Thromboxane A2/prostaglandin H2 Research Articles

The Effects of AT1 Receptor Blockade on Skin Microcirculatory Blood Flow and Thromboxane A2 (TXA2) Production in Young Healthy Women

AT1 receptor activation has an important role in maintaining the vascular release of prostaglandins responsible for mediating hypoxic dilation in skeletal muscle microvessels in normotensive rats. High salt diet leads to impairment of vasodilator responses that could be restored by infusion of subpressor dose of ANG II via its interaction with AT1 receptors. In addition to AT1 receptors blockade, losartan blocks thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. The aim of this study was to determine the effect of one week AT1 receptor blockade by losartan in 10 healthy normotensive women (20±2 years old) on law salt diet (LSD) on post occlusive hyperemic blood flow response in skin microcirculation by Laser Doppler Flowmetry (LDF) and to determine the plasma renin activity (PRA) and plasma thromboxane A2 (TXA2) concentration. Participants maintained LSD (<40 mmol of Na/day) and took 50 mg of losartan/day for 7 days. After one week of losartan administration the post occlusive hyperemic tissue blood flow was unchanged. However, plasma PRA and TXA2 levels were significantly increased compared to pre‐losartan values. Our results indicate that although one week of AT1 receptor blockade increases the plasma TXA2 concentration and PRA, the skin microcirculatory blood flow remained unchanged, possibly due to losartan blockade of TXA2 receptors and ANG II interaction with AT2 receptors.

Dilator NO, prostaglandins (PGs) and constrictor PGH2/thromboxane A2 mediate flow‐induced dilation of venules.

Mediation of flow dependent responses of venules is still not well characterized. We hypothesized that nitric oxide (NO) and prostanoids contribute to the mediation of responses. Thus, changes in diameter of pressurized venules (active diam.: 259±11 μm, and passive diam.: 412±11 μm, at 10 mmHg) isolated from rat gracilis muscle to increases in perfusate flow were measured, before and after incubation of the vessels with the prostaglandin H2/thromboxane A2 (PGH2/TXA2) receptor blocker SQ 29,548, or the NO synthase inhibitor Nω‐nitro‐L‐arginine‐methyl‐esther (L‐NAME), or the non‐specific cyclooxygenase (COX‐1 and COX‐2) inhibitor indomethacin (INDO), or the specific COX‐1 inhibitor SC 560, or COX‐2 inhibitor NS 398, or with their combination. Increases in flow elicited dilations (max. ΔD: 18±3 %), which were significantly augmented in the presence of SQ 29,548 (37±3 %). Additional presence of L‐NAME, or INDO significantly decreased the dilations (max. ΔD: 23±4 and 15±5, respectively), whereas SC 560 significantly reduced the dilations (max. ΔD: 12±6 %), and NS 398 was without effect. Thus, in isolated skeletal muscle venules, the final response to increases in flow is dilation, due to the combined effect of dilator NO and COX‐1‐derived PGE2/PGI2 and constrictor PGH2/TXA2. We propose that flow‐induced release of dilator and constrictor mediators contribute to the regulation of peripheral venular resistance. (Grants: Hungarian Sci. Res. Funds/OTKA: T48376, T767984 and Health Sci. Council/ETT 364/2006; and Am. Heart Assoc. NE Aff. 0555897T, USA)

Contribution of polyol pathway to arteriolar dysfunction in hyperglycemia. Role of oxidative stress, reduced NO, and enhanced PGH2/TXA2 mediation

Hyperglycemia increases glucose metabolism via the polyol pathway, which results in elevations of intracellular sorbitol concentration. Thus we hypothesized that elevated level of sorbitol contributes to the development of hyperglycemia-induced dysfunction of microvessels. In isolated, pressurized (80 mmHg) rat gracilis muscle arterioles (approximately 150 microm), high glucose treatment (25 mM) induced reduction in flow-dependent dilation (from maximum of 39 +/- 2% to 15 +/- 1%), which was significantly mitigated by an aldose reductase inhibitor, zopolrestat (maximum 27 +/- 2%). Increasing doses of sorbitol (10(-10)-10(-4) M) elicited dose-dependent constrictions (maximum 22 +/- 3%), which were abolished by endothelium removal, a prostaglandin H(2)/thromboxane A(2) (PGH(2)/TXA(2)) receptor (TP) antagonist SQ-29548, or superoxide dismutase (SOD) plus catalase (CAT). Incubation of arterioles with sorbitol (10(-7) M) reduced flow-dependent dilations (from maximum of 39 +/- 2% to 20 +/- 1.5%), which was not further affected by inhibition of nitric oxide synthase by N(omega)-nitro-l-arginine methyl ester but was prevented by SOD plus CAT and mitigated by SQ-29548. Nitric oxide donor sodium nitroprusside-induced (10(-9)-10(-6) M) dilations were also decreased in a SQ-29548 and SOD plus CAT-reversible manner, whereas adenosine dilations were not affected by sorbitol exposure. Sorbitol significantly increased arterial superoxide production detected by lucigenin-enhanced chemiluminescence, which was inhibited by SOD plus CAT. Sorbitol treatment also increased arterial formation of 3-nitrotyrosine. We suggest that hyperglycemia by elevating intracellular sorbitol induces oxidative stress, which interferes with nitric oxide bioavailability and promotes PGH(2)/TXA(2) release, both of which affect regulation of vasomotor responses of arterioles. Thus increased activity of the polyol pathway may contribute to the development of microvascular dysfunction in diabetes mellitus.

