Activation of peroxisome proliferators-activated receptor δ (PPARδ) promotes blastocyst hatching in mice.

Prostaglandins participate in a variety of female reproductive processes, including ovulation, fertilization, embryo implantation and parturition. In particular, maternal prostacyclin (PGI(2)) is critical for embryo implantation and the action of PGI(2) is not mediated via its G-protein-coupled membrane receptor, IP, but its nuclear receptor, peroxisome-proliferator-activated receptor δ (PPARδ). Recently, several studies have shown that PGI(2) enhances blastocyst development and/or hatching rate in vitro, and subsequently implantation and live birth rates in mice. However, the mechanism by which PGI(2) improves preimplantation embryo development in vitro remains unclear. Using molecular, pharmacologic and genetic approaches, we show that PGI(2)-induced PPARδ activation accelerates blastocyst hatching in mice. mRNAs for PPARδ, retinoid X receptor (heterodimeric partners of PPARδ) and PGI(2) synthase (PGIS) are temporally induced after zygotic gene activation, and their expression reaches maximum levels at the blastocyst stage, suggesting that functional complex of PPARδ can be formed in the blastocyst. Carbaprostacyclin (a stable analogue of PGI(2)) and GW501516 (a PPARδ selective agonist) significantly accelerated blastocyst hatching but did not increase total cell number of cultured blastocysts. Whereas U51605 (a PGIS inhibitor) interfered with blastocyst hatching, GW501516 restored U51605-induced retarded hatching. In contrast to the improvement of blastocyst hatching by PPARδ agonists, PPAR antagonists significantly inhibited blastocyst hatching. Furthermore, deletion of PPARδ at early stages of preimplantation mouse embryos caused delay of blastocyst hatching, but did not impair blastocyst development. Taken together, PGI(2)-induced PPARδ activation accelerates blastocyst hatching in mice.

Cerebral arteriolar damage by arachidonic acid and prostaglandin G2.

Application of arachidonic acid or prostaglandin G(2) to the brain surface of anesthetized cats induced cerebral arteriolar damage. Scavengers of free oxygen radicals inhibited this damage. Prostaglandin H(2), prostaglandin E(2), and 11,14,17-eicosatrienoic acid did not produce arteriolar damage. It appears that increased prostaglandin synthesis produces cerebral vascular damage by generating free oxygen radicals.

Cyclooxygenase-2-derived endogenous prostacyclin reduces apoptosis and enhances embryo viability in mouse.

The role of prostaglandins (PGs) in apoptosis in preimplantation mice embryo development is reported in this study. It is known that apoptosis plays a very important role in normal mice embryo development. Very few reports are available on this subject. Embryos (6-8 cells) were cultured in the presence of a selective cyclooxygenase (COX)1 inhibitor (SC560), a selective COX2 inhibitor (NS398) and a selective prostacyclin synthase (PGIS) inhibitor (U51605) in a 48-h culture. In another experiment, culture media were supplemented with prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2 or prostacyclin) analogues. The apoptosis was evaluated by detection of active caspase-3. It was strongly detected in the presence of selective COX-2 and PGIS inhibitors, which can be decreased by a PGI2 analogue. In our embryo transfer experiment, the implantation rate decreased with exposure to either the COX2 or the PGIS inhibitor which is increased further after PGI2 supplementation. The level of PGI2 is also higher at the 8-16-cell stage, compaction and blastocyst stage than PGE2. All these results indicate that COX2-derived PGI2 plays an important role in preimplantation embryo development and acts as an antiapopetic factor in in vitro culture.

Oxygen modulates prostacyclin synthesis in ovine fetal pulmonary arteries by an effect on cyclooxygenase.

