Heterologous desensitization of platelet-derived growth factor-mediated arachidonic acid release and prostaglandin synthesis.

Prolonged treatment of quiescent Swiss 3T3 cells with vasopressin induced heterologous desensitization of specific early signals stimulated by platelet-derived growth factor (PDGF). PDGF caused a striking dose-dependent release of [3H]arachidonic acid (EC50 = 2 ng/ml) and prostaglandin E2 (EC50 = 5 ng/ml). These responses are severely attenuated (greater than 85%) by prior exposure to vasopressin in a dose-dependent manner (IC50 = 1.5 nM). Maximal loss of responsiveness occurred after 40 h of vasopressin treatment with a half-maximal desensitization after 11-13 h. The desensitization is dependent upon binding to the V1 receptor, since it can be prevented by the antagonist [Pmp1,O-Me-Tyr2,Arg8]vasopressin. In contrast, stimulation of inositol phosphate accumulation and production of diacylglycerol and phosphatidic acid by PDGF are unchanged. Thus, the observed heterologous desensitization cannot be attributed to an inability to activate phospholipase C. Furthermore, prior exposure to vasopressin did not affect the ability of PDGF to evoke tyrosine phosphorylation of cellular substrates, demonstrating that vasopressin-induced heterologous desensitization causes a block at a point distal to activation of receptor tyrosine kinase activity. Other downstream responses including transient induction of c-fos expression and stimulation of DNA synthesis were attenuated by vasopressin pretreatment. The findings demonstrate a novel mechanism of heterologous cellular desensitization namely, persistent occupancy of a guanine nucleotide-binding protein-coupled receptor, like the V1 type vasopressin receptor, attenuates responsiveness to a polypeptide growth factor like PDGF that initiates responses through a tyrosine kinase receptor.     

Luteolysis and termination of early pregnancy in the rhesus monkey with prostalene,a synthetic prostaglandin analog

Prostalene, a synthetic prostaglandin (PG) analog was compared to PGF₂α for ability to both shorten the luteal phase and to terminate early, confirmed,pregnancy in the rhesus macaque. 15mg of either prostalene or PGF2a administered as a single intramuscular injection 30 days from last menstrual bleed aborted all treated animals. In contrast, while a single dose of 15mg of prostalene administered 7 days before expected menstruation to regularly cycling, non-pregnant monkeys shortened the luteal phase by 5.0 ± 0.7 days, the same dose regimen of PGF2a was ineffective whether judged by circulating levels of progesterone or by menstrual cycle length. Mean, peripheral, plasma progesterone levels in the non-pregnant monkeys declined abruptly following prostalene treatment. Although menstrual flow was of normal duration, the cycle length following the prostalene treated cycle was greater than pretreatment cycle lengths, suggesting that follicular maturation was not accelerated after treatment. Experiments in the mare and pregnant hamster demonstrated that a two fold increase in dose above the luteolytic dose of prostalene was required to ensure immediate return of ovulation. It is suggested that prostalene demonstrates both luteolytic and hypothalamo-pituitary stimulating properties but at different dose levels. The compound may thus be useful in dissecting the differing biological mechanisms concerned.     

Bioconversion of arachidonic acid by human uterine cervical tissue and endocervix in late pregnancy

Prostaglandins (PGs) may play an important role on cervical ripening in late pregnancy, namely cervical dilatation and softening. To investigate this, arachidonic acid metabolites of cervical tissue and endocervix were studied. To separate and identify the metabolites, silicic acid chromatography, thin layer chromatography, reversed phase chromatography, gas-liquid chromatography and GC-MS were used. In cervical tissue, arachidonic acid was converted to 6-ketoPGF1 alpha, PGF2 alpha, PGE2, thromboxane B2, and 12-HETE. In endocervix, arachidonic acid was converted to PGF2 alpha, PGE2, thromboxane B2, 12-hydroxy-5, 8, 10-heptadecatrienoic acid, and 12-HETE. There was no relation between the arachidonic acid conversion rate and the Bishop score (points of cervical ripening).     

Luteolytic action of 15-keto prostaglandin F2alpha in the rhesus monkey.