Vascular effects of 15-F2t-isoprostane in spontaneously hypertensive rats

F2-isoprostanes are a family of compounds derived from arachidonic acid by free radical-catalyzed peroxidation. Among F2-isoprostanes, 15-F2t-IsoP is a vasoconstrictor in animal and human vascular beds. Several recent studies found increased 15-F2t-IsoP levels in animal models of hypertension. However, no data is available on the vascular effect of 15-F2t-IsoP in such models. The contractile responses of 15-F2t-IsoP (10(-9) to 3 x 10(-5) mol/L) were tested on rat thoracic aortic rings in spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto (WKY) rats. The contraction induced by 15-F2t-IsoP was not significantly different in aortic rings from WKY rats and SHR (Emax 139% +/- 5% vs. 134% +/- 6%, respectively) and was mediated through thromboxane A2-prostaglandin H2 receptor activation as shown by the rightward shift of the concentration-contraction curves in presence of GR 32191, a specific thromboxane A2-prostaglandin H2 receptor antagonist. Endothelial denudation increased the maximal contraction compared to intact rings induced by 15-F2t-IsoP in both WKY rats (170% +/- 20% vs. 139% +/- 5%, p < 0.05) and SHR (194% +/- 11% vs. 134% +/- 6%, p < 0.01), whereas pretreatment with Nomega-nitro-L-arginine (10(-4) mol/L) or with indomethacin (10(-5) mol/L) increased the maximal contraction to 15-F2t-IsoP in WKY rats but not in SHR. SHRs treated with an angiotensin-converting enzyme inhibitor, enalapril, for four weeks showed decreased maximal contraction to 15-F2t-IsoP in vessels with and without endothelium compared with untreated SHR. In conclusion, 15-F2t-IsoP-induced vasoconstriction is similar in SHR compared with WKY rats. Endothelium modulates 15-F2t-IsoP contraction in both strains. However, whereas this effect is mediated through nitric oxide- and cyclooxygenase-dependent pathways in WKY rats, other mediators are implicated in SHR.

Argan (Argania spinosa) oil lowers blood pressure and improves endothelial dysfunction in spontaneously hypertensive rats

Traditionally hand-pressed argan oil, obtained from Argania spinosa seeds, is eaten raw in south-west Morocco; its rich composition of tocopherols, MUFA and PUFA make a study of its actions on risk factors for CVD, such as hypertension, interesting. The effects of 7 weeks of treatment with argan oil (10 ml/kg) on the blood pressure and endothelial function of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats were investigated. Systolic blood pressure and heart rate were measured every week by the tail-cuff method and endothelial function was assessed by carbachol (10(-8) to 10(-4) M)-induced relaxations of aortic rings and small mesenteric arteries pre-contracted with phenylephrine. Argan-oil administration reduced the mean blood pressure of SHR after the fifth week of treatment (P<0.05) and increased (P<0.01) the endothelial responses of arteries from SHR. The NO synthase inhibitor, L-N-omega-nitroarginine (3 x 10(-5) M) revealed a greater participation of NO in the relaxant effect after the treatment. When cyclooxygenase (COX) was blocked with indomethacin (10(-5) M), an involvement of COX products in the endothelium-dependent response was characterized. Enzyme immunoassay of thromboxane B2 showed a significant decrease (P<0.05) in the release of thromboxane A2 in both aorta and small mesenteric artery after argan-oil treatment of SHR. Experiments in the presence of the thromboxane A2-prostaglandin H2 receptor antagonist ICI 192,605 (10(-5) M) confirmed this result. Results after incubation with the antioxidants superoxide dismutase and catalase suggested that a decreased oxidative stress might contribute to explain the beneficial effects of argan-oil treatment.

