A comparison of the effects of PGI2, PGE2 and PGH2 on the cyclic nucleotide levels in rat anterior pituitary glands

Rat anterior pituitary explants were incubated with PGI₂, PGH₂ and PGE₂ in the presence of theophylline (1mM) and the production of cyclic AMP was measured. PGE₂ was found to be about 20 times more potent than PGI₂ while PGH₂ was slightly more effective than PGI₂. The results suggest that PGI₂ does not play a physiological role in cyclic AMP mediated events in the rat anterior pituitary.     

Metabolism and effect of prostaglandin H2 in adipose tissue.

Prostaglandin H2 (PGH2) inhibited noradrenaline induced cyclic AMP accumulation in isolated rat fat cells in a dose-dependent manner. IC50 was 10-25 ng/ml both in the absence and in the presence of theophylline. The degree of inhibition produced by PGH2 increased with time of incubation. A stable PGH2 analog did not inhibit cyclic AMP accumulation. PGH2 was rapidly converted by isolated fat cells to PGD2, PGE2 and PGF2alpha' but no formation of thromboxane B2 was found either in vitro or in vivo. PGE2 was a more potent inhibitor than PGH2 of noradrenaline induced cyclic AMP accumulation. PGD2 enhanced cyclic AMP accumulation in a limited concentration interval, while PGF2alpha was essentially uneffective. Our results suggest that PGH2 is an inhibitor of cyclic AMP formation in isolated rat fat cells only after conversion to PGE2. A physiological role for PGH2 as a modulator of lipolysis is considered unlikely.     

A comparison of the effects of PGI2, PGE2 and PGH2 on the cyclic nucleotide levels in rat anterior pituitary glands in vitro

Rat anterior pituitary explants were incubated with PGI₂, PGH₂ and PGE₂ in the presence of theophylline (1mM) and the production of cyclic AMP was measured. PGE₂ was found to be about 20 times more potent than PGI₂ while PGH₂ was slightly more effective than PGI₂. The results suggest that PGI₂ does not play a physiological role in cyclic AMP mediated events in the rat anterior pituitary.     

Prostacyclin stimulation of dog arterial cyclic AMP levels.

Prostacyclin (PGI2) dose-dependently increases the adenosine 3',5'-cyclic monophosphate (cyclic AMP) levels in canine femoral, carotid, and canine and bovine coronary arteries. The prostacyclin-stimulation is enhanced by phosphodiesterase inhibitors, and is readily measurable after 60 sec incubation. The prostaglandin endoperoxide PGH2, but not PGH1, also elevates cAMP levels in femoral arteries. Inhibition of arterial prostacyclin synthetase with 28 microM 9,11-azoprosta-5,13-dienoic acid (azo analog I) blocks the PGH2-stimulation of cAMP accumulation. Azo analog I does not attenuate a direct PGI2 stimulation, indicating that the PGH2 dependent elevation of cAMP is due to conversion of PGH2 to PGI2 by the artery. PGI2 and PGE1 increase cyclic AMP levels and relax dog femoral and bovine coronary arteries, while PGE2, which actually contracts bovine coronary arteries, has no effect on arterial cyclic AMP levels. The significance of the PGI2-stimulation of arterial cyclic AMP is not known, but it is probably related to relaxation of arterial strips.     

6-keto-prostaglandin E1 is not equipotent to prostacyclin (PGI2) as an antiaggregatory agent

A direct comparison of the relative potencies of the two antiaggregatory prostaglandins PGI2 and 6-keto-PGE1 showed PGI2 was at least 20 times more potent than 6-keto-PGE1 when tested against ADP-induced human platelet aggregation. This marked difference in potency was even more evident when the ability of PGI2 and 6-keto-PGI2 to stimulate platelet cyclic AMP levels was determined. When cyclic AMP levels were measured direct comparisons were difficult because the respective dose response curves were not parallel, but 10 ng of PGI2 was equivalent to 300 ng of 6-keto-PGE1. PGI2 was also more potent (10-20 times) than 6-keto-PGE1 as a disaggregatory agent, and the disaggregatory activity of both prostaglandins was enhanced by the phosphodiesterase inhibitor 1-methyl-3-isobutylmethylxanthine. PGI2 was also more active than 6-keto-PGE1 as an inhibitor of thrombus formation in dog coronary arteries in vivo. In vivo, 6-keto-PGE1 was at least 10 times less potent thatn PGI2, the exact difference could not be determined because 6-keto-PGE1 caused significant falls in blood pressure before anti-platelet activity could be detected. PGI2 is an intrinsically more potent anti-aggregatory molecule than 6-keto-PGE1, but these data do not rule out the possibility that some of the activities attributed to PGI2 could be the result of the conversin of PGI2 and/or 6-keto-PGF1 alpha to 6-keto-PGE1.     

