretion. These results in saponin-permeabilized platelets demonstrate that IP3-induced aggregation is a secretion-mediated process which requires both TXA2 and secreted ADP. Taken together, the findings suggest that IP3 is not capable of directly causing platelet aggregation, but may function in platelets to amplify an initial agonist response through TXA2 production and secretion.     

Chronic administration of genistein improves endothelial dysfunction in spontaneously hypertensive rats: involvement of eNOS, caveolin and calmodulin expression and NADPH oxidase activity

The soya-derived phytoestrogen genistein has been suggested to be protective in cardiovascular diseases. In the present study, we have analysed whether chronic oral genistein might influence endothelial function in male SHRs (spontaneously hypertensive rats) via ERs (oestrogen receptors), changes in eNOS (endothelial NO synthase) activity and vascular O(2)(-) (superoxide) production. Rats (23-weeks old) were divided into the following groups: WKY (Wistar-Kyoto)-vehicle, SHR-vehicle, WKY-genistein (10 mg.kg(-1) of body weight.day(-1)); SHR-genistein; SHR-genistein-faslodex (ICI 182780; 2.5 mg.kg(-1) of body weight.day(-1)). Vascular expression of eNOS, caveolin-1 and calmodulin-1 were analysed by Western blotting, eNOS activity by conversion of [(3)H]arginine into L-[(3)H]citrulline and O(2)(-) production by chemoluminescence of lucigenin. In SHRs, after 5 weeks of treatment, genistein reduced systolic blood pressure and enhanced endothelium-dependent aortic relaxation to acetylcholine, but had no effect on the vasodilator responses to sodium nitroprusside. Compared with WKY rats, SHRs had up-regulated eNOS and down-regulated caveolin-1 and calmodulin-1 expression, increased NADPH-induced O(2)(-) production, but reduced eNOS activity. Genistein increased aortic calmodulin-1 protein abundance and eNOS activity, and reduced NADPH-induced O(2)(-) production in SHRs. The pure ERalpha and ERbeta antagonist faslodex did not modify any of the changes induced by genistein in SHRs, suggesting that these effects are unrelated to ER stimulation. In conclusion, genistein reduced the elevated blood pressure and endothelial dysfunction in SHRs. This latter effect appears to be related to increased eNOS activity associated with increased calmodulin-1 expression and decreased O(2)(-) generation.     

Effects of the beta 3-adrenergic receptor agonist disodium 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL-316243) on bladder micturition reflex in spontaneously hypertensive rats

The present study investigated whether beta3-adrenoceptor activation acts on the bladder afferent pathway by examination of the visceromotor reflex (VMR) and pressor responses to urinary bladder distension (UBD) and whether beta3-adrenoceptor activation produces urinary bladder relaxation in hyperactive spontaneously hypertensive rats (SHRs) in comparison with their normotensive control rats [Wistar-Kyoto (WKY)]. Using the VMR responses to noxious UBD as a measure of bladder afferent signal transmission, SHRs did not present a sensitized bladder phenotype. However, reduced bladder compliance accompanied by a reduced void threshold was detected in the SHR detrusor. Furthermore, the selective beta3-adrenoceptor agonist disodium 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL-316243) (i.v.) failed to attenuate VMR or pressor responses to UBD in either SHRs or WKY rats, but it dose-dependently inhibited rhythmic contraction (RC) in SHRs. The minimal effective dose was 0.001 mg/kg. Using the same model in WKY rats, CL-316243 did not elicit significant inhibition of contractions in the bladder RC assay. These results suggest that SHRs represent abnormal efferent/detrusor function (detrusor overactivity) without mechanosensory afferent hypersensitivity. The beta3-adrenoceptor agonist CL-316243 acts on the detrusor muscle to increase urine storage in SHRs.     