In the presence of L-NAME SERCA blockade induces endothelium-dependent contraction of mouse aorta through activation of smooth muscle prostaglandin H2/thromboxane A2 receptors.

1. The mechanism of transient contractions induced by the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) blocker cyclopiazonic acid (CPA) in the presence of L-NAME was investigated in mouse aorta. 2. The contractions elicited by 10 micro M CPA required an intact endothelium, were dependent upon external Ca(2+) and were prevented by 10 micro M indomethacin, the inhibitor of prostaglandin synthesis, or 1 micro M SQ29548, the specific prostaglandin H2/thromboxane A2 (PGH2/TXA2) receptor blocker. 3. A blocker of receptor/store operated Ca(2+) channels and voltage gated calcium channels (VGCC), SK&F 96365 (10 micro M), completely abolished the contractions, while a specific blocker of VGCC nifedipine (1 micro M) inhibited them by one third. 4. Dichlorobenzamyl hydrochloride, a blocker of Na(+)/Ca(2+) exchange effectively prevented return of tension to baseline value. 5. At higher concentrations (30-100 micro M) CPA induced indomethacin-resistant tonic contractions of mouse aorta. The CPA dose response curve for tonic contractions is shifted to the right compared to the transient contractions suggesting that smooth muscle is less sensitive to CPA than endothelium. 6. PGH2/TXA2 receptors in mouse aorta are highly sensitive to the thromboxane analogue U46619 (EC(50) : 1.93 nM). This compound stimulates contractions even in the absence of external Ca(2+), which are abolished by the Rho-kinase inhibitor HA-1077. 7. The results suggest that 10 micro M CPA induced capacitive Ca(2+) entry in endothelial cells stimulating the release of PGH2/TXA2, which subsequently caused smooth muscle contraction dependent on Ca(2+) influx and myofilament sensitization by Rho-kinase. Higher concentrations of CPA (30-100 micro M) directly induced contraction of mouse aortic smooth muscle.

Role of thromboxane receptors in the dipsogenic response to central angiotensin II.

Central angiotensin II (ANG II) regulates thirst. Because thromboxane A2-prostaglandin H2 (TP) receptors are expressed in the brain and mediate some of the effects of ANG II in the vasculature, we investigated the hypothesis that TP receptors mediate the drinking response to intracerebroventricular (icv) injections of ANG II. Pretreatment with the specific TP-receptor antagonist ifetroban (Ifet) decreased water intake with 50 ng/kg icv ANG II (ANG II + Veh, 7.2 +/- 0.7 ml vs. ANG II + Ifet, 2.8 +/- 0.8 ml; n = 5 rats; P < 0.001) but had no effect on water intake induced by hypertonic saline (NaCl + Veh, 8.4 +/- 1.1 ml vs. NaCl + Ifet, 8.9 +/- 1.8 ml; n = 5 rats; P = not significant). Administration of 0.6 microg/kg icv of the TP-receptor agonist U-46,619 did not induce drinking when given alone but did increase the dipsogenic response to a near-threshold dose of 15 ng/kg icv ANG II (ANG II + Veh, 1.1 +/- 0.7 vs. ANG II + U-46,619, 4.5 +/- 0.9 ml; n = 5 rats; P < 0.01). We conclude that central TP receptors contribute to the dipsogenic response to ANG II.

Vascular reactivity to angiotensin II alone or combined with a thromboxane A2 mimetic in the isolated perfused kidney of Lyon hypertensive rats

The aim of this study was to evaluate whether thromboxane A2-prostaglandin H2 (TP) receptor activation potentiates the renal vasoconstrictor effect of Angiotensin II (Ang II) in genetically hypertensive rats of the Lyon strain (LH). Concentration-response curves (CRCs) to Ang II (5 pM to 10 nM), to the specific TP receptor agonist U46619 (7.5-960 nM) and to a mixture of Ang II + U46619 (fixed molar ratio of 1 : 9) were obtained in single-pass perfused kidneys isolated from 8 week-old LH and low blood pressure (LL) control rats. Baseline vascular resistance was significantly increased in LH compared to LL kidneys. Comparison of the CRCs obtained for Ang II and U46619 showed that, in both strains, Ang II was about 100 times more potent than U46619. For both drugs, the pD2 or slope values did not differ among the two strains. Co-activation of TP receptors, analyzed with the method of Pöch and Holzmann, tended to potentiate the effects of Ang II in LH but not in LL kidneys. In conclusion, isolated perfused kidneys of LH rat did not exhibit an increased vascular sensitivity to acute infusion of Ang II or U46619 compared to control LL ones. In addition, the results suggest that the interactions between Ang II and TP receptor agonist may differ among the two strains.

