Gq pathway regulates proximal C-type lectin-like receptor-2 (CLEC-2) signaling in platelets.

Platelets play a key role in the physiological hemostasis or pathological process of thrombosis. Rhodocytin, an agonist of the C-type lectin-like receptor-2 (CLEC-2), elicits powerful platelet activation signals in conjunction with Src family kinases (SFKs), spleen tyrosine kinase (Syk), and phospholipase γ2 (PLCγ2). Previous reports have shown that rhodocytin-induced platelet aggregation depends on secondary mediators such as thromboxane A2 (TxA2) and ADP, which are agonists for G-protein-coupled receptors (GPCRs) on platelets. How the secondary mediators regulate CLEC-2-mediated platelet activation in terms of signaling is not clearly defined. In this study, we report that CLEC-2-induced Syk and PLCγ2 phosphorylation is potentiated by TxA2 and that TxA2 plays a critical role in the most proximal event of CLEC-2 signaling, i.e. the CLEC-2 receptor tyrosine phosphorylation. We show that the activation of other GPCRs, such as the ADP receptors and protease-activated receptors, can also potentiate CLEC-2 signaling. By using the specific Gq inhibitor, UBO-QIC, or Gq knock-out murine platelets, we demonstrate that Gq signaling, but not other G-proteins, is essential for GPCR-induced potentiation of Syk phosphorylation downstream of CLEC-2. We further elucidated the signaling downstream of Gq and identified an important role for the PLCß-PKCα pathway, possibly regulating activation of SFKs, which are crucial for initiation of CLEC-2 signaling. Together, these results provide evidence for novel Gq-PLCß-PKCα-mediated regulation of proximal CLEC-2 signaling by Gq-coupled receptors.

Inhibition of thromboxane A2-induced arrhythmias and intracellular calcium changes in cardiac myocytes by blockade of the inositol trisphosphate pathway.

We have recently reported that left atrial injections of the thromboxane A(2) (TXA(2)) mimetic, (5Z)-7-[(1R,4S,5S,6R)-6-[(1E,3S)-3-hydroxy-1-octenyl]-2 -oxabicyclo[2.2.1]hept-5-yl]-5-heptenoic acid (U46619), induced ventricular arrhythmias in the anesthetized rabbit. Data from this study led us to hypothesize that TXA(2) may be inducing direct actions on the myocardium to induce these arrhythmias. The aim of this study was to further elucidate the mechanism responsible for these arrhythmias. We report that TXA(2)R is expressed at both the gene and protein levels in atrial and ventricular samples of adult rabbits. In addition, TXA(2)R mRNA was identified in single, isolated ventricular cardiac myocytes. Furthermore, treatment of isolated cardiac myocytes with U46619 increased intracellular calcium in a dose-dependent manner and these increases were blocked by the specific TXA(2)R antagonist, 7-(3-((2-((phenylamino)carbonyl)hydrazino)methyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid (SQ29548). Pretreatment of myocytes with an inhibitor of inositol trisphosphate (IP(3)) formation, gentamicin, or with an inhibitor of IP(3) receptors, 2-aminoethoxydiphenylborate (2-APB), blocked the increase in intracellular calcium. In vivo pretreatment of anesthetized rabbits with either gentamicin or 2-APB subsequently inhibited the formation of ventricular arrhythmias elicited by U46619. These data support the hypothesis that TXA(2) can induce arrhythmias via a direct action on cardiac myocytes. Furthermore, these arrhythmogenic actions were blocked by inhibitors of the IP(3) pathway. In summary, this study provides novel evidence for direct TXA(2)-induced cardiac arrhythmias and provides a rationale for IP(3) as a potential target for the treatment of TXA(2)-mediated arrhythmias.

Regional heterogeneity of substance P-induced endothelium-dependent contraction, relaxation, and -independent contraction in rabbit pulmonary arteries.