Prostacyclin (PGI2) plays an integral role in O2 mediation of pulmonary vasomotor tone in the fetus and newborn. We hypothesized that O2 modulates PGI2 synthesis in vitro in ovine fetal intrapulmonary arteries, with decreasing O2 causing attenuated synthesis. A decline in PO2 from 680 to 40 mmHg caused a 26% fall in basal PGI2 synthesis. PGI2 synthesis maximally stimulated by bradykinin, A23187, and arachidonic acid were also attenuated at low PO2, by 35%, 33%, and 35%, respectively. PGE2 synthesis was equally affected. In contrast, varying O2 did not alter PGI2 synthesis with exogenous PGH2, which is the product of cyclooxygenase and the substrate for prostacyclin synthetase. Prostaglandin-mediated effects of O2 on cAMP production were also examined. Decreasing PO2 to 40 mmHg caused complete inhibition of basal cAMP production, whereas cAMP production stimulated by exogenous PGI2 was not affected. In parallel studies of mesenteric arteries, PGI2 synthesis and cAMP production were enhanced at low O2. Thus, PGI2 synthesis in fetal intrapulmonary arteries is modulated by changes in O2, with decreasing O2 causing attenuated synthesis. This process is due to an effect on cyclooxygenase activity, it causes marked parallel alterations in cAMP production, and it is specific to the pulmonary circulation.

Regulation of endothelial cell cyclic nucleotide metabolism by prostacyclin.

An analysis of prostaglandin-stimulated adenosine 3',5'-cyclic monophosphate (cyclic AMP) accumulation in cultured human umbilical vein endothelial cells showed prostacyclin (PGI2) to be the most potent agonist followed by prostaglandin (PG)H2, which was more potent than PGE2, while PGD2 was essentially inactive. The endothelial cells studied apparently have a high rate of cyclic AMP phosphodiesterase activity because significant PGI2-mediated increases in cyclic AMP could not be shown in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (MIX). Endoperoxide PGH2-stimulation of cyclic AMP accumulation was inhibited 75--80% by the prostacyclin synthetase inhibitors 12-hydroperoxyeicosatetraenoic acid or 9,11-azoprosta-5,13-dienoic acid. These data indicate that the PGH2-stimulation is due primarily to conversion to PGI2. The beta-adrenergic agonist L-isoproterenol stimulated cyclic AMP accumulation in the endothelial cells. This accumulation was completely blocked by propranolol. However, stimulation of cyclic AMP accumulation by the beta-adrenergic agent did not equal that induced by PGI2. Furthermore, the PGI2 response could not be blocked by propranolol. Thrombin-stimulated PGI2 biosynthesis was attenuated by PGE1 or isoproterenol in the presence of MIX. MIX alone was less effective than a combination of PGE1 or isoproterenol plus MIX. These data suggest two potential effects of PGI2 biosynthesis by endothelial cells: first, the PGI2 can elevate cyclic AMP in platelets, and second, endothelial cell cyclic AMP can be elevated as well, so that subsequent PGI2 synthesis will be attenuated.

Synergistic activation by collagen and 15-hydroxy-9 alpha,11 alpha-peroxidoprosta-5,13-dienoic acid (PGH2) of phosphatidylinositol metabolism and arachidonic acid release in human platelets.