Simultaneously increased TxA(2) activity in isolated arterioles and platelets of rats with hyperhomocysteinemia.

We aimed to elucidate the effect of hyperhomocysteinemia (HHcy) on the synthesis of prostaglandins in rat skeletal muscle arterioles and platelets. Male Wistar rats were divided into 2 groups: (1) control rats, with plasma Hcy levels of 6.5+/-0.5 micromol/L (n=50) and (2) rats with HHcy, induced by daily intake of 1 g/kg body weight methionine in the drinking water for 4 weeks (plasma Hcy levels were 20.6+/-3.0 micromol/L, P<0.01 versus controls; n=50). Arterioles (diameter approximately 130 micrometer) were isolated from the gracilis muscle, cannulated, and pressurized (at 80 mm Hg), and changes in their diameters were followed by video microscopy. Constrictions to bradykinin (BK; 10(-10) to 10(-7) mol/L) were significantly greater in HHcy than in control rat arterioles (at 10(-9) mol/L BK, changes were 11+/-3% in control and 41+/-9% in HHcy rats). The cyclooxygenase inhibitor indomethacin (10(-5) mol/L), the prostaglandin H(2)/thromboxane A(2) (PGH(2)/TxA(2)) receptor antagonist SQ 29,548 (10(-6) mol/L), or the TxA(2) synthase inhibitor furegrelate (5x10(-6) mol/L) significantly decreased constrictions to BK in both groups but more so in HHcy arterioles, thus eliminating the difference between responses of HHcy and control arterioles. Constrictions to U46619 (a TxA(2) analogue) were significantly greater in HHcy than in control arterioles (at 10(-8) mol/L U46619, values for controls were 33+/-2% and 54+/-3% for HHcy). Endothelium removal or indomethacin treatment attenuated constrictions to U46619 in HHcy arterioles and eliminated the difference in responses. Also, aggregation of platelets from HHcy rats to collagen and ADP was significantly enhanced compared with controls (with 5 microgram/mL collagen: controls, 23+/-5%; HHcy, 49+/-5%; with 10(-7) mol/L ADP: controls, 25+/-3%; HHcy, 35+/-3%). Indomethacin or SQ 29,548 caused greater inhibition of aggregation of HHcy platelets compared with controls, thereby eliminating the differences between the 2 groups. Thus, HHcy enhances TxA(2) synthesis both in the arteriolar endothelium and platelets. By promoting vascular constriction and platelet aggregation simultaneously, these alterations are likely to contribute to the atherothrombotic vascular diseases described in HHcy.

Shear stress-induced release of prostaglandin H(2) in arterioles of hypertensive rats.

The nitric oxide-mediated portion of shear stress-induced dilation of rat gracilis muscle arterioles was shown to be impaired in spontaneously hypertensive rats (SHR). Because shear stress-induced dilation is primarily mediated by endothelium-derived prostaglandins in rat cremasteric arterioles, we hypothesized that in the cremasteric vascular bed the mediation of shear stress-induced dilation by prostaglandins is altered in hypertension. At a constant intraluminal pressure of 80 mm Hg, the active diameters of isolated rat cremasteric arterioles of normotensive 30-week-old Wistar-Kyoto rats (WKY) and SHR were 58.0+/-3.1 and 51.7+/-3.6 microm, respectively, whereas their passive diameters were 109.4+/-4.4 and 101.9+/-6.7 microm, respectively. Dilations to increases in shear stress elicited by increases in intraluminal flow (from 0 to 25 microL/min) were significantly less (P<0.05) in cremasteric arterioles isolated from SHR than from WKY. Arachidonic acid (10(-5) mol/L) elicited constrictions in SHR arterioles but dilations in WKY arterioles. The prostaglandin H(2)/thromboxane A(2) (PGH(2)/TxA(2)) receptor antagonist SQ 29,548 (10(-6) mol/L) significantly increased basal diameter by 11% and normalized the attenuated shear stress-induced dilation in SHR, whereas it did not affect basal diameter and arteriolar responses of WKY. Furegrelate, a specific inhibitor of TxA(2) synthase, did not affect the response in SHR. Also, SQ 29,548 reversed the arachidonic acid-induced constriction to dilation in SHR arterioles, whereas it did not affect the dilator response in WKY arterioles. Constrictions of arterioles of WKY and SHR to U46,619 (a PGH(2)/TxA(2) receptor agonist) were not different. These results demonstrate that in cremasteric arterioles of hypertensive rats, shear stress elicits an enhanced release of PGH(2), resulting in a reduced shear stress-dependent dilation. Thus, augmented hemodynamic forces can alter the shear stress-induced synthesis of prostaglandins, which may contribute to the elevated vascular resistance in hypertension.