AIMS: The present study examined whether substance P (SP)-induced endothelium-dependent TXA(2)-mediated contraction (EDC), nitric oxide (NO)-mediated relaxation (EDR), and endothelium-independent contraction (EIC) are different between the rabbit proximal and distal intrapulmonary arteries. MAIN METHODS: The helically cut strips of isolated proximal and distal arteries were fixed vertically between hooks in organ bath, and changes in isometric tension were measured. KEY FINDINGS: SP-induced EDC was greater in the distal than proximal arteries, and EDR was greater in the proximal than distal arteries. However, under the complete blockade of NK(2) receptors and NO production, SP (10(-9)-3x10(-7) M)-induced EDC did not differ between proximal and distal arteries. Under the complete blockade of NK(2) receptors and TXA(2) production, SP (3x10(-10)-3x10(-8) M)-induced EDR was greater in the proximal than distal arteries. Neither contraction induced by U-46619, a TXA(2) agonist, nor relaxation by sodium nitroprusside, an NO donor, was different between both portions of the arteries. Both ionomycin (10(-8) M)- and l-arginine (1 mM)-induced EDRs were also significantly greater in the proximal than distal arteries. Under the blockade of NK(1) receptors and NO and TXA(2) production, SP (10(-7) M)-induced EIC was greater in the distal than proximal arteries. In summary, the capacity for NO production is higher in the proximal than distal arteries, resulting in SP-induced higher EDR and lower EDC in the proximal arteries. SIGNIFICANCE: These regional differences in responses to SP may play important roles in maintaining the homogenous distribution of blood flow in the lung.

Role of brain prostanoids in glucagon-like peptide-1-induced central activation of sympatho-adrenomedullary outflow in rats.

1. The aim of the present study was to characterize the source of plasma catecholamines induced by centrally administered glucagon-like peptide-1 (GLP-1), with regard to brain prostanoids, in urethane-anaesthetized rats. 2. Glucagon-like peptide-1 and other compounds were administered intracerebroventricularly (i.c.v.) and blood samples were collected via a cannula inserted into the femoral artery. Catecholamines were extracted from plasma with activated alumina and were assayed electrochemically using high-performance liquid chromatography. 3. At 0.3, 1.0 and 3.0 nmol/animal, GLP-1 dose-dependently elevated plasma levels of noradrenaline and adrenaline and the 1.0 nmol GLP-1-induced response was dose-dependently reduced by 5 and 10 nmol/animal exendin (5-39), a selective GLP-1 receptor antagonist. The GLP-1-induced elevation of concentrations of both catecholamines was abolished by 1.2 micromol/animal indomethacin, an inhibitor of cyclo-oxygenase, whereas 1.2 micromol/animal baicalein, a lipoxygenase inhibitor, had no effect. 4. Both furegrelate (1.8 micromol/animal; an inhibitor of thromboxane A(2) synthase) and (+)S-145 (625 nmol/animal; a thromboxane A(2) receptor antagonist) attenuated the GLP-1-induced increases in plasma adrenaline concentrations, but had no effect on the increases in plasma noradrenaline. The GLP-1-induced increase in plasma adrenaline concentrations was abolished by acute bilateral adrenalectomy, but the procedure had no effect on increases in plasma noradrenaline. 5. These results suggest that, in rats, centrally administered GLP-1 induces the secretion of adrenaline from the adrenal medulla by brain thromboxane A(2)-mediated mechanisms, whereas the peptide evokes the release of noradrenaline from sympathetic nerves by brain prostanoids via mechanisms other than those mediated by thromboxane A(2).

Postnatal maturational shift from PKCzeta and voltage-gated K+ channels to RhoA/Rho kinase in pulmonary vasoconstriction.

OBJECTIVE: The neonate is at high risk of developing pulmonary hypertension, which may reflect a misbalance between vasodilator and vasoconstrictor agents. Thromboxane A(2) (TXA(2)) is involved in several forms pulmonary hypertension, but the signaling pathways mediating its pulmonary vasoconstrictor responses during postnatal maturation have not been analyzed. We therefore investigated the role of L-type Ca(2+) channels, protein kinase C (PKC) zeta, voltage-gated K(+) channels (K(V)), and RhoA/Rho kinase in TXA(2)-induced pulmonary vasoconstriction during postnatal maturation. METHODS: Changes in contractility and intracellular calcium were analyzed in 1 day (newborn) and 2-week-old piglets' pulmonary arteries (PA). K(V) currents were investigated in freshly isolated smooth muscle cells using the whole-cell configuration of the patch clamp technique. RESULTS: The contractile responses to the TXA(2) mimetic U46619 were similar at both ages but the L-type Ca(2+) channel blocker nifedipine and a PKCzeta pseudosubstrate inhibitor only attenuated the contraction in newborn PA. K(V) currents were similarly inhibited by U46619, although their density was dramatically reduced in 2-week-old as compared to newborn PA smooth muscle cells. This was consistent with a greater contraction to the K(V) inhibitor, 4-aminopyridine, and with a leftward shift in the increase in intracellular Ca(2+) by U46619 in newborn versus older animals. On the other hand, the Rho kinase inhibitor Y-27632 induced a stronger inhibitory effect on the contraction induced by U46619 in 2-week-old than in newborn PA and this was accompanied with minor effects on intracellular calcium levels. CONCLUSION: TXA(2)-induced pulmonary vasoconstriction involves PKCzeta-K(V)-L-type Ca(2+) channel and RhoA/Rho kinase signaling pathways, which are downregulated and upregulated, respectively, during postnatal maturation. The different contribution of these pathways could be of relevant importance for the vasodilator therapy choice in the treatment of pulmonary hypertension.