Soy isoflavones improve endothelial function in spontaneously hypertensive rats in an estrogen-independent manner: role of nitric-oxide synthase, superoxide, and cyclooxygenase metabolites

The aim of this study was to analyze the effects of the isoflavones genistein and daidzein, and the mammalian estrogen 17beta-estradiol on endothelial function in isolated aortic rings from male spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). Relaxation to acetylcholine on precontracted rings was impaired and endothelium-dependent contraction to acetylcholine in aortic rings was increased in SHR compared with WKY. Aortic NADPH-stimulated O(2)(-) release and prostaglandin (PG)H(2) production evoked by acetylcholine were increased, whereas nitric-oxide synthase activity was reduced in SHR versus WKY. Genistein, daidzein, or 17beta-estradiol enhanced the relaxant response to acetylcholine and decreased the endothelium-dependent vasoconstrictor responses to acetylcholine in SHR, but not in WKY, and these effects were not modified by the estrogen receptor antagonist ICI 182,780 (7alpha,17beta-[9[(4,4,5,5,5-pentafluoropentyl)-sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol). Moreover, isoflavones enhanced nitric-oxide (NO) synthase activity and inhibited NADPH-stimulated O(2)(-) roduction and endothelial release of PGH(2). The contractions induced by the TP receptor agonist U46619 (9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F(2alpha)) in denuded aortic rings were inhibited by genistein, daidzein, and 17beta-estradiol in both strains. In conclusion, the isoflavones genistein and daidzein and 17beta-estradiol restore endothelial function in male SHR through estrogen receptor-independent mechanisms. Increased NO production and protection of NO from O(2)(-)-driven inactivation might be involved in the improvement of vascular relaxation to acetylcholine in aortic rings from SHR. Moreover, isoflavones and 17beta-estradiol inhibited aortic endothelium-dependent contraction to acetylcholine in SHR by reducing the endothelial PGH(2) release and its vasoconstrictor response.     

ROLE OF ENDOTHELIUM ON PHENYLEPHRINE-TRIGGERED CONTRACTILE EVENTS IN AORTA OF SPONTANEOUSLY HYPERTENSIVE RATS

The ability of basal release of endothelium derived relaxing factor (EDRF) to alter contractile events in phenylephrine (PE)-triggered contraction was tested on ring segments of the thoracic aorta removed from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). In normal medium, PE (1 microM) elicited similar whole contractions in endothelium denuded arteries of SHR and WKY. The presence of endothelium only reduced the WKY response. On aorta incubated in a Ca2+ free-medium, PE (1 microM) induced an initial phasic contraction due to intracellular Ca2+ release. This was followed by a tonic contraction after Ca2+ (2.5 mM) was restored to the bath. This sustained contraction was dependent on extracellular calcium influx. Identical phasic and tonic contractions were observed in endothelium denuded rings of SHR and WKY. However, the presence of endothelium only reduced the sustained contraction of WKY arteries. When experiments were carried out in medium containing D600 (1 microM), the presence of endothelium diminished the whole contraction of both SHR and WKY rings whereas the sustained but not the phasic contractions of WKY was also inhibited. This inhibitory effect of endothelium on WKY sustained contraction was significantly higher in the presence of D600. The calcium antagonist reduced both the whole and the tonic contractions of all preparations but was ineffective on the phasic one. The D600 inhibitory action on the sustained contraction was more pronounced in denuded SHR rings than in the corresponding WKY arteries. Thus it is concluded that there is a basal influence of endothelium in both SHR and WKY. Under our conditions, the endothelial function inhibited the extracellular Ca2+ influx and especially the part of Ca2+ influx insensitive to D600. This part of Ca2+ influx is diminished in SHR and thus the efficacy of endothelium products (e.g. EDRF) is reduced in this strain.     