PGE1 and prostacyclin suppression of NK-cell mediated cytotoxicity and its relation to cyclic AMP

The capacity of three prostanoids (PGE1, 6-beta-PGI1, PGI2 or prostacyclin) and a phosphodiesterase inhibitor (rolipram) to inhibit NK ("natural killer") cell cytotoxicity and to raise cyclic AMP levels in purified NK cells was compared. PGE1 was about 200 times more potent than prostacyclin both in its ability to raise cyclic AMP and to inhibit NK cell cytotoxicity. The stable prostacyclin analogue, 6-beta-PGI1, had an intermediate potency. A 50% inhibition of cytotoxicity was obtained at approximately 10(-8) M for PGE1, 10(-7) M for 6-beta-PGI1, and 10(-6) M for both prostacyclin and rolipram. These doses raised the level of cyclic AMP by approximately 100%. These results suggest that PGE1 is likely to be more important as an endogenous regulator of lymphocyte cytotoxicity than prostacyclin. The results also provide further evidence that cyclic AMP is the mediator of prostanoid-induced reduction in NK cell activity.     

Prostaglandin I2 stimulation of granulosa cell cyclic AMP production.

The ability of prostaglandin I2 (PGI2) to stimulate cyclic AMP production by granulosa cells, isolated from intact immature rats, has been demonstrated in vitro. The minimal effective dose was 15 ng/ml, which was comparable to the minimal effective dose for PGE2. However, a concentration of 15 microgram/ml PGI2 was required to stimulate cyclic AMP production maximally, compared to a concentration of 1 microgram/ml PGE2, which produced the maximum response. It therefore appears that PGI2 is not more effective than PGE2 in stimulating cyclic AMP production in granulosa cells, and is possibly less effective. Submaximal concentrations of PGI2 appeared to be able to modify the stimulation of cyclic AMP production by follicle-stimulating hormone (FSH), but whether or not PGI2 plays any role in follicular function remains to be established.     

Regulation of macrophage-mediated tumor cell killing by prostaglandins: comparison of the effects of PGE2 and PGI2

Mouse resident peritoneal macrophages stimulated in vitro by purified bacterial lipopolysaccharide (LPS) produced both prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2), the latter detected as its stable metabolite, 6-keto PGF1 alpha. Maximum production, induced in each case by 1 ng/ml purified LPS, was in the range of 10(-7)M for PGI2 and 3 x 10(-8)M for PGE2. A quantitatively similar increase in intracellular levels of macrophage cyclic AMP (measured on a whole cell basis), with a similar duration of effect, was stimulated by PGE2 and PGI2; however, only PGE2 had a negative regulatory effect on macrophage activation for tumor cell killing. These data confirm that more than a whole cell increase in the concentration of cyclic AMP is needed to shut off nonspecific tumor cell killing mediated by LPS-activated resident peritoneal macrophages.     

Correlation between cyclic AMP and LH release following prostaglandin E2 administration.

Five min following a single iv injection of PGE2 into ovariectomized mature rats pretreated with estrogen and progesterone, plasma LH and plasma and pituitary cyclic AMP levels were raised significantly. A close correlation was observed between increased pituitary cyclic AMP contents and release of plasma LH. The average level of cyclic AMP in the anterior pituitary and plasma cyclic AMP increased significantly, while the circulating plasma LH level was not changed at 1 min after PGE2 injection. Plasma LH le-el increased at 2 min after PGE2 and reached a maximum level at the above-mentioned time. This is consistent with hypothesis that increased release of hormone is a consequence of increased pituitary cyclic AMP content.     

Factors regulating the production of prostaglandin E2 and prostacyclin (prostaglandin I2) in rat and human adipocytes.