The naturally-occurring metabolite of prostaglandin F2alpha, 15-keto prostaglandin F2alpha (15-keto PGF2alpha), elicited rapid and sustained declines in serum progesterone concentrations when administered to rhesus monkeys beginning on day 22 of normal menstrual cycles. Evidence for luteolysis of a more convincing nature was obtained in studies where a single dose of 15-keto PGF2alpha was given on day 20 of ovulatory menstrual cycles in which intramuscular injections of hCG were also given on days 18-20; serum progesterone concentrations fell precipitously in monkeys within 24 hours following intramuscular administration of 15-keto PGF2alpha. However, corpus luteum function was impaired in only 4 of 11 early pregnant monkeys when 15-keto PGF2alpha was administered on days 30 and 31 from the last menses, a time when the ovary is essential for the maintenance of pregnancy. Gestation failed in 2 additional monkeys 32 and 60 days after treatment with 15-keto PGF2alpha, but progressed in an apparently normal manner in the remaining 5 animals. Two pregnant monkeys treated with 15-keto PGF2alpha on day 42 from the last menstrual period, a time when the ovary is no longer required for gestation, continued their pregnancies uneventfully. Corpus luteum function was not impaired in 9 control monkeys which received injections of vehicle or hCG at appropriate times during the menstrual cycle or pregnancy.     

Epidermal growth factor family in rhesus monkey uterus during the menstrual cycle and early pregnancy

This study examines immunohistochemically the presence of EGF, TGFalpha, HB-EGF, AR, and EGFR, members of the EGF family in the monkey uterus during the menstrual cycle and early pregnancy. EGF, TGFalpha, HB-EGF, AR, and EGFR were mainly localized in glandular and luminal epithelium. TGFalpha, HB-EGF, and AR staining were stronger in the glandular epithelium closer to the myometrium than in that closer to the luminal epithelium. The level of EGF, TGFalpha, HB-EGF, AR, and EGFR staining was low on days 1 and 6, and began to increase on day 9 of the menstrual cycle. A high level of EGF, and EGFR staining was maintained on days 16, 20, and 25 of the menstrual cycle. The highest levels of TGFalpha, AR, and HB-EGF staining were seen on days 16 and 20 of the menstrual cycle. In early pregnancy, a low level of EGF, TGFalpha, HB-EGF, AR, and EGFR staining appeared on days 1 and 2 of pregnancy, and then gradually increased from day 3 of pregnancy. The highest levels of EGF, TGFalpha, HB-EGF, and EGFR were detected on days 9, and 11 of pregnancy. Our data suggest that the EGF family may play a role in monkey implantation. Mol. Reprod. Dev. 55:164-174, 2000.     

Polycations as prostaglandin synthesis inducers. Stimulation of arachidonic acid release and prostaglandin synthesis in cultured fibroblasts by poly(L-lysine) and other synthetic polycations

Poly(L-lysine) hydrobromide stimulates arachidonic acid release with concomitant synthesis and release of prostaglandins and lipoxygenase-mediated metabolites (hydroxyeicosatetraenoic acids) in cultures of 3T3 Swiss mouse fibroblasts biosynthetically labeled with [3H]arachidonic acid. The response is rapid, reversible with trypsin and persists for at least 50 min. An evaluation of the calcium dependence of the hydrolytic process was consistent with the rate-limiting step involving a cell-surface, calcium-dependent enzyme. The response involves stimulated hydrolysis of arachidonic acid-containing phospholipids, implying the activation of a phospholipase. Arachidonic acid release is stimulated only by poly(L-lysine) hydrobromide preparations with a molecular weight greater than 30 000, which corresponds to a polypeptide chain of more than 140 lysine hydrobromide residues. A variety of other polycations (Mr greater than 30 000), but not polyanions or neutral polymers, stimulated arachidonic acid release and prostaglandin synthesis. The results are consistent with an activation mechanism involving cross-linking of anionic sites on the cell surface. Poly(L-lysine) hydrobromide is also cytotoxic, but the cytotoxic response occurs at 10-fold higher concentrations than arachidonic acid release.     