Collagen stimulates the activation of phosphatidylinositol (PI)-specific phospholipase C (EC 3.1.4.10) in human platelets, as manifested by the disappearance of PI, the transient formation of diacylglycerol (DG), and release of myoinositol. Platelets exposed to collagen also form lysophosphatidylinositol (LPI). Maximum formation of DG occurs within 60 s of the addition of collagen and is in proportion to the concentration of collagen provided, up to 100 micrograms/2 x 10(9) platelets/ml. Hydrolysis of PI, formation of DG, and release of arachidonic acid are all inhibited approximately 68% by aspirin or indomethacin, both of which inhibit platelet cyclooxygenase. This inhibition is reversed by the product of cyclooxygenase activity, 15-hydroxy - 9 alpha,11 alpha - peroxidoprosta - 5,13 - dienoic acid (PGH2), or by the PGH2 analogue and agonist, U-46619. The counteracting effects of either PGH2 or the PGH2 analogue can be blocked, in turn, by a PGH2 antagonist, U-51605. Neither PGH2 nor its stable analogue is, by itself, an efficient stimulus for PI breakdown to DG and LPI in platelets. However, in conjunction with collagen, these agents synergistically promote the net breakdown of PI and the release of arachidonic acid in aspirin-treated platelets. Our findings thereby imply that PGH2 has an important role in regulating both the release of its precursor, arachidonic acid, and the metabolism of PI induced by collagen. Dibutyryl cyclic AMP or prostaglandin D2 (PGD2), a prostaglandin that elevates concentrations of cAMP in platelets by stimulating adenylate cyclase, inhibits the hydrolysis of PI induced by collagen by 70%. The activation of PI metabolism by collagen appears to be inhibited by cAMP independently of any effects of this inhibitor on the formation of PGH2.

Cardiac and renal prostaglandin I2. Biosynthesis and biological effects in isolated perfused rabbit tissues.

Both the isolated perfused rabbit heart and kidney are capable of synthesizing prostaglandin (PG) I(2). The evidence that supports this finding includes: (a) radiochemical identification of the stable end-product of PGI(2), 6-keto-PGF(1alpha), in the venous effluent after arachidonic acid administration; (b) biological identification of the labile product in the venous effluents which causes relaxation of the bovine coronary artery assay tissue and inhibition of platelet aggregation; and (c) confirmation that arachidonic acid and its endoperoxide PGH(2), but not dihomo-gamma-linolenic acid and its endoperoxide PGH(1), serve as the precursor for the coronary vasodilator and the inhibitor of platelet aggregation. The rabbit heart and kidney are both capable of converting exogenous arachidonate into PGI(2) but the normal perfused rabbit kidney apparently primarily converts endogenous arachidonate (e.g., generated by stimulation with bradykinin, angiotensin, ATP, or ischemia) into PGE(2); while the heart converts endogenous arachidonate primarily into PGI(2). Indomethacin inhibition of the cyclo-oxygenase unmasks the continuous basal synthesis of PGI(2) by the heart, and of PGE(2) by the kidney. Cardiac PGI(2) administration causes a sharp transient reduction in coronary perfusion pressure, whereas the intracardiac injection of the PGH(2) causes an increase in coronary resistance without apparent cardiac conversion to PGI(2). The perfused heart rapidly degrades most of the exogenous endoperoxide probably into PGE(2), while exogenous PGI(2) traverses the heart without being metabolized. The coronary vasoconstriction produced by PGH(2) in the normal perfused rabbit heart suggests that the endoperoxide did not reach the PGI(2) synthetase, whereas the more lipid soluble precursor arachidonic acid (exogenous or endogenous) penetrated to the cyclooxygenase, which apparently is tightly coupled to the PGI(2) synthetase.

Effects of prostaglandin cyclic endoperoxides on the lung circulation of unanesthetized sheep.

Although prostaglandins E(2) and F(2alpha) have been suggested as mediators of the pulmonary hypertension seen after endotoxin infusion or during alveolar hypoxia, their precursors, the endoperoxides (prostaglandins G(2) and H(2)) are much more potent vasoconstrictors in vitro. In this study we compared the effects of prostaglandin (PG)H(2), a stable 9-methylene ether analogue of PGH(2) (PGH(2)-A), PGE(2), and PGF(2alpha) on pulmonary hemodynamics in awake sheep. The animals were prepared to allow for measurement of (a) lung lymph flow; (b) plasma and lymph protein concentration; (c) systemic and pulmonary vascular pressures; and (d) cardiac output. We also determined the effect of prolonged PGH(2)-A infusions on lung fluid balance and vascular permeability by indicator dilution methods, and by assessing the response of lung lymph. Both PGH(2) and PGH(2)-A caused a dose-related increase in pulmonary artery pressure: 0.25 mug/kg x min tripled pulmonary vascular resistance without substantially affecting systemic pressures. Both were 100 times more potent than PGE(2) or PGF(2alpha) in this preparation. PGH(2)-A, as our analysis of lung lymph and indicator dilution measurements show, does not increase the permeability of exchanging vessels in the lung to fluid and protein. It does, however, augment lung fluid transport by increasing hydrostatic pressure in the pulmonary circulation. We conclude: (a) that PGH(2) is likely to be an important mediator of pulmonary vasoconstriction; (b) its effects are probably not a result of its metabolites PGE(2) or PGF(2alpha).