Cytochrome P-450 arachidonate metabolite inhibition improves renal function in Lyon hypertensive rats

The present study evaluated the effects of miconazole, a selective inhibitor of epoxygenase activity, on renal hemodynamics and the pressure-natriuresis response of saline-drinking, uninephrectomized Lyon hypertensive (LH) and Lyon low blood pressure (LL) rats. Infusion of miconazole (final concentration, 1 μmol/L) into the renal artery had no effect on the renal function of LL rats over a range of renal perfusion pressures (RPP) from 100 to 140 mm Hg. In contrast, miconazole lowered renal vascular resistance (RVR, 17.9 ± 1.1 v 26.3 ± 1.5 mm Hg/mL/min/g, P < .01) and increased urinary sodium excretion (6.4 ± 1.2 v 4.2 ± 0.8 μmol /min/g, P < .05) in LH rats at a RPP of 140 mm Hg. To determine whether the effects of epoxyeicosatrienoic acids were dependent on activation of the thromboxane A2-prostaglandin H2 (TP) receptor, we studied the effects of a TP receptor antagonist, GR 32191B (0.1 mg/kg/min), on the renal response to an infusion of miconazole into the renal artery in LH rats. GR 32191B decreased basal RVR and prevented the dilation induced by miconazole. It did not, however, alter its natriuretic effect. The renal metabolism of arachidonic acid was also compared in LH and LL rats. The production of epoxygenase metabolites was 25% lower in microsomes prepared from the renal cortex of LH versus LL rats. Miconazole (1 μmol /L) reduced epoxygenase activity similarly, by approximately 60%, in both strains. These results suggest that endogenously formed P450 metabolites of arachidonic acid may serve as a substrate for the formation of vasoconstrictor endoperoxides that interact with TP receptors in LH rats and contribute to the enhanced renal vascular tone but not the blunted pressure-natriuresis response.

Role of AVP in pressor responses during activation of central TxA2/PGH2 receptors.

Administration of thromboxane A2/prostaglandin H2 (TxA2/PGH2)-receptor agonist U-46619 (2.86 nmol/kg i.v.) to conscious rats increased mean arterial pressure (MAP) by 17 +/- 2 mmHg (n = 6; P < 0.001) and plasma arginine vasopressin (AVP) by 3.5 +/- 1.1 IU/ml (n = 6; P < 0.001). Ifetroban (TxA2/PGH2 antagonist; intracerebroventricularly) prevented both responses. Intracerebroventricular U-46619 increased MAP in Long-Evans rats (n = 6) more than in AVP-deficient Brattleboro rats. AVP V1-receptor antagonist d(CH2)5Tyr(Me)AVP (3 microg/kg i.v.) blocked 67 +/- 5% and 69 +/- 7% of pressor response to intravenous AVP and intracerebroventricular U-46619, respectively. AVP (10 ng/kg i.v.) increased AVP by 4.7 +/- 0.5 pg/ml, comparable to the increase of 3.5 +/- 1.2 pg/ml with intracerebroventricular U-46619 (2.86 nmol/kg), but the rise in MAP was only one-half as great (+8 +/- 3 mmHg for AVP vs. +17 +/- 2 mmHg for U-46619; P < 0.05). In conclusion, U-46619 raises blood pressure and releases AVP by activating brain receptors. AVP explains approximately one-half of the pressor response.

Role of endothelium in the endothelin-1-mediated potentiation of the norepinephrine response in the aorta of hypertensive rats.