Thromboxane A2 regulates vascular tone via its inhibitory effect on the expression of inducible nitric oxide synthase.

BACKGROUND: Circulatory failure in sepsis arises from vascular hyporesponsiveness, in which nitric oxide (NO) derived from inducible NO synthase (iNOS) plays a major role. Details of the cross talk between thromboxane (TX) A2 and the iNOS-NO system, however, remain unknown. We intended to clarify the role of TXA2, via the cross talk, in vascular hyporesponsiveness. METHODS AND RESULTS: We examined cytokine-induced iNOS expression and NO production in cultured vascular smooth muscle cells (VSMCs) and cytokine-induced hyporesponsiveness of the aorta from mice lacking the TXA2 receptor (TP-/- mice). The cytokine-induced iNOS expression and NO production observed in wild-type VSMCs were significantly augmented in TP-/- VSMCs, indicating an inhibitory effect of endogenous TXA2 on iNOS expression. Furthermore, in indomethacin-treated wild-type VSMCs, U-46619, a TP agonist, inhibited cytokine-induced iNOS expression and NO production in a concentration-dependent manner, effects absent from TP-/- VSMCs. In an ex vivo system, the cytokine-induced hyporesponsiveness of aortas to phenylephrine was significantly augmented in TP-/- aorta but was almost completely canceled by aminoguanidine, an iNOS inhibitor. Accordingly, cytokine-induced NO production was significantly higher in TP-/- aorta than in wild-type aorta. Moreover, U-46619 significantly suppressed lipopolysaccharide-induced NO production in vivo only in wild-type mice. CONCLUSIONS: These results suggest that TXA2 has a protective role against the development of vascular hyporesponsiveness via its inhibitory action on the iNOS-NO system under pathological conditions such as sepsis.

Effects of BM-573, a thromboxane A2 modulator on systemic hemodynamics perturbations induced by U-46619 in the pig.

The aim of our study was to evaluate the effects of thromboxane A2 (TXA2) agonist, U-46619, on systemic circulatory parameters in the pigs before and after administration of a novel TXA2 receptor antagonist and synthase inhibitor (BM-573). Twelve anesthetized pigs were randomly assigned in two groups: in Ago group (n=6), the animals received six consecutive injections of U-46619 at 30 min interval, while in Anta group (n=6) they received an increasing dosage regimen of BM-573 10 min before each U-46619 injection. The effects of each dose of BM-573 on ex vivo platelet aggregation induced by arachidonic acid, collagen or ADP were also evaluated. Vascular properties such as characteristic impedance, peripheral resistance, compliance, arterial elastance were estimated using a windkessel model. Intravenous injections of 0.500 mg/ml of BM-573 and higher doses resulted in a complete inhibition of platelet aggregation induced by arachidonic acid. In the same conditions, BM-573 completely blocked the increase of arterial elastance, and stabilized both mean aortic blood pressure and mean systemic blood flow. In conclusion, BM-573 could therefore be a promising therapeutic approach in pathophysiological states where TXA2 plays a main role in the increase of vascular resistance like in pathologies such as systemic hypertension.

Chronic therapy with an ET(A/B) receptor antagonist in conscious dogs during progression of congestive heart failure. Intracellular Ca(2+) regulation and nitric oxide mediated coronary relaxation.