A role for prostaglandins and thromboxanes in the exposure of platelet fibrinogen receptors.

Exposure of fibrinogen receptors by a variety of agonists is a prerequisite for platelet aggregation. Because the synthesis of prostaglandins and thromboxane A2 also occurs during platelet aggregation we wondered whether these agents participate in the exposure of platelet fibrinogen receptors. Therefore, we measured the binding of human 125I-fibrinogen to gel-filtered normal human platelets after prostaglandin and thromboxane synthesis had been inhibited by aspirin or indomethacin. The fibrinogen binding assay was performed at 37 degrees C but without stirring to prevent the formation of platelet aggregates. Platelet secretion, measured with [14C]serotonin, did not occur during the procedure. Aspirin or indomethacin inhibited fibrinogen binding stimulated by 10 microM epinephrine by 53%, and inhibited fibrinogen binding stimulated by 1-2 microM ADP by 37.1%. However, ADP at concentrations greater than 2 microM returned fibrinogen binding toward control values. Scatchard analysis demonstrated that aspirin decreased the number but not the affinity of the exposed fibrinogen receptors. To determine whether prostaglandins are capable of directly exposing fibrinogen receptors, prostaglandin H2 was used to stimulate platelets in the fibrinogen binding assay. Prostaglandin H2 exposed approximately 54,000 fibrinogen receptors/platelet and corrected the deficit in receptor exposure induced by aspirin. These studies demonstrate that platelet prostaglandins or thromboxane A2 can play a direct role in the exposure of platelet fibrinogen receptors. In addition, they suggest that the synthesis of prostaglandins and thromboxane A2 by stimulated platelets may be all that is required for optimal secondary platelet aggregation.

Mechanisms underlying ATP-induced endothelium-dependent contractions in the SHR aorta.

In mature spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats, acetylcholine, the calcium ionophore A 23187 and ATP release endothelium-derived contracting factor (EDCF), cyclooxygenase (COX) derivatives that activate thromboxane-endoperoxide (TP) receptors on vascular smooth muscle. The EDCFs released by acetylcholine have been identified as prostacyclin and prostaglandin (PG) H(2) while in response to A 23187 thromboxane A(2), along with the two other prostaglandins, contributes to the endothelium-dependent contractions. The purpose of the present study was to identify the EDCFs produced by ATP. Isometric tension and the release of prostaglandins were measured in isolated aortic rings of WKY rats and SHR. ATP produced the endothelium-dependent release of prostacyclin, thromboxane A(2) and PGE(2) (PGI(2)>>TXA(2)> or =PGE(2)>PGF(2alpha)) in a similar manner in aorta from WKY rats and SHR. In SHR aortas, the release of thromboxane A(2) was significantly larger in response to ATP than to acetylcholine while that to prostacyclin was significantly smaller. The inhibition of cyclooxygenase with indomethacin prevented the release of prostaglandins and the occurrence of endothelium-dependent contractions. The thromboxane synthase inhibitor dazoxiben selectively abolished the ATP-dependent production of thromboxane A(2) and partially inhibited the corresponding endothelium-dependent contractions. U 51605, a non-selective inhibitor of PGI-synthase, reduced the release of prostacyclin elicited by ATP but induced a parallel increase in the production of PGE(2) and PGF(2alpha), suggestive of a PGH(2)-spillover, which was associated with the enhancement of the endothelium-dependent contractions. Thus, in the aorta of SHR, endothelium-dependent contractions elicited by ATP involve the release of thromboxane A(2) and prostacyclin with a possible contribution of PGH(2).