Contractile effects of Bay k 8644, a dihydropyridine calcium agonist, on isolated femoral arteries from spontaneously hypertensive rats

Agonist actions of methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)- pyridine-5-carboxylate (Bay k 8644) were investigated in femoral and mesenteric arteries from 6-week-old spontaneously hypertensive rats (SHRs), and data compared with findings in normotensive Wistar-Kyoto rats (WKYs). The addition of Bay k 8644 produced a dose-dependent contraction in SHR femoral artery with a pD2 value of 8.55. Maximum contraction induced by this agonist (1 X 10(-7) M) was comparable to the maximum developed by K+-depolarization. Bay k 8644 was much less effective in eliciting the contractile responses on WKY femoral artery. Contractile responses of mesenteric and tail arteries to Bay k 8644 were weak and were not significantly different between SHR and WKY. Thoracic aorta was sensitive to the contractile response to Bay k 8644, but the sensitivity was not significantly different between SHR and WKY. Increased responsiveness to exogenously applied K+ was also observed in SHR femoral artery as compared to WKY. Contractile responses of SHR femoral artery to Bay k 8644 were antagonized competitively by nifedipine (pA2 = 8.36), a dihydropyridine Ca++ antagonist, but noncompetitively by diltiazem, a non-dihydropyridine Ca++ antagonist. When the effect of nifedipine on the dose-response curve for Bay k 8644 was determined in WKY femoral artery, there was a similar extent of rightward displacement of the dose-response curve to that seen in SHR. Nifedipine was less efficacious in relaxing the contractile response to Bay k 8644 compared to the contractile response to K+ in SHRs femoral arteries.(ABSTRACT TRUNCATED AT 250 WORDS)     

alpha 2-Adrenoceptors potentiate angiotensin II- and vasopressin-induced renal vasoconstriction in spontaneously hypertensive rats

Hypertension in spontaneously hypertensive rats (SHRs) is due in part to enhanced effects of vasoactive peptides on the renal vasculature. We hypothesize that the G(i) signal transduction pathway enhances renovascular responses to vasoactive peptides in SHRs more so than in normotensive Wistar-Kyoto (WKY) rats. To test this hypothesis, we examined in isolated perfused kidneys from SHRs and WKY rats the renovascular responses (assessed as changes in renal perfusion pressure in mm Hg) to angiotensin II (10 nM) and vasopressin (3 nM) in the presence and absence of UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; an agonist that selectively activates the G(i) pathway by stimulating alpha(2)-adrenoceptors]. In SHR, but not WKY, kidneys, UK-14,304 (10 nM) enhanced (P < 0.05) renovascular responses to angiotensin II (control WKY, 43 +/- 6; UK-14,304-treated WKY, 52 +/- 19; control SHR, 66 +/- 17; UK-14,304-treated SHR, 125 +/- 16) and vasopressin (control WKY, 42 +/- 17; UK-14,304-treated WKY, 36 +/- 11; control SHR, 16 +/- 8; UK-14,304-treated SHR, 83 +/- 17). Pretreatment of SHRs with pertussis toxin (30 microg/kg, intravenously, 3-4 days before study) to inactivate G(i) blocked the effects of UK-14,304. Western blot analysis of receptor expression in whole kidney and preglomerular microvessels revealed similar levels of expression of AT(1), V(1a), and alpha(2A) receptors in SHRs compared with WKY rats. We conclude that activation of alpha(2)-adrenoceptors selectively enhances renovascular responses to angiotensin II and vasopressin in SHRs via an enhanced cross talk between the G(i) signal transduction pathway and signal transduction pathways activated by angiotensin II and vasopressin.     

Acute and chronic captopril, but not prazosin or nifedipine, normalize alterations in adrenergic intracellular Ca2+ handling observed in the mesenteric arterial tree of spontaneously hypertensive rats