BACKGROUND: Although it is known that endothelin (ET-1) is elevated in heart failure (HF), it remains unclear if chronic ET(A/B) receptor antagonism affects the progression of HF, particularly by affecting coronary vasoactivity and left ventricular (LV) diastolic function. METHODS: We examined the effects of an ET(A/B) receptor antagonist, L-753,037 (oral bid for 6 weeks, n=7), and vehicle (n=8) in conscious dogs with previously implanted aortic, coronary sinus and left atrial catheters, LV pressure gauge, aortic flow probe, LV dimension crystals and pacers. RESULTS: Baseline hemodynamics were similar in the two groups. During the development of rapid pacing-induced HF, treatment with the ET(A/B) antagonist significantly reduced total peripheral resistance and increased cardiac output compared to vehicle. After 2 weeks of pacing, LV diastolic function (tau) was improved (P<0.05) in the ET(A/B) antagonist group (+6+/-2 ms) compared to the vehicle group (+12+/-2 ms). In addition, ET(A/B) antagonist treatment attenuated the increase in mean left atrial pressure and LV end-diastolic pressure that occurred during heart failure in vehicle-treated animals. However, LV systolic function (LV dP/dt, fractional shortening and Vcfc) neither at rest nor in response to dobutamine was altered by ET(A/B) antagonist treatment. Also, ET(A/B) antagonist treatment did not affect the progressive increases in LV dimension. After 6 weeks of pacing, maximal Ca(2+) transport in isolated cardiac sarcoplasmic reticulum (SR) was reduced (P<0.02) in the vehicle-treated compared to the ET(A/B) antagonist-treated dogs (1.34+/-0.09 vs. 1.60+/-0.06 micromol/mg/min, respectively). The improvement in SR function in the ET(A/B) antagonist-treated dogs was associated with a significant attenuation of the reduction in protein expression of SERCA2a and calsequestrin observed in the vehicle-treated dogs. Coronary arteries isolated from the dogs treated with the ET(A/B) antagonist exhibited enhanced (P<0.01) coronary endothelium-dependent relaxation compared to the vehicle group, while coronary responses to an NO donor were identical in the two groups. Plasma NO levels in the coronary sinus during the late stage of HF were higher (P<0.05) in the ET(A/B) antagonist group (40+/-2 microM) compared to the vehicle group (18+/-2 microM). CONCLUSIONS: We conclude that in conscious dogs during the development of HF induced by rapid pacing, chronic inhibition of ET(A/B) receptors does not affect resting myocardial contractile function nor reserve, but reduces vascular resistance and improves LV diastolic function. After 6 weeks of pacing, the reduction in intracellular Ca(2+) regulation by the SR is also attenuated, and endothelium-dependent coronary relaxation is improved, which appears to be related to the preservation of coronary NO levels.

New trends in thromboxane and prostacyclin modulators.

Thromboxane A2 (TXA2) and prostacyclin (PGI2) are two labile products formed from arachidonic acid by the way of cyclooxygenase. An overproduction of thromboxane A2 has been detected in a series of diseases whereby this prostanoid is assumed to contribute to the underlying pathomechanisms by its potent stimulation of platelet aggregation and smooth muscle contraction. This increased TXA2 biosynthesis is frequently accompanied by a stimulation of prostacyclin formation which is one of the most potent inhibitors of platelet aggregation and smooth muscle contraction. Therefore, TXA2 / prostaglandin endoperoxide H2 receptor antagonists, thromboxane synthase inhibitors and drugs which combine both activities have been developed with the aim to suppress the formation and/or the action of thromboxane A2. Since prostacyclin has been demonstrated to counterbalance the pathological effects of TXA2, several PGI2 agonists have also been developed. This review will highlight the evolution and some of the latest findings in the field of prostacyclin and thromboxane A2 modulators mainly those which are under clinical evaluation or marketed.

The promoting effect of epinephrine on lipopolysaccharide-induced interleukin-8 production in whole blood may be mediated by thromboxane A2.

Epinephrine is known to enhance lipopolysaccharide (LPS)-induced interleukin (IL)-8 secretion in a platelet dependent manner. To determine whether thromboxane A2 (TxA2; a product from activated platelets) is involved in this process, blood samples drawn either before or 2 h after oral administration of 440 mg acetylsalicylic acid (ASA) were stimulated with LPS (5 ng mL(-1)) and different concentrations of epinephrine were added (0.1-100.0 micromol L(-1)). ASA ingestion significantly (global P < 0.05) reduced the enhancing effect of epinephrine on LPS-induced IL-8 release by 15-28%. To further explore whether TxA2 may be involved in this process, a TxA2 agonist (U46619) was added to whole blood together with LPS instead of epinephrine. U46619 mimicked the epinephrine effect: 20 ng mL(-1) U46619 enhanced LPS-induced IL-8 release by 39% (P < 0.05). Furthermore, preincubation of whole blood with 75 micro mol L-1 or 150 micromol L(-1) SQ29548, a TxA2 receptor antagonist, completely blocked epinephrine's promoting effect on LPS-induced IL-8 release. Since thrombin-activated platelets have been reported to be important in the production of IL-8 in monocytes through the activation of monocytes by exposed RANTES in a P-selectin-dependent reaction, we suggest that the epinephrine effect is mediated by enhanced TxA2 production and subsequent rise in the exposure of RANTES and P-selectin on the platelets of whole blood.