Evidence of protein kinase C involvement in phorbol diester-stimulated arachidonic acid release and prostaglandin synthesis

Many stimulators of prostaglandin production are thought to activate the Ca2+- and phospholipid-dependent protein kinase first described by Nishizuka and his colleagues (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. In this paper we report evidence that the activation of protein kinase C caused by 12-O-tetradecanoylphorbol-13-acetate (TPA) is involved in the increased prostaglandin production induced by 12-O-tetradecanoylphorbol-13-acetate in Madin-Darby canine kidney (MDCK) cells. We have shown that TPA activates protein kinase C in MDCK cells with similar dose response curve as observed for TPA induction of arachidonic acid release in MDCK cells. Activation of protein kinase C was associated with increased phosphorylation of proteins of 40,000 and 48,000 daltons. We used two compounds (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OMe) and 1-(5-isoquinolinesulfonyl)piperazine) known to inhibit protein kinase C by different mechanisms to further examine if activation of protein kinase C was involved in the increased synthesis of prostaglandins in TPA-treated MDCK cells. We found that both compounds inhibited protein kinase C partially purified from MDCK cells and that ET-18-OMe inhibited the phosphorylation of proteins by protein kinase C in the intact cells. Addition of either compound during or after TPA treatment decreased both release of arachidonic acid from phospholipids and prostaglandin synthesis. Release of [3H]arachidonic acid from phosphatidylethanolamine in TPA-treated cells was blocked by ET-18-OMe or 1-(5-isoquinolinesulfonyl)piperazine addition. However, arachidonic acid release stimulated by A23187 is not blocked by Et-18-OMe. When assayed in vitro, treatment of cells with Et-18-OMe did not prevent the enhanced conversion of arachidonic acid into prostaglandins induced by pretreatment of cells with TPA. Our results suggest that the stimulation of phospholipase A2 activity by TPA occurs via activation of protein kinase C by TPA.     

Metabolism of prostacyclin. Oxidation by rhesus monkey lung 15-hydroxyl prostaglandin dehydrogenase.

Partially purified rhesus monkey lung 15-hydroxyl prostaglandin dehydrogenase (15-OH PGDH) catalyzed the NAD-dependent oxidation of prostacyclin (PGI₂) and 6-keto-PGF_1a to 6,15-diketo-PGF_1a. The product was identified by gas chromatography-mass spectroscopy. Prostacyclin was oxidized four to six times faster than 6-keto-PGF_1a under identical reaction conditions, suggesting that the metabolism of prostacyclin probably proceeds through a bicyclic 15-keto intermediate before chemically decomposing to the final stable product, 6,15-diketo-PGF_1a. Prostacyclin has a good affinity for the 15-OH PGDH enzyme. A Lineweaver-Burke plot gave an apparent K_m value of 7.4 μm, which compares very favorably with the K_m values for PGE₁ and PGE₂.     

Metabolism of arachidonic acid and prostaglandin synthesis in the preadipocyte clonal line OB17

Biosynthesis of prostaglandins in ob₁₇ preadipose cells was studied in culture. Dihomo-γ-linolenic acid is exclusively converted to PGE₁. Arachidonic acid behaves quantitatively as a more potent precursor, leading to the synthesis of PGE₂ and 6-keto-PGF_1α (stable product of prostacyclin). In all cases prostaglandin synthesis was confirmed directly by radioimmunoassay. This synthesis is maximal during the growth phase and decreases dramatically after confluence at a time where adipose conversion occurs, suggesting a possible relationship between both events.     

Acute suppression of FSH secretion by oestradiol in the ovariectomized rhesus monkey

Using long-term ovariectomized rhesus monkeys, we examined the ability of oestradiol to decrease circulating FSH concentrations in the absence of other ovarian factors. Daily blood samples were obtained from untreated monkeys for 8 days, followed by insertion of oestradiol capsules after the Day-8 sample was taken. Samples were then taken on Days 9-15, the capsules were removed after the Day-15 sample, and samples were obtained on Days 16-19. Serum was assayed for concentration of oestradiol, FSH and LH by RIA. The concentration of FSH (ng/ml) in serum did not change during the first 8 days before oestradiol treatment (overall mean = 356 +/- 55) but decreased from the Day-8 value of 320 +/- 8 to 190 +/- 42 on Day 9 and by Day 15, after 7 days of oestradiol treatment, had reached a nadir of 20 +/- 5. By Day 17, i.e. 2 days after removal of the oestradiol capsules, serum FSH had increased (P less than 0.05) to 92 +/- 23 with a further increase (P less than 0.05) on Day 19 (171 +/- 16). This study demonstrates that, unlike in rats, mice, and sheep, administration of oestradiol alone to ovariectomized rhesus monkeys reduces immunoreactive serum FSH to concentrations measured in intact animals.     