The effect of hypertension and acute (36-h) or chronic (from age 6 to 16 weeks) antihypertensive treatment with prazosin (2 mg kg(-1) per day), nifedipine (50 mg kg(-1) per day), or captopril (50 mg kg(-1) per day) on Ca2+ mobilization due to alpha1-adrenoceptor activation was analyzed in functional studies using arterial rings [four conductance/distributing vessels: aorta, main mesenteric, iliac, and tail arteries and two resistance vessels; first and second small mesenteric artery branches obtained from spontaneously hypertensive rats (SHR, 6 and 16 weeks old) and age-matched Wistar Kyoto rats (WKY)]. Maximal response to noradrenaline in the presence of extracellular Ca2+ is not affected by hypertension or by the antihypertensive treatment. The extracellular Ca2+-independent contractile responses increased with age in iliac, tail, and small mesenteric arteries (SMA) and were further increased in SHR in SMA from both young and adult animals and in the main mesenteric artery of adult SHR. In main mesenteric artery, this increased contraction in SHR was associated with a higher increase in cytosolic [Ca2+] mobilized by noradrenaline without changes in the total stored Ca2+. Acute or chronic treatment with captopril abolished the differences observed between WKY and SHR in the noradrenaline-induced contraction in mesenteric arteries loaded in Ca2+-free medium. In contrast, animals acutely treated with prazosin or chronically treated with either prazosin or nifedipine exhibit the same differences in Ca2+ handling than untreated rats. In conclusion, these differences are not a consequence of increased blood pressure but precede it and can only be normalized by inhibition of the rennin-angiotensin system.     

Interleukin 1beta-induced production of H2O2 contributes to reduced sigmoid colonic circular smooth muscle contractility in ulcerative colitis

We have shown that neurokinin A-induced contraction of human sigmoid circular muscle (HSCM) is reduced in patients with ulcerative colitis and that interleukin (IL)-1beta may play a role in this change. We now examine changes in the signal transduction pathway mediating neurokinin A-induced contraction of HSCM and explore the role of IL-1beta and of H(2)O(2) in these changes. In Fura 2-AM-loaded ulcerative colitis HSCM cells, neurokinin A- and caffeine-induced peak Ca(2+) increase and cell shortening were significantly reduced. In normal cells, neurokinin A-induced contraction was decreased by protein kinase C inhibitor chelerythrine and by calmodulin inhibitor CGS9343B [1,3-dihydro-1-[1-[(4-methyl-4H,6H-pyrrolo[1,2-a][4,1]-benzoxazepin-4-yl)methyl]-4-piperidinyl]-2H-benzimidazol-2-one (1:1) maleate]. In ulcerative colitis muscle cells, contraction was inhibited only by chelerythrine but not by CGS9343B. IL-1beta treatment of normal HSCM strips and cells reproduced the changes observed in ulcerative colitis. IL-1beta-induced reduction in caffeine-induced peak Ca(2+) increase and contraction was reversed by catalase, suggesting a role of H(2)O(2). IL-1beta-induced H(2)O(2) production was inhibited by mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 (2'-amino-3'-methoxyflavone) and by cytosolic phospholipase A2 (cPLA(2)) inhibitor AACOCF3 (arachidonyltrifluoromethyl ketone), but neither by p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole] nor by nuclear factor-kappaB (NF-kappaB) inhibitory peptide NF-kappaB SN50 (H-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-OH). IL-1beta significantly increased the phosphorylation of extracellular signal-regulated kinase 1 (ERK1)/ERK2 MAPKs and cPLA(2) and IL-1beta-induced cPLA(2) phosphorylation was blocked by PD98059. We conclude that Ca(2+) stores of HSCM cells may be reduced in ulcerative colitis and that the signal transduction pathway of neurokinin A-induced contraction switches from calmodulin- and protein kinase C-dependent in normal cells to protein kinase C-dependent in ulcerative colitis cells. IL-1beta reproduces these changes, possibly by production of H(2)O(2) via sequential activation of MAPKs (ERK1/ERK2) and cPLA(2).     