Comparison of maitotoxin with thromboxane A2 in rabbit platelet activation.

Maitotoxin (MTX), a Ca2+ channel-activating marine toxin, caused shape change followed by aggregation in rabbit platelets, like U46619, a thromboxane A2 analogue. Although both drugs failed to cause aggregation in the absence of external Ca2+, U46619, but not maitotoxin, elicited shape change in the absence of external Ca2+. The observations of platelets with a scanning electron microscope showed that both drugs caused contraction of platelets and extension of pseudopodia (shape change) followed by aggregation with a clot in the presence of Ca2+. It is noteworthy that long term exposure to MTX caused the lysis of platelets in the presence of Ca2+. While U46619 transiently increased the internal Ca2+ concentration ([Ca2+]i), maitotoxin slowly but irreversibly increased [Ca2+]i in an external Ca2(+)-dependent manner. MTX-induced phosphoinositide hydrolysis was totally dependent on the presence of external Ca2+, but U46619-induced phosphoinositide hydrolysis was still observed in the absence of external Ca2+. MTX-induced phosphoinositide hydrolysis was partly inhibited by SK&F96365, a voltage-independent Ca2+ channel antagonist, or by genistein, a tyrosine kinase inhibitor. MTX caused phosphorylation of tyrosine residues of several proteins, like U46619. Thus, MTX is similar to U46619 in functions of Ca2+ mobilization, phosphoinositide hydrolysis and tyrosine phosphorylation, but MTX-induced actions are strictly dependent on the presence of external Ca2+.

Prostanoid stimulation of cytokine production in an amnion-derived cell line: evidence of a feed-forward mechanism with implications for term and preterm labor.

OBJECTIVE: To test the hypothesis that amnion cytokine production might be regulated by prostanoids. METHODS: Amnion-derived WISH cells were treated with a range of prostanoids and their effects on production of interleukin (IL)-6 and IL-8 were determined by enzyme-linked immunosorbent assay and Northern analysis. The effects of thromboxane inhibitors on cytokine production by term primary amnion explants also were examined. RESULTS: Prostaglandin (PG)A2, PGD2, PGF2 alpha, PGE2, PGJ2, and the PGI2 analogue carbaprostacyclin (1-1000 nmol/L) exhibited no significant effects on cytokine production. However, the thromboxane A2 (TXA2) agonist U46619 and carbocyclic (c)TXA2 both stimulated WISH cytokine production with similar potencies under basal or cytokine-stimulated conditions. Significant stimulation of IL-6 production was observed at concentrations > or = 8 nmol/L (P < .05 by analysis of variance), whereas IL-8 production was stimulated significantly but to a lesser extent. The effects of U46619 and cTXA2 were rapid; maximal stimulation of cytokine production occurred within 4 to 8 hours of treatment. U46619 augmented IL-1 beta-stimulated IL-6 and IL-8 mRNA expression within 2 hours of treatment. In amnion explants inhibitors of TX synthesis and action abrogated the stimulatory effects of IL-1 beta on cytokine production. CONCLUSION: These results are consistent with the presence of a feed-forward loop in amnion involving TXA2 and cytokines, which could play a significant role in the progression of the inflammatory response involved in the mechanism of infection-driven preterm labor.

Effects of U-46619 on pulmonary hemodynamics before and after administration of BM-573, a novel thromboxane A2 inhibitor.