Evaluation of cyclooxygenase 2 derived endogenous prostacyclin in mouse preimplantation embryo development in vitro.

Cyclooxygenase (COX) plays an important role in prostaglandin (PG) synthesis and has two isoforms, COX1 and COX2. PGI synthase (PGIS) catalyzes the isomeization of PGH(2) to prostacyclin (PGI(2)). It is reported that COX2 derived PGI2(2) plays a critical role in blastocyst implantation and decidualization and PGI2 mediates its function via PPARdelta receptor. It is also known that cyclooxygenase derived prostaglandins play an important role in mouse blastocyst hatching in vitro. In this study we hypothesized that COX2 derived PGI2 plays an important role in preimplantation embryonic development by increasing the cell number. To examine this hypothesis, 8-cell stage mouse embryos were cultured in the presence of selective inhibitors of COX1 (SC560), COX2 (NS398) and PGIS (U51605) respectively. COX2 and PGIS inhibitor significantly reduced the blastocyst development and presence of PGI2 analogue along with these inhibitors restored the blastocyst development by increasing the total number of embryonic cells. Our immunohistochemical analysis showed that COX1 is expressed at 2-cell, 8-cell, compaction and blastocyst stage whereas COX2 expression starts from eight cell stage embryos. PGIS and PPARdelta expression starts at 2-cell stage of development. Our results suggest that PGI(2) may affect blastomeres number via the so called hypothesis of PPARdelta nuclear receptor in autocrine manner.

Vasomotor action of insulin on the rabbit normal cavernous smooth muscle.

Investigations on the effects of insulin on the normal vasculature have produced conflicting results. This study was aimed at establishing the vasomotor actions of insulin on normal cavernous smooth muscle. Insulin produced dose-dependent (10(-10)-10(-5) M) relaxation of the norepinephrine-precontracted strips of cavernosum, and of Bay K8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-2(trifluoromethylphenyl)pyridine-5-carboxylate]-precontracted strips. Endothelial denudation or indomethacin (10 microM) pre-treatment significantly reduced these insulin-induced relaxations, whereas NG-nitro-L-arginine methyl ester (L-NAME, 5 mM) did not. Moreover, the pre-treatment of the cavernosum strips with a prostacyclin synthesis inhibitor [9,11-diazo-15-deoxy-prostaglandin H2 (U-51605), 10 microM] significantly reduced insulin-induced response, whereas pretreatment with a cyclooxygenase-2 (COX-2) inhibitor (NS-398, 10 microM) did not. In addition, responses to insulin were not inhibited by K+ channel blockers, i.e., tetraethylammonium (TEA, 10 mM) or 4-aminopyridine (4-AP, 10 microM). Moreover, L-type Ca2+ currents were reduced by prostacyclin (2 microM) but not by insulin (10 microM). We conclude that insulin induces the endothelium-dependent relaxation of cavernous smooth muscles and that this relaxation response may emanate from the direct inhibition of L-type Ca2+ channels by prostacyclin.

Regulatory role of cyclic adenosine 3',5'-monophosphate on the platelet cyclooxygenase and platelet function.