The regulation of PGE2 (prostaglandin E2) and PGI2 (prostaglandin I2; prostacyclin) formation was investigated in isolated adipocytes. The formation of both PGs was stimulated by various lipolytic agents such as isoproterenol, adrenaline and dibutyryl cyclic AMP. During maximal stimulation the production of PGE2 and PGI2 (measured as 6-oxo-PGF1 alpha) was 0.51 +/- 0.04 and 1.21 +/- 0.09 ng/2 h per 10(6) cells respectively. Thus PGI2 was produced in excess of PGE2 in rat adipocytes. The production of the PGs was inhibited by indomethacin and acetylsalicylic acid in a concentration-dependent manner. The half-maximal effective concentration of indomethacin was 328 +/- 38 nM and that of acetylsalicylic acid was 38.5 +/- 5.3 microM. The PGs were maximally inhibited by 70-75% after incubation for 2 h. In contrast with their effect on PG production, the two agents had a small potentiating effect on the stimulated lipolysis (P less than 0.05). The phospholipase inhibitors mepacrine and chloroquine inhibited both PG production and triacylglycerol lipolysis and were therefore unable to indicate whether the PG precursor, arachidonic acid, originates from phospholipids or triacylglycerols in adipocytes. Angiotensin II significantly (P less than 0.05) stimulated both PGE2 and PGI2 production in rat adipocytes without affecting triacylglycerol lipolysis. Finally, it was shown that PGE2 and PGI2 were also produced in human adipocytes, although in smaller quantities than in rat adipocytes. It is concluded that the production of PGs in isolated adipocytes is regulated by various hormones. Moreover, at least two separate mechanisms for PG production may exist in adipocytes: (1) a mechanism that is activated concomitantly with triacylglycerol lipolysis (and cyclic AMP) and (2) an angiotensin II-sensitive, but lipolysis (and cyclic AMP)-independent mechanism.     

Inhibition of PGE1-stimulated cAMP accumulation in human platelets by thromboxane a2.

The prostaglandin endoperoxide PGH2, HHT, HETE, thromboxane A2, and thromboxane B2, which are all products of arachidonic acid metabolism of human platelets, were tested for their ability to modulate platelet cyclic nucleotide levels. None of the compounds tested altered the basal level of cAMP or cGMP, and only PGH2 and thromboxane A2 inhibited PGE1-stimulated cAMP accumulation. Thromboxane A2 was found to be a more potent inhibitor of PGE1-stimulated cAMP accumulation and inducer of platelet aggregation than PGH2.     

Biochemical evidence for PGI2 and PGE2 receptors in the rabbit renal preglomerular microvasculature

It has been proposed that a portion of the biologic actions of vasodilator prostaglandins occurs via an interaction with specific adenylate cyclase-linked receptors. This hypothesis was explored further in the renal microvasculature by examining the effects of PGI2, PGE1, and PGE2 on rabbit preglomerular microvascular adenylate cyclase. A membrane preparation derived from freshly isolated rabbit renal preglomerular microvessels was used in these studies. NaF, forskolin, or 5'-guanylyl imidodiphosphate were found to be effective in increasing adenylate cyclase activity in the absence of exogenous guanosine-5'-triphosphate. A dose-dependent stimulation of adenylate cyclase was also observed with guanosine-5'-triphosphate. PGE1, PGE2, and PGI2 produced a dose-dependent stimulation of adenylate cyclase activity only in the presence of guanosine-5'-triphosphate suggesting that this nucleotide is essential for prostaglandin-induced stimulation of the enzyme. PGI2 exhibited a time-dependent increase in adenylate cyclase activity and this increased activity reached a plateau at 20-25 min. When PGE1 and PGE2 were added together, no additive effect on adenylate cyclase stimulation was noted whereas PGI2 and PGE2 when added together produced an additive stimulatory effect. When viewed together, these data suggest the presence of separate PGI2 and PGE adenylate cyclase-linked receptors in rabbit renal preglomerular microvessels. These findings also suggest that in the renal microvasculature, cyclic AMP may be a second messenger mediating the vasodilatory effects of both PGI2 and PGE2.     

Thromboxane synthase inhibition: implications for prostaglandin endoperoxide metabolism. I. Characterisation of an acute intravenous challenge model to measure prostaglandin endoperoxide metabolism