We studied the effects on pulmonary hemodynamics of U-46619, a thromboxane A2 (TXA2) agonist, before and after administration of a novel TXA2 receptor antagonist and synthase inhibitor (BM-573). Six anesthetized pigs (Ago group) received 6 consecutive injections of U-46619 at 30-min interval and were compared with six anesthetized pigs (Anta group) which received an increasing dosage regimen of BM-573 10 min before each U-46619 injection. Consecutive changes in pulmonary hemodynamics, including characteristic resistance, vascular compliance, and peripheral vascular resistance, were continuously assessed during the experimental protocol using a four-element Windkessel model. At 2 mg/kg, BM-573 completely blocked pulmonary hypertensive effects of U-46619 but pulmonary vascular compliance still decreased. This residual effect can probably be explained by a persistent increase in the tonus of the pulmonary vascular wall smooth muscles sufficient to decrease vascular compliance but not vessel lumen diameter. Such molecule could be a promising therapeutic approach in TXA2 mediated pulmonary hypertension as it is the case in pulmonary embolism, hyperacute lung rejection and endotoxinic shock.

The role of inhibition of nitric oxide synthesis in the aggregation of platelets due to the stimulated production of thromboxane A2.

The aggregation of platelets by ADP is reported to be mediated through prostaglandin synthesis. In contrast, nitric oxide is known to inhibit platelet aggregation through the synthesis of cyclic AMP and cyclic GMP. Studies were conducted to determine the role of ADP, if any, on the synthesis of nitric oxide in platelets. Both normal male and female volunteers between the ages of 30 and 45 years participated in the study. Thromboxane A2 (TXA2) was measured as thromboxane B2 by ELISA. Nitric oxide was measured by methhaemoglobin method. It was found that the treatment of platelet-rich plasma (PRP) with different concentrations of ADP (0-8.0 µmol/l) resulted in increased platelet aggregation, and at 8.0 µmol/l ADP, the basal nitric oxide level was found to be maximally decreased from 0.3 ± 0.10 nmol/10 platelets to 0 nmol/10 platelets in PRP (P < 0.0001; n = 10). Line-weaver-Burk plot of nitric oxide synthase (NOS) activity in the presence of 2.0 µmol/l ADP reduced the Vmax from 6.662 to 2.22 nmol nitric oxide/h per mg protein compared with control. Inhibition of nitric oxide synthesis by N-methyl-L-arginine acetate ester (L-NAME), an inhibitor of NOS, was found to aggregate platelets due to the reduction of platelet nitric oxide level (Pearson's coefficient of correlation, r =  -0.986, P < 0.001, n = 10). The treatment of PRP to L-NAME was found to increase TXA2 synthesis to 1.679 ± 0.05 from 0 pmol/10 platelets. These results suggested that inhibition of NOS in platelets resulted in platelet aggregation through TXA2 synthesis in PRP through a novel pathway.

Interaction of angio-associated migratory cell protein with the TPα and TPß isoforms of the human thromboxane A2 receptor.

In humans, thromboxane (TX) A2 signals through the TPα and TPß isoforms of its G-protein coupled TXA2 receptor (TP) to mediate a host of (patho)physiologic responses. Herein, angio-associated migratory cell protein (AAMP) was identified as a novel interacting partner of both TPα and TPß through an interaction dependent on common (residues 312-328) and unique (residues 366-392 of TPß) sequences within their carboxyl-terminal (C)-tail domains. While the interaction was constitutive in mammalian cells, agonist-stimulation of TPα/TPß led to a transient dissociation of AAMP from immune complexes which coincided with a transient redistribution of AAMP from its localization in an intracellular fibrous network. Although the GTPase RhoA is a downstream effector of both AAMP and the TPs, AAMP did not influence TP-mediated RhoA or vice versa. Small interfering RNA (siRNA)-mediated disruption of AAMP expression decreased migration of primary human coronary artery smooth muscle cells (1° hCoASMCs). Moreover, siRNA-disruption of AAMP significantly impaired 1° hCoASMC migration in the presence of the TXA2 mimetic U46619 but did not affect VEGF-mediated cell migration. Given their roles within the vasculature, the identification of a specific interaction between TPα/TPß and AAMP is likely to have substantial functional implications for vascular pathologies in which they are both implicated.

Mechanisms of U46619-induced contraction of rat pulmonary arteries in the presence and absence of the endothelium.