Endogenous vasoconstrictor prostanoids: role in serotonin and vasopressin-induced coronary vasoconstriction

A vasoconstrictor-induced prostacyclin (PGI2) production in a perfused rat heart was found, suggesting a mitigating role of PGI2 on coronary vasoconstriction. Treatment of the heart with cyclooxygenase inhibitors (aspirin or indomethacin) decreased PGI2 production by more than 90% and paradoxically reduced the vasoconstriction response. The attenuating effect of cyclooxygenase blockade suggested that endogenous prostanoids contribute to serotonin-, vasopressin- or U46619-induced vasoconstriction. Two prostaglandin (PG) H2/thromboxane A2 (TXA2) receptor antagonists, i.e., 13-azaprostanoic acid (13-APA) and SQ 29,548 were used to investigate putative endogenous vasoconstrictor prostanoids on the exogenously induced vasoconstriction. Retrogradely perfused (5-6 ml/min) rat hearts were rendered guiescent, yet responsive to stimuli, by local injection of lidocaine to the atrioventricular node. Changes in coronary vascular resistance (i.e., perfusion pressure at constant flow) were monitored and the cardiac effluent was collected for analysis of 6-keto PGF1 alpha (the stable metabolite of PGI2) as well as PGF2 alpha by radioimmunoassay. Three vasoconstrictors, i.e., serotonin, vasopressin and the TXA2/PGH2 analog U46619, as well as authentic PGD2, PGE2 and PGF2 alpha were infused. PGD2, PGE2 and PGF2 alpha exerted a dose-related coronary vasoconstriction, as did U46619, serotonin and vasopressin. Treatment with 13-APA (100 microM) or SQ 29,548 (100 nM) almost abolished U46619-induced vasoconstriction. The addition of PGH2/TXA2 receptor antagonists also significantly reduced the pressor effect of exogenously administered PGs, serotonin and vasopressin, with the exception that SQ 29,548 did not significantly antagonize PGE2-induced vasoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel angiotensin II AT(1) receptor antagonist irbesartan prevents thromboxane A(2)-induced vasoconstriction in canine coronary arteries and human platelet aggregation

This study was conducted to investigate whether the novel orally active nonpeptide angiotensin II (Ang II) AT(1) receptor antagonist irbesartan interacts with the thromboxane A(2)/prostaglandin endoperoxide H(2) (TxA(2)/PGH(2)) receptor in canine coronary arteries and human platelets. Coronary artery rings were isolated from male dog hearts (n = 18) and isometric tension of vascular rings was measured continuously at optimal basal tension in organ chambers. Autoradiographic binding of [(3)H]SQ29,548, a TxA(2) receptor antagonist, in canine coronary sections was determined. Blood for platelet aggregation studies was collected by venous puncture from healthy human volunteers (n = 6) who were free of aspirin-like agents for at least 2 weeks. Vascular reactivity and platelet aggregation in response to the TxA(2) analogs U46619 and autoradioagraphic receptor binding to the TxA(2) receptor antagonist [(3)H]SQ29,548 were studied with and without irbesartan. The TxA(2) analog U46619 produced dose-dependent vasoconstriction in coronary rings (EC(50) = 11.6 +/- 1.5 nM). Pretreatment with irbesartan inhibited U46619-induced vasoconstriction, and the dose-response curve was shifted to the right in a dose-dependent manner. The EC(50) of U46619 was increased 6- and 35-fold in the presence of 1 and 10 microM of irbesartan without a change of maximal contraction. At 1 microM, irbesartan is 2-fold more potent than the AT(1) receptor antagonist losartan in the inhibition of U46619-induced vasoconstriction in canine coronary arteries. In contrast, neither AT(1) receptor antagonists (CV11974 and valsartan), the AT(2) receptor antagonist PD123319, nor the angiotensin converting enzyme inhibitor lisinopril had any effect on U46619-induced coronary vasoconstriction. Irbesartan did not change potassium chloride-induced vasoconstriction; however, irbesartan did inhibit the vasoconstriction mediated by another TxA(2)/PGH(2) receptor agonist prostaglandin F(2alpha) (PGF(2alpha)). Neither the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester nor the cyclooxygenase inhibitor indomethacin had any effect on irbesartan's attenuation of U46619-induced vasoconstriction. Irbesartan specifically reversed U46619-preconstricted coronary artery rings with and without endothelium in a dose-dependent manner. Irbesartan at high concentrations significantly competed for [(3)H]SQ29,548 binding in canine coronary sections. U46619 stimulated dose-dependent human platelet aggregation of platelet-rich plasma. Preincubation with irbesartan significantly inhibited platelet aggregation in a concentration-dependent manner. In conclusion, the dual antagonistic actions of irbesartan by acting at both the AT(1) and TxA(2) receptors in blood vessels and platelets may overall enhance its therapeutic profile in the treatment of hypertension, atherosclerosis, and arterial thrombosis.     