Thromboxane A2 plays an important role in arachidonic acid- and prostaglandin H2-induced platelet aggregation. Agents that stimulate platelet adenylate cyclase (prostaglandin I2, prostaglandin I1 and prostaglandin E1) and dibutyryl cyclic AMP inhibit both thromboxane A2 formation and arachidonate-induced aggregation in platelet-rich plasma. Despite complete suppression of aggregation with agents that elevate cyclic AMP, considerable thromboxane A2 is still formed. Prostaglandin H2-induced aggregations which bypass the cyclooxygenase regulatory step are also inhibited by agents that elevate cyclic AMP without any measurable effect on thromboxane A2 production. These data demonstrate that cyclic AMP can inhibit platelet aggregation by a mechanism independent of its ability to suppress the cyclooxygenase enzyme. Parallel experiments with washed platelet preparations suggest that they may be an inadequate model for studying the relationship between the platelet cyclooxygenase and platelet function.

Transformations of 5,8,11,14,17-eicosapentaenoic acid in human platelets.

5,8,11,14,17-[1-14C]Eicosapentaenoic acid was prepared biosynthetically from [1-14C]arachidonic acid using the fungus, Saprolegnia parasitica. Incubation of 5,8,11,14,17-[1-14C]eicosapentaenoic acid with suspensions of human platelets led to the formation of three labeled compounds which were identified as thromboxane B3 (2-5% yield), 12-hydroxy-5,8,10,14-heptadecatetraenoic acid (2-5% yield), and 12-hydroxy-5,8,10,14,17-eicosapentaenoic acid (7-59% yield).

A comparison of imidazole and 9,11-azoprosta-5,13-dienoic acid. Two selective thromboxane synthetase inhibitors.

Two selective thromboxane A2 synthetase inhibitors, imidazole and 9,11-azoprosta-5,13-dienoic acid (azo analog I) were compared to determine their effects on the quantitative formation of thromboxane B2 and prostaglandin E2 accompanying human platelet aggregation. Azo analog I was at least 200 times more potent, on a molar basis, than imidazole in suppressing thromboxane B2 formation in either platelet-rich plasma or washed platelet suspensions aggregated with arachidonic acid or prostaglandin H2. The inhibitors differed in their effect on the aggregation response itself. Azo analog I selectively suppressed thromboxane A2 formation with an accompanying, parallel, suppression of the platelet aggregation. Imidazole selectively suppressed thromboxane A2 formation, but only suppressed the accompanying aggregation in platelet rich plasma, and not washed platelet suspensions. The results indicate that azo analog I functions by competitive inhibition of prostaglandin H2 on the thromboxane synthetase, and that imidazole, while it suppresses thromboxane A2 formation, may have an associated agonist activity that enhances platelet aggregation. The data presented support this hypothesis, and they emphasize the importance of thromboxane A2 in arachidonate mediated platelet aggregation.

Regulation of 3T3-L1 fibroblast differentiation by prostacyclin (prostaglandin I2).

Prostaglandin biosynthesis and prostaglandin-stimulated cyclic AMP accumulation were studied in 3T3-L1 fibroblasts as they differentiated into adipocytes. Incubation of 3T3-L1 membranes with [1-14C]prostaglandin H2, and subsequent radio-TLC analysis, showed that prostacyclin (prostaglandin I2) is the principal enzymatically synthesized prostaglandin in this cell line. Confirmation of the radiochemical data was obtained by demonstrating the presence of 6-keto-prostaglandin F1 alpha, the stable hydrolysis product of prostaglandin I2, by gas chromatography-mass spectrometry. In support of previous work, indomethacin, the prostaglandin endoperoxide synthetase (EC 1.14.99.1) inhibitor, accelerated 3T3-L1 differentiation. More importantly, the incubation of 3T3-L1 cells with insulin and the prostaglandin I2 synthetase inhibitor 9,11-azoprosta-5,13-dienoic acid (azo analog I) also enhanced the rate of cellular differentiation, even though this compound does not inhibit the synthesis of other prostaglandins. The repeated addition of exogenous prostaglandin I2 to 3T3-L1 cells inhibited insulin- and indomethacin-mediated differentiation. When 3T3-L1 cells were exposed to various prostaglandins and the cyclic AMP levels were measured, prostaglandin I2 proved to be the most potent stimulator of cyclic AMP accumulation, followed by prostaglandin E1 greater than prostaglandin H2 much greater than prostaglandin E2, while prostaglandin D2 was inactive. As 3T3-L1 cells differentiate, the ability of prostaglandin I2 or prostaglandin H2 to stimulate cyclic AMP accumulation progressively diminishes. It is suggested that 3T3-L1 differentiation may be controlled by the rate of prostaglandin I2 synthesis and/or sensitivity of the adenylate cyclase to prostaglandin I2.