It has been proposed that thromboxane synthase inhibition (TXSI) may be a useful form of anti-thrombotic therapy and that this is due, in part, to redirection of PGH2 metabolism in favour of PGI2, a potent vasodilator and anti-platelet agent. While redirection has been observed ex vivo there are conflicting reports of its occurrence in vivo. We now describe the characterisation of an acute intravenous challenge model using thrombin, collagen, arachidonic acid (AA) and PGH2 for the study of PGH2 metabolism. Following challenge, plasma concentrations of TXB2, 6-oxo-PGF1 alpha, alleged metabolites of PGI2 (PGI2m) and PGE2 were measured by radioimmunoassay (RIA). Thrombin and collagen challenge resulted in a dose-related increase in plasma TXB2 while AA and PGH2, in addition, elevated 6-oxo-PGF1 alpha and PGI2m. Injection of PGH2 elevated 6-oxo-PGF1 alpha, PGI2m, TXB2 and PGE2 levels. Experimental conditions were defined such that challenge with thrombin (40 NIH units kg-1), collagen (100 micrograms kg-1), AA (1 mg kg-1) and PGH2 (5 micrograms kg-1) and measurement of eicosanoids 0.5 min following challenge were optimal for detection of redirection of PGH2 metabolism in vivo. The identity of immunoreactive TXB2 and 6-oxo-PGF1 alpha was further supported by experiments in which the extracted immunoreactive eicosanoids co-eluted with authentic [3H]standards when subject to reverse phase high performance liquid chromatography (RPHPLC). Evidence is also presented that the levels of plasma eicosanoids measured in this model reflect in vivo biosynthesis.     

II - Prostaglandin hyperalgesia: the peripheral analgesic activity of morphine, enkephalins and opioid antagonists.

Morphine, enkephalins, nalorphine, naloxone and pentazocine are shown to have a peripheral analgesic effect. In our modification of the Randall-Selitto test these substances were 50--100 times more potent than a standard local anaesthetic, lidocaine. At this peripheral site, naloxone did not antagonize the effect of morphine. Morphine had a marked analgesic effect on the hyperalgesia induced by PGE2 and PGI2, BaCl2, Ca2+ ionophore A23187, isoprenaline but not on that induced by dibutyryl cyclic AMP. It was suggested that the peripheral analgesic effect of morphine is due to an inhibition of adenylate-cyclase activity.     

Endogenous synthesis of prostaglandins E1 and I2 in 3T3 fibroblasts transformed by polyoma virus. Effects on adenosine 3':5'-monophosphate production

Prostaglandins (PG)E1, E2 and I2 were produced by polyoma virus transformed (py) 3T3 fibroblasts. The levels of PGE1, PGE2 and 6-keto-PGF1 alpha (degradation product of PGI2) were 22.7, 225 and 33.2 ng/ml medium, respectively, 72 h after medium change. The stimulatory potencies of exogenous PGE1, PGE2 and PGI2 on adenosine 3':5'-monophosphate (cyclic AMP) formation were similar. Therefore, the prostaglandin mediated increase in cyclic AMP levels observed during growth of these cells (Claesson, H.-E., Lindgren, J.A. and Hammarstr?m, S. (1977) Eur. J. Biochem. 74, 13) is largely (greater than 80%) mediated by PGE2 and to lesser extents by PGE1 and PGI2.     

Two Possibly Distinct Prostaglandin E1 Receptors in N1E-115 Clone: One Mediating Inositol Trisphosphate Formation, Cyclic GMP Formation, and Intracellular Calcium Mobilization and the Other Mediating Cyclic AMP Formation

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.     

Increase in concentrations of prostaglandin endoperoxide synthase and prostacyclin synthase in human myometrium in late pregnancy

Concentrations of prostaglandin endoperoxide synthase (i.e. cyclooxygenase; PGH synthase) and prostacyclin synthase (PGI synthase) were quantified with specific radioimmunometric assays in human myometrium during the last trimester of pregnancy (n = 23) and in non-pregnant controls (n = 8). Pregnant myometrium contained 3 times more PGH synthase per mg microsomal protein than non-pregnant myometrium (p less than 0.01) but there was no increase with increasing gestational age in the third trimester nor with the onset of labor. In pregnancy, as compared to the non-pregnant state, there was no significant change in the PGI synthase content of myometrial microsomes, but significantly more PGI synthase was recovered in other subcellular fractions (p less than 0.01). This suggests that pregnancy affects preferential changes in the subcellular distribution of PGI synthase in myometrial cells. Relative to its PGI synthase content pregnant myometrium contained twice as much PGH synthase as non-pregnant myometrium (p less than 0.01). This may offer further evidence that PGH synthase rather than PGI synthase itself is the rate limiting factor in myometrial PGI2 production. On the other hand, the much larger increase in PGH synthase than in PGI synthase in pregnant as compared to non-pregnant myometrium, may serve to promote preferential synthesis of prostaglandins that are potent myometrial stimulants and of critical importance in human parturition.     