Mechanism for homologous downregulation of thromboxane A2 receptors in cultured human chronic myelogenous leukemia (K562) cells.

Desensitization of the biologic response to thromboxane A2 (TXA2) mimetics has been observed ex vivo in human platelets due to TXA2 receptor uncoupling and downregulation. To define more clearly the mechanisms of homologous TXA2 receptor downregulation, the effects of the TXA2 mimetics U44069 ([15S)-hydroxy-9,11- (epoxymethano) prostadienoic acid] and I-BOP ([1S-(1 alpha 2 beta(5Z),3 alpha(1E,3S),4 alpha))-7-[3-(3-hydroxy-4- (p-iodophenoxy)-1-butenyl)-7-oxabicyclo[2.2.1]heptan-2-yl]-5 -heptenoic acid) on receptor-mediated calcium fluxes and on ligand binding to TXA2 receptors were studied in the K562 cultured human leukemic cell line which possesses many platelet characteristics. Incubation with U44069 resulted in a time-dependent decrease in the amplitude of TXA2 receptor-mediated intracellular free calcium transients. Under the same conditions, binding of [125I] BOP demonstrated a concurrent loss of K562 plasma membrane binding sites to approximately one-third the original number. The loss of [125I]BOP binding was prevented by coincubation with the TXA2 antagonist SQ29548 ([1S-1 alpha,2 beta (5Z), 3 beta,4 alpha]-7- (3-[2-[phenylamino)-carbonyl) hydrazino) methyl)-7-oxabicyclo-(2.2.1)- heptan-2-yl)-5-heptenoic acid]) and was reversed upon removal of U44069 from the culture medium. SQ29548 alone had no affect on receptor density or affinity. Loss of surface receptors was demonstrated to be mediated by agonist-occupied receptor internalization which was inhibited by incubation at 4 degrees C and did not occur with antagonist occupation. The results indicate that homologous downregulation of TXA2 receptors in K562 cells occurs by agonist-mediated active internalization of plasma membrane TXA2 receptors.     

Characterization of an 125I-labeled thromboxane A2/prostaglandin H2 receptor agonist

Stable synthetic mimetics of thromboxane (TX) A2 and prostaglandin (PG) H2 have been synthesized and reported to stimulate platelets and vascular smooth muscle. The synthetic agonists induce aggregation of isolated platelets and contraction of vascular tissue. The tritiated agonists [3H]U46619 and [3H]U44069 have been used in radioligand binding studies to characterize platelet and vascular smooth muscle TXA2/PGH2 receptors, but have limited usefulness due to their low specific activities and variable specific binding. In an attempt to overcome these problems, we have synthesized a stable, high affinity, 125I-radiolabeled TXA2/PGH2 receptor agonist, [1S-(1 alpha, 2 beta (5Z), 3 alpha(1E,3S*), 4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo - [2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP). I-BOP induced shape change, increased intracellular free calcium concentrations and aggregated isolated human platelets (EC50 = 0.21 +/- 0.05 nM, n = 3; 4.1 +/- 1.1 nM, n = 4; 10.8 +/- 3 nM, n = 9, respectively). The kinetically determined Kd was 1.02 +/- 0.33 nM (kobs = 0.19 +/- 0.05 min-1, k-1 = 0.097 +/- 0.02 min-1, k1 = 0.119 +/- 0.03 min-1 M, n = 4). Equilibrium binding studies of [125I]BOP to isolated human platelets indicated one class of high affinity sites, Kd = 2.2 +/- 0.3 nM and a maximum binding of 0.028 +/- 0.002 x 10(-12) mol/10(7) platelets (1699 +/- 162 sites/platelet, n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)     