To investigate the role of the endothelium in the functional interaction between endothelin-1 and norepinephrine in the contractile response of aortas from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Thoracic aorta rings with and without endothelium from SHR and from WKY rats were suspended in an organ bath to record the isometric tension. After an equilibration period of 120 min, the preparations with and without endothelin-1 were subjected to single and cumulative additions of norepinephrine in different experiments. To characterize the mechanisms involved in the interaction between endothelin-1 and norepinephrine, the aortic rings were pretreated with a cyclooxygenase pathway inhibitor (piroxicam, SO29548), an inhibitor of NO synthase [NG-nitro-L-arginine (NLA)], or selective endothelin receptor blockers (BQ-123 or BQ-788). In some experiments we examined the contractile responses to norepinephrine in aortas pretreated either with angiotensin II (AII) or with U46619, an agonist of prostaglandin H2-thromboxane A2 receptors. Finally, we examined the effect of the combination of calcium-entry blockade by administration of nifedipine and treatment with either endothelin-1 or U46619 on the norepinephrine reactivity. Administration of 3 x 10(-10) mol/l endothelin-1 potentiated the contractile response to norepinephrine in SHR aortas with endothelium, irrespective of whether they had been treated with NLA. No endothelin-1-mediated enhancement of the response to norepinephrine was observed in SHR denuded rings and in untreated and NLA-treated WKY rat aortas. All did not affect the response to norepinephrine in SHR rings with endothelium. The amplification by endothelin-1 of the response to (1-100) x 10(-9) mol/l norepinephrine was abolished by blockade of the cyclooxygenase pathway with piroxicam or SO29548. In WKY rat and SHR denuded aortas, 10(-8) mol/l U46619 potentiated the contractile responses to norepinephrine. Administration of 3 x 10(-6) mol/l BQ-123 abolished the increase in reactivity to norepinephrine evoked by endothelin-1 in intact SHR aorta, whereas 3 x 10(-6) mol/l BQ-788 failed to modify this potentiating effect. Administration of 10(-8) mol/l nifedipine inhibited the potentiation of the norepinephrine-induced contractions evoked both by endothelin-1 in SHR aortic rings with endothelium and by U46619 in SHR denuded rings. Our results show that a low concentration of endothelin-1 induced potentiation of the contractile response to norepinephrine in SHR aortas but not in WKY rat aortas. This response was endothelium-dependent. Furthermore, our study affords functional arguments that both endothelial and smooth muscle pathways are involved in the potentiating interaction. We propose that endothelin-1 stimulates the production of endothelium- and cyclooxygenase-generated vasoconstrictor factors, which in turn may serve directly as priming stimuli at the vascular smooth muscle level, to activate the Ca(2+)-signal pathway and consequently to increase locally the vascular sensitivity to norepinephrine.

Release of nitric oxide and prostaglandin H2 to acetylcholine in skeletal muscle venules.

The role of endothelium in regulating venular resistance is not well characterized. Thus we aimed to elucidate the endothelium-derived factors involved in the mediation of responses of rat gracilis muscle venules to acetylcholine (ACh) and other vasoactive agents. Changes in diameter of perfusion pressure (7.5 mmHg)- and norepinephrine (10(-6) M)-constricted venules (approximately 225 microns in diam) to cumulative doses of ACh (10(-9) to 10(-4) M) and sodium nitroprusside (SNP, 10(-9) to 10(-4) M), before and after endothelium removal or application of various inhibitors, were measured. Lower doses of ACh elicited dilations (up to 42.1 +/- 4.7%), whereas higher doses of ACh resulted in smaller dilations or even constrictions. Endothelium removal abolished both ACh-induced dilation and constriction. In the presence of indomethacin (2.8 x 10(-5) M), a cyclooxygenase blocker, or SQ-29548 (10(-6) M), a thromboxane A2-prostaglandin H2 (PGH2) receptor antagonist, higher doses of ACh caused further dilation (up to 72.7 +/- 7%) instead of constriction. Similarly, lower doses of arachidonic acid (10(-9) to 10(-6) M) elicited dilations that were diminished at higher doses. These reduced responses were, however, reversed to substantial dilation by SQ-29548. The nitric oxide (NO) synthase blocker, N omega-nitro-L-arginine (L-NNA, 10(-4) M), significantly reduced the dilation to ACh (from 30.6 +/- 5.5 to 5.4 +/- 1.4% at 10(-6) M ACh). In contrast, L-NNA did not affect dilation to SNP. Thus ACh elicits the release of both NO and PGH2 from the venular endothelium.

Platelet-derived thromboxane A2 decreases microvascular perfusion after arterial repair.