The inhibition of arachidonic acid metabolism in human platelets by RHC 80267, a diacylglycerol lipase inhibitor.

The diacylglycerol lipase inhibitor, RHC 80267, 1,6-di(O-(carbamoyl)cyclohexanone oxime)hexane, was tested for its ability to block the release of arachidonic acid from human platelets. At a concentration (10 microM) reported to completely inhibit diacylglycerol lipase in fractions of broken platelets, RHC 80267 had no effect on diacylglycerol lipase activity or the release of arachidonic acid from washed human platelets stimulated with collagen. At a high concentration (250 microM), the compound inhibited the formation of arachidonyl-monoacylglycerol by 70% and the release of arachidonate by 60%. However, at this concentration RHC 80267 was found to inhibit cyclooxygenase activity, phospholipase C activity and the hydrolysis of phosphatidylcholine (PC) (presumably by inhibiting phospholipase A2). The phospholipase C inhibition was attributed to the inhibition of prostaglandin H2 formation, as it was alleviated by the addition of the endoperoxide analog, U-46619. PC hydrolysis was only partially restored with U-46619, suggesting that RHC 80267 directly alters phospholipase A2 activity. The inhibition of arachidonate release observed was accounted for by the inhibition of PC hydrolysis. We conclude that RHC 80267, because of its lack of specificity at concentrations needed to inhibit diacylglycerol lipase, is an unsuitable inhibitor for studying the release of arachidonic acid in intact human platelets.

Prostaglandin I2 synthesis and elevation of cyclic AMP levels in 3T3 fibroblasts.

Arachidonic acid and prostaglandin H2 elevate the levels of adenosine 3':5'-monophosphate (cyclic AMP) in Balb/c 3T3 fibroblasts. This effect was inhibited by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid, an inhibitor of prostaglandin I2 synthase (Claesson, H.-E., Lindgren, J.A. and Hammarstr!om, S. (1977) FEBS Lett. 81, 415-418). After addition of arachidonic acid to 3T3 cultures, cellular cyclic AMP levels and growth medium concentrations of 6-ketoprostaglandin F1 alpha (degradation product of prostaglandin I2) were quantitatively determined. The stimulatory effect of exogenously-added prostaglandin I2 on cellular cyclic AMP levels was also determined. The results indicate that the endogenous production of prostaglandin I2 is sufficient to explain the stimulatory action of arachidonic acid on cyclic AMP formation in 3T3 fibroblasts.

Effects of chronic PGHS-2 inhibition on PGHS-dependent vasoconstriction in the aged female rat.