An endothelium-dependent contraction induced by A-23187, a Ca++ ionophore in canine basilar artery

In most isolated canine basilar arteries tested, Ca++ ionophore A-23187 induced a small relaxation followed by a transient contraction. Both contraction and relaxation were abolished by removal of endothelium. The endothelium-dependent contraction induced by A-23187 was attenuated by a phospholipase A2 inhibitor (quinacrine), cyclooxygenase inhibitors (aspirin and indomethacin), a thromboxane A2 (TXA2) synthetase inhibitor (OKY-046) and a TXA2 antagonist (ONO-3708). The A-23187-induced contraction was abolished by lowering the Ca++ concentration of medium to 10%, whereas the contraction induced by 9,11-epithio-11,12-methano-TXA2 (STA2) was attenuated slightly by lowering [Ca++]. The A-23187-induced contraction was reduced markedly by nifedipine (10(-9) to 10(-7) M), but the STA2-induced contraction was only attenuated slightly by nifedipine. Bay K 8644 did not affect the A-23187- and STA2-induced contractions. The present experiments demonstrate that A-23187 induced an endothelium-dependent contraction in canine basilar artery, and suggest that Ca++ might play a key role in production of an endothelium-derived contracting factor (probably TXA2).     

Proteinase-activated receptor-2 (PAR2): vascular effects of a PAR2-derived activating peptide via a receptor different than PAR2

We studied the actions of the proteinase-activated receptor-2-activating peptide (PAR2-AP) trans-cinnamoyl-LIGRLO-amide (tc-LI) in femoral (FA), renal, and small mesenteric (MA) arterial vessels from C57BL/6 [PAR2 (+/+)] and PAR2 (-/-) mice. The actions of tc-LI were compared with those of the parent PAR2-AP Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-amide; SLI-NH2). Either SLI-NH2 or tc-LI (0.1-10 microM) induced relaxation of either 9,11-dideoxy-9alpha,11alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619)- or cirazoline-precontracted FA from PAR2 (+/+) in endothelium-intact preparations but did not relax vessels from PAR2 (-/-) mice. This FA relaxation by SLI-NH2 and by tc-LI was inhibited by 1) pretreatment with a combination of L-N(G)-nitroarginine methyl ester (L-NAME) and 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 2) precontraction with 30 mM KCl, or 3) removal of the endothelium. In contrast, tc-LI caused an L-NAME/ODQ/indomethacin-resistant relaxation of MA from PAR2 (+/+) mice. In contrast with SLI-NH2, tc-LI (>30 microM) contracted arteries from both PAR2 (-/-) and PAR2 (+/+) mice. Pretreatment of tissues with a combination of cyclopiazonic acid plus caffeine reduced significantly tc-LI-induced contractions, whereas nifedipine, CdCl2, and Ca2+-free conditions did not. Inhibitors of vascular muscarinic, alpha1-adrenergic, neurokinin, thromboxane A2, histamine, angiotensin II, or endothelin-1 receptors failed to inhibit contractions by 50 microM tc-LI. At resting tension, SLI-NH2 (>10 microM) contracted all arteries in an endothelium-independent manner but only from PAR2 (+/+) mice. We conclude that the endothelium-dependent vasodilation initiated by SLI-NH2 and tc-LI, but not the endothelium-independent contraction initiated by tc-LI, are due to the activation of PAR2. Indeed, the data from PAR2 (-/-) mice indicate that tc-LI, in addition to activating PAR2, is an agonist of vascular smooth muscle contraction via a receptor different than PAR2.