Previous work suggests that cod liver oil helps to protect the microcirculation from the consequence of thromboembolic events. The possibility that altered synthesis of thromboxane A2 accounts for the protective effects seen with cod liver oil was investigated in the present study. This was done using the combined thromboxane A2 synthetase inhibitor and thromboxane A2-prostaglandin H2 receptor blocker R68070 (Ridogrel). A standardized microvascular injury was inflicted on the right iliac artery of the rat to generate emboli. The downstream cremaster muscle was used to visualize the passage of the ensuing emboli and to assess the effects of this arterial injury on capillary perfusion and arteriole diameters. The number of visible emboli was not changed by either cod liver oil diet or Ridogrel administration. However, capillary perfusion was preserved by using cod liver oil (n = 7) and was significantly increased by using Ridogrel (n = 7) in comparison with untreated controls (n = 7) in which capillary perfusion was decreased because of the emboli. The administration of Ridogrel to cod liver oil-treated animals (n = 7) provided no additive benefit. The percentage change in A-2 vessel diameters in cod liver oil-treated (n = 7) animals was no different from the control group (n = 7). Ridogrel (n = 7), on the other hand, produced a significant increase in A-3 vessel (n = 21) diameters, but its effects were comparatively less in the cod liver oil-treated animals (n = 7). The formation of platelet aggregates (emboli) appears relatively independent of thromboxane A2 in the rat. Ridogrel is very effective in protecting the microcirculation, and these effects appear to be mediated by A-3 vasodilatation, which, therefore, is at least partially thromboxane A2-dependent. The positive effects of cod liver oil may be mediated by a mechanism that reduces thromboxane A2 synthesis, but further studies are necessary.

P-77 - Pharmacological profile of Z-335, a novel thromboxane A2prostaglandin H2 receptor antagonist.

P-77 - Pharmacological profile of Z-335, a novel thromboxane A2prostaglandin H2 receptor antagonist.

Mechanism of the contractile effect of diaspirin-cross-linked hemoglobin in rat isolated aorta strip denuded of endothelium as revealed using an oil-immersion procedure

Diaspirin-cross-linked hemoglobin (DCLHb) is a chemically modified hemoglobin (Hb) (i.e., alpha-subunits are cross-linked by a covalent bond) currently being tested as a potential oxygen-carrying blood substitute. It was examined for possible vasoactive properties, using the rat isolated aorta strip denuded of endothelium. In this experimental model, DCLHb (1.6-155 microM) was found to be inactive as a vasoconstrictor when added to the Krebs medium but to elicit contractile responses once the Krebs medium containing DCLHb was replaced by mineral oil, a procedure that favors the sequestration of a fixed amount of DCLHb within a substantially reduced volume of extracellular fluid. The contractile activity of DCLHb in our experimental model (i.e., prior exposure of tissues to drugs in the Krebs medium followed by replacement of the Krebs medium by mineral oil) was mimicked by methemoglobin and metmyoglobin, but not by cytochrome c, albumin, hemin, hematin, Fe2+, and a variety of hemorphins. It was abolished by indomethacin, SQ-29548 (prostaglandin H2-thromboxane A2 receptor antagonist), thiourea, or N-2-mercaptopropionylglycine (MCPG), reduced partially by verapamil, but not affected by dazmegrel, MK-886 (leukotriene biosynthesis inhibitor), dimethylsulfoxide, vitamin C or E, deferoxamine, NG-nitro-L-arginine, naloxone, and a variety of other drug receptor antagonists (e.g., prazosin) and protease inhibitors (e.g., pepstatin). Rat aorta strips denuded of endothelium exhibited contractile responses to arachidonic acid added in the Krebs medium (i.e., with no mineral oil added afterwards). Such contractile activity was reduced by SQ-29548, thiourea, or MCPG. Addition of U-46619 (prostaglandin H2-thromboxane A2 mimetic) to the Krebs medium also elicited contractile responses in rat aorta strips denuded of endothelium. Such contractile activity was reduced by SQ-29548, thiourea, or verapamil but not by MCPG. Within the limitations of our experimental approach, these results suggest that (1) the contractile activity of DCLHb in rat aorta strips denuded of endothelium following replacement of the Krebs medium by mineral oil involves the participation of a secondary mediator, which could be a vasoconstrictor metabolite of arachidonic acid; (2) the participation of reactive oxygen species, potential degradation products of DCLHb (e.g., heme, Fe2+, hemorphins), or other mediators in the contractile activity of DCLHb is unlikely; and (3) Ca2+ entry into target cells might be involved in the process by which DCLHb elicits its contractile activity in our experimental model.

Specificity of G alpha q and G alpha 11 gene expression in platelets and erythrocytes. Expressions of cellular differentiation and species differences.