OBJECTIVE: In menopause, aging and decreased estrogen levels are risk factors contributing to impaired vascular function. Previously, in a young ovariectomized rat model, we demonstrated an increase in prostaglandin H synthase (PGHS)-dependent vasoconstriction that could be prevented by estrogen replacement. Subsequently, we found that, with aging, estrogen acts through suppression of the PGHS-2 isoform. HYPOTHESIS: Chronic PGHS-2 inhibition reduces PGHS-dependent vasoconstriction in aging. METHODS: Ovariectomized, aged (12 months) Sprague-Dawley rats were treated with a placebo (n=7), or the PGHS-2 inhibitor (NS-398, s.c. 3 mg/kg) for 1 week (n=6) or 4 weeks (n=6). Methacholine (endothelium-dependent dilator) and phenylephrine (adrenergic constrictor) were used to assess vascular function in the absence or presence of the nonselective PGHS inhibitor, meclofenamate (1 micromol/l) or the specific PGHS-2 inhibitor NS-398 (10 micromol/l). RESULTS: One week of chronic PGHS-2 inhibition abolished a PGHS-dependent shift in methacholine-induced relaxation, while modulation was still observed in phenylephrine constriction. Surprisingly, 4 weeks of PGHS-2 inhibition enhanced PGHS-dependent modulation of vasoconstriction (P<0.05). PGH2/thromboxane inhibition (U-51605, 50 micromol/l) mimicked the results observed with PGHS inhibition among the groups. PGHS-2 expression increased with chronic PGHS-2 inhibition compared to control (P<0.05). CONCLUSIONS: Our data indicate a paradoxical increase in PGHS-dependent vasoconstriction and PGHS-2 expression with prolonged inhibition of PGHS-2 activity. Hence, inhibitors of PGHS-2 activity may not be beneficial in counteracting the vascular dysfunction seen with aging and menopause.

Prostacyclin (PGI2) induces coronary vasodilatation in anaesthetised dogs.

Prostacyclin (PGI2), the predominant metabolite of arachidonic acid in isolated hearts, relaxes strips of bovine coronary artery and is a potent vasodilator in isolated perfused hearts. We have examined the actions of prostacyclin on coronary blood flow in open chest dogs anaesthetised with chloralose. An electromagnetic flow probe was fitted to the left circumflex artery and phasic coronary flow, mean coronary flow (a measure of coronary volume flow over 4 s intervals), and coronary vascular resistance were recorded together with aortic pressure and heart rate. Intravenous infusion of prostacyclin (0.05 to 1.0 microgram.kg.1.min.1), reduced coronary vascular resistance and aortic pressure according to dose, but had only small effects on phasic coronary flow or mean coronary flow. Both tachycardia and bradycardia occurred during infusion of prostacyclin, but 6-oxo-prostaglandin F1alpha (infused at 10 micrograms.kg-1.min-1), the stable degradation produce of prostacyclin, had no cardiovascular effects. The coronary vasodilator effects of prostacyclin were clear when it was injected into the left circumflex artery via a fine catheter distal to the flow probe. Prostacyclin (0.05 to 0.5 microgram) increased phasic coronary flow and mean coronary flow up to 3 fold and reduced coronary vascular resistance without affecting aortic pressure or heart rate, although higher doses had systemic effects. Prostaglandin E1 (0.1 to 0.5 microgram), which also dilated the coronary vessels, had a longer lasting effect and was 1 to 4 times more potent than prostacyclin. Prostaglandin E2, (0.5 to 4 microgram) was less potent than prostacyclin. In four dogs prostacyclin (20 to 500 micrograms) applied epicardially to the left ventricle caused marked and prolonged coronary vasodilatation. Epicardial application of prostacyclin (10 to 25 micrograms) to the right ventricle increased coronary sinus oxygen content with minimal changes in blood pressure. The endoperoxide prostaglandin H2 was a coronary vasodilator of similar potency to prostacyclin, but its analogue U46619 is a vasoconstrictor. Inhibition of cyclo-oxygenase with indomethacin (5 mg.kg-1 i.v.) or sodium meclofenamate (2 mg.kg-1 i.v.) potentiated the coronary dilator effects of prostacyclin given intravenously or into the coronary artery. Cyclo-oxygenase inhibition did not alter the hypotensive effects and increased the coronary vasodilator potency of prostacyclin relative to prostaglandin E2. Thus the sensitivity of the coronary vascular bed to prostacyclin is enhanced when endogenous biosynthesis of prostaglandin-like substances is inhibited. Although the importance of arachidonic acid metabolites in the coronary circulation still requires validation in vivo, it is clear that prostacyclin, and not prostaglandin E2, is the prostaglandin most likely to be involved.