BACKGROUND AND PURPOSE: Thromboxane A(2) and endothelial dysfunction are implicated in the development of pulmonary hypertension. The receptor-transduction pathway for U46619 (9,11-dideoxy-9 alpha, 11 alpha-methanoepoxy prostaglandin F(2 alpha))-induced contraction was examined in endothelium-intact (E+) and denuded (E-) rat pulmonary artery rings. EXPERIMENTAL APPROACH: Artery rings were mounted on a wire myograph under a tension of 7-7.5 mN at 37 degrees C and gassed with 95% O(2)/5% CO(2). Isometric recording was made by using Powerlab data collection and Chart 5 software. KEY RESULTS: Both E+ and E- contractile responses were sensitive to Rho-kinase inhibition and the chloride channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)benzoic acid]. The E+ response was sensitive to the store-operated calcium channel blockers SKF-96365 {1-[B-[3-(4-methoxyphenyl)propoxy]-4-methoxy-phenethyl]-1H-imidazole hydrochloride} and 2-APB (2-amino ethoxy diphenylborate) (75-100 micromol x L(-1)). The E- response was sensitive to 2-APB (10-30 micromol x L(-1)), a putative IP(3) receptor antagonist, and the calcium and chloride channel blockers nifedipine, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid) and niflumic acid but was insensitive to SKF-96365. Inhibiting K(V) with 4-AP in E+ rings exposed a contraction sensitive to nifedipine, DIDS and niflumic acid, whereas inhibiting BK(Ca) exposed a contraction sensitive to mibefradil, DIDS and niflumic acid. This indicates that removal of the endothelium allows the TP receptor to inhibit K(V), which may involve coupling to phospholipase C, because inhibition of phospholipase C with U73122 (1-[6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-y]amino]hexyl]- 1H-pyrrole-2,5-dione) switched the E- pathway to the E+ pathway. CONCLUSIONS AND IMPLICATIONS: The results from this study indicate that distinct transduction pathways can be employed by the TP receptor to produce contraction and that the endothelium is able to influence the coupling of the TP receptor.

Ciglitazone, a peroxisome proliferator-activated receptor gamma inducer, ameliorates renal preglomerular production and activity of angiotensin II and thromboxane A2 in glycerol-induced acute renal failure.

Peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear transcription factor, modulates vascular responses to angiotensin II (AII) or thromboxane A(2) (TxA(2)) via regulation of their gene/receptor. Increased vasoconstriction and deteriorating renal function in glycerol-induced acute renal failure (ARF) may be attributed to down-regulation of PPARgamma. In this study, we investigated the effect of ciglitazone (CG), a PPARgamma inducer, on AII and TxA(2) production and activity in glycerol-induced ARF. Vascular responses to AII or 9,11-dideoxy-11alpha,9alpha-epoxymethano prostaglandin F(2alpha) (U46619), a TxA(2) mimetic, were determined in preglomerular vessels following induction of ARF with glycerol. Renal damage and function were assessed in CG-treated (9 nmol/kg for 21 days) rats. PPARgamma protein expression and activity, which were significantly lower in ARF rats, were enhanced by CG (26 and 30%). CG also increased PPARgamma mRNA by 67 +/- 6%, which was reduced in ARF. In ARF, there was significant tubular necrosis and apoptosis, a 5-fold increase in proteinuria and a 2-fold enhancement in vasoconstriction to AII and U46619. CG reduced proteinuria (49 +/- 3%), enhanced Na(+) (124 +/- 35%) and creatinine excretion (92 +/- 25%), markedly diminished tubular necrosis, and reduced ARF-induced increase in AII (40 +/- 3%) and TxA(2) (39 +/- 2%) production, the attending increase in vasoconstriction to AII (36 +/- 2%) and U46619 (50 +/- 11%), and the increase in angiotensin receptor-1 (AT(1)) (23 +/- 3%) or thromboxane prostaglandin (TP) receptor (13 +/- 1%). CG reduced free radical generation by 55 +/- 14% while elevating nitrite excretion (65 +/- 13%). Our results suggest that enhanced activity of AII and TxA(2), increased AT(1) or TP receptor expression, and renal injury in glycerol-induced ARF are consequent to down-regulation of PPARgamma gene. CG ameliorated glycerol-induced effects through maintaining PPARgamma gene.

Novel prostanoid thromboxane A2 agonists.

The chemistry and biology of novel TXA2(TP)-receptor agonists based on the prostanoid skeleton is described and structure-activity-relationships are discussed. One compound,(5Z,13E), (9R,15R)-9-fluoro-15-hydroxy-16-phenoxy-17,18,19,20-tetranor- 5,13-prostadienoic acid (33), was identified which is 10 times more potent than the standard TP-receptor against U 46619.