G alpha q and G alpha 11, members of the Gq family of G-proteins, transduce signals from receptors to the beta isoenzymes of phosphatidyl-inositol-specific phospholipase C (PI-PLC). The receptor specificity of these alpha subunits is unknown. G alpha q and G alpha 11 are ubiquitously expressed in tissues; however, there have been conflicting reports of the presence or absence of G alpha 11 protein in haematopoietic cells. Platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors activate PI-PLC via G alpha q, but the role of G alpha 11 is uncertain. To define their roles in platelet activation we studied G alpha q and G alpha 11 gene expression by immunotransfer blotting and by reverse transcription of mRNA followed by PCR (RT-PCR) and direct sequencing. An antiserum specific for mouse G alpha 11 failed to identify G alpha 11 in dog or human platelets or in dog liver, a tissue known to contain G alpha 11. RT-PCR performed with gene-specific primers demonstrated G alpha q mRNA, but not G alpha 11 mRNA, in normal human and mouse platelets and in thromboxane-sensitive and thromboxane-insensitive dog platelets. Studies of mouse and dog liver and human retina confirmed that the cDNA, primers and probes used could amplify and recognize G alpha 11 in other tissues. However, species-specific oligonucleotide primers and probes were essential to demonstrate G alpha 11, but not G alpha q, mRNA. Compared with mouse cDNA, dog and human G alpha 11 cDNA had twice as many nucleotide substitutions (approx. 12% compared with approx. 6%) as G alpha q, G alpha q mRNA was also found in mature erythrocytes but G alpha 11 mRNA was not identified, whereas both G alpha q and G alpha 11 mRNAs were found in bone marrow stem cells. Therefore G alpha 11 gene expression in haematopoietic cells is linked with cellular differentiation. The lack of G alpha 11 indicates that signal transduction from platelet TXA2/PGH2 receptors to PI-PLC occurs via G alpha q, and that G alpha 11 deficiency is not responsible for defective activation of PI-PLC in thromboxane-insensitive dog platelets. Despite the high degree of similarity that exists between G alpha q and G alpha 11, significantly greater species-specific variation in nucleotide sequence is present in G alpha 11 than in G alpha q. Cellular specificity and species specificity are important characteristics of these Gq family G-proteins.

Two mechanisms for platelet‐mediated killing of tumour cells: one cyclo‐oxygenase dependent and the other nitric oxide dependent

We have tried to identify the cytotoxic effectors in platelet-mediated tumour cell killing, using two tumour cell lines K562 (a chronic myelogenic leukaemic cell line) and LU99A (a lung cancer cell line), which are both sensitive to platelet cytotoxicity. Cyclo-oxygenase inhibitors, acetylsalicylic acid (ASA) and indomethacin, effectively inhibited the platelet-mediated killing of K562 cells, but not that of LU99A cells. In contrast, inhibitors of the nitric oxide (NO) pathway. NG-nitro-1-arginine (L-NA), haemoglobin and methylene blue, reduced the cytotoxic activity of platelets against LU99A, but not against K562. Synthetic analogues of platelet cyclo-oxygenase products thromboxane A2/ prostaglandin H2(TXA2/PGH2) exerted cytotoxicity against K562 cells but not against LU99A cells. Electron microscopic study showed that TXA2/PGH2 analogues induced bleb formation and disruption of the plasma membrane of K562 cells. K562 cells enhanced the production of TXA2 by platelets, as inferred from the accumulation of thromboxane B2 (TXB2), a spontaneous hydrolysis product of TXA2. LU99A cells had no such effects. These results indicate that platelets kill these two tumour cell lines through different mechanisms. In K562, the cyclooxygenase products TXA2/PGH2 possibly play a significant role but in LU99A the NO pathway seems to be involved.

Interactions between nitric oxide and prostanoids in isolated perfused kidneys of the rat.

1. The present study was aimed to assess the interaction between nitric oxide (NO) and thromboxane (Tx) A2-prostaglandin (PG) H2 in single-pass perfused isolated kidneys of the rat. 2. Noradrenaline (NA, 63 and 110 nM) dose-dependently elevated the renal vascular resistance (RVR), the glomerular filtration rate (GFR) and the urinary excretion of sodium (UNa V). Infusion of N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM), an inhibitor of NO synthesis, enhanced the effects of NA on RVR and on UNa V, but decreased those on GFR. The TxA2-PGH2 (TP) receptor blockade by GR32191B (10 microM) attenuated this potentiating effect of L-NAME. 3. When renal perfusion pressure was stepwise increased from 90 to 150 mmHg, L-NAME similarly decreased renal perfusion flow rate and GFR. 4. The venous excretion of TxB2 and 6-keto-PGF1 alpha was increased by L-NAME in baseline conditions as well as after NA or increasing renal perfusion pressure (RPP). 5. These results suggest that: (1) TxA2 and PGH2 play an important role in the overall effect of the renal prostanoids, (2) NO strongly interacts with the cyclo-oxygenase pathway and reduces the prostanoid synthesis in the kidney, and (3) the pressor effect of L-NAME partly relies upon the vasoconstrictor effect of TxA2 and PGH2.