Histamine, endogenous prostaglandins and cyclic nucleotides in the regulation of airway muscle responses in the guinea pig

Indomethacin (30 mg/kg, i.p.) reduced pulmonary resistance in guinea pigs but did not affect their sensitivity to histamine. This treatment preferentially reduced the generation of PGE2 by isolated tracheal preparations. The ratios of PGF2 alpha/PGE2 before and after treatment were 1/1 and 6/1, respectively. Chronic indomethacin treatment (30 mg/kg, i.p., twice a day for 4 days) increased histamine sensitivity in vivo 2 fold while a longer treatment (10 days) was without effect. The efficacy of histamine and the potency of isoproterenol in tracheal tissues were unaffected by either treatment. Indomethacin (17 microM for 30 min) relaxed tracheal tissues but not bronchial tissues. Responses of both tissues to contractile agonists were potentiated after indomethacin treatment. The efficacy of histamine was smaller in bronchi than in tracheas. Similarly, PGE2, PGI2 and isoproterenol were less potent in bronchi. Basal amounts of cyclic AMP were higher in bronchi than in tracheas; indomethacin did not affect the basal amounts of cyclic AMP in tracheal tissues but reduced them in bronchial preparations. Histamine elevated cyclic AMP content in both preparations; this elevation was reduced by indomethacin. While prostaglandins play a role in modulating airway responses in vitro, their role in airways in normal animals in vivo is more difficult to demonstrate.     

Inhibition of GSH-dependent PGH2 isomerase in mammary adenocarcinoma cells by allicin

We have reported that allicin, a constituent of garlic oil, has no effect on the activities of platelet cyclooxygenase or thromboxane synthase, or vascular PGI2 synthase. The effect of allicin on glutathione (GSH) dependent PGH2 to PGE2 isomerase is unknown. We therefore studied the effect of allicin on PGE2 biosynthesis in a murine mammary adenocarcinoma cell line (No 4526). Intact or sonicated cells were incubated with either 14C-arachidonic acid (AA) or 14C-PGH2, respectively. Following metabolism, products were extracted, separated by TLC and analyzed by radiochromatographic scan. PGE2 was predominantly formed with minimal amounts of PGF2 alpha and PGD2. Formation of 6-keto-PGF1 alpha or TXB2 was not detected indicating the absence of TXA2 and PGI2 synthase activity. Indomethacin and ibuprofen inhibited the PGE2 formation (p less than 0.05). The enzymatic PGE2 formation in sonicates was blocked by depletion of the cellular non-protein thiols by buthionine sulfoximine and was shown to be dependent on GSH. Allicin, over the range of 10-1000 microM, inhibited the formation of PGE2 in cells exposed to 2.0 microM 14C-AA for 20 min. and in sonicated cells incubated with 20.0 microM 14C-PGH2 for 2 min (p less than 0.05). Allicin did not alter cyclooxygenase-mediated oxygen utilization in ram seminal vessicle microsomes, suggesting that allicin selectively inhibits the GSH-dependent PGH2 to PGE2 isomerase in this adenocarcinoma cell line.     

A possible role of thromboxane A2 (TXA2) and prostacyclin (PGI2) in circulation.

Recently two local hormones, thromboxane A2 (TXA2) and prostacyclin (PGI2) have been discovered. These hormones are labile metabolites of arachidonic acid. TXA2 is generated by blood platelets, while PGI2 is produced by vascular endothelium. TXA2 is a potent vasoconstrictor. It also initiates the release reaction, followed by platelet aggregation. PGI2 is a vasodilator, especially potent in coronary circulation. It also inhibits platelet aggregation by virtue of stimulation of platelet adenyl cyclase. Common precursors for both hormones are cyclic endoperoxides PGG2 and PGH2, being formed by cyclooxygenation of arachidonic acid. This last enzymic reaction is more efficient in platelets than in vascular endothelium, and therefore the generation of PGI2 by vasuclar wall is accelerated by an interaction between platelets and endothelial cells. During this interaction platelets supply the endothelial PGI2 synthetase with their cyclic endoperoxides. The newly formed PGI2 repels the platelets from the intima. When PGI2 synthetase is irreversibly inactivated by low concentration of lipid peroxides, then the platelets are not rejected but stick to the endothelium, generate TXA2 and mature thrombi are formed. A balance between formation and release of PGI2, TXA2 and/or cyclic endoperoxides in circulation is of utmost importance for the control of intra-arterial thrombi formation and possibly plays a role in the pathogenesis of atherosclerosis.