The role of iron in prostaglandin synthesis: Ferrous iron mediated oxidation of arachidonic acid

Arachidonic acid (AA) is the essential substrate for production of platelet endoperoxides and thromboxanes. Iron or heme is an essential cofactor for the peroxidase, lipoxygenase and cyclo-oxygenase enzymes involved in formation of these products. The present study has examined the direct interactions between iron and arachidonic acid. Iron caused the oxidation of AA into more polar products which could be detected by UV absorbtion at 232 nM or the thiobarbituric acid (TBA) reaction. High pressure liquid chromatography, chem-ionization and electron-impact mass spectrometry and nuclear magnetic resonance spectroscopy suggest that the major product was a hydroperoxide of AA. Ferrous iron (Fe⁺⁺) and oxygen were absolute requirements. Fe⁺⁺ was converted to the ferric iron (Fe⁺⁺⁺) state during oxidation of AA, but Fe⁺⁺⁺ could not substitute for Fe⁺⁺. No other enzymes, cofactors or ions were involved. Conversion of AA to a hydroperoxide by Fe⁺⁺ was inhibited by the antioxidant, 2, (3)-Tert-butyl-4-hydroxyanisole, the radical scavenger, nitroblue tetrazolium, and iron chelating agents, including EDTA, imidazole and dihydroxybenzoic acid. The reaction was not affected by superoxide dismutase, catalase or aspirin. These findings and preliminary studies of the Fe⁺⁺ induced oxidation product of AA as a substrate for prostaglandin synthesis and inhibitor of prostacyclin production indicate the critical role of Fe⁺⁺ in AA activation.     

Existence of acyl-CoA hydrolase-mediated pathway supplying arachidonic acid for prostaglandin synthesis in microsomes from rabbit kidney medulla.

We have previously shown that acyl-coenzyme A (CoA) hydrolase that hydrolyzes arachidonoyl-CoA (AA-CoA) to arachidonic acid (AA) and CoA is present in the cytosol of rabbit kidney medulla and that this enzyme can supply AA for prostaglandin (PG) synthesis in this region. In the present study, the existence of the acyl-CoA hydrolase-mediated pathway that supplies AA available for PG synthesis in microsomes from the kidney medulla was examined. AA-CoA (20 microM) was preincubated with the 105,000 g pellet (microsomes, 0.5 mg of protein) from the medulla for 5 min at 37 degrees C followed by incubation with the medulla microsomes (0.5 mg of protein) (the source of PG synthesizing enzymes) in the presence of hydroquinone and reduced glutathione for 5 min at 37 degrees C. The PGs formed were measured by high-pressure liquid chromatography using 9-anthryldiazomethane for derivatization. The addition of the microsomal fraction from the medulla in the preincubation mixture increased total PG formation from 3.86 to 8.70 nmol, and this stimulatory effect was somewhat weaker than that of the cytosolic fraction. On the other hand, the microsomal fraction in the kidney cortex has an extremely lower capacity to supply AA for PG synthesis than do medulla microsomes. These results suggest that, in kidney medulla, the microsomes as well as the cytosol have the potential route that supplies AA from AA-CoA for PG synthesis and that this pathway is mediated by acyl-CoA hydrolase.     

Anti-apoptotic effects of arachidonic acid and prostaglandin E2 in pancreatic beta-cells.

BACKGROUND/AIMS: The polyunsaturated fatty acid arachidonic acid (AA) has been implicated in beta-cell defence mechanisms and prostaglandin (PG) products of cyclooxygenase (COX) 2 action confer resistance to alloxan-induced apoptosis in insulin-secreting RIN cells. We have now investigated the anti-apoptotic effects of AA and its metabolite, PGE(2), in the MIN6 mouse insulin-secreting beta-cell line and mouse islets. METHODS: Apoptosis was determined in MIN6 beta-cell and mouse islet extracts by measurement of capase-3 activity, and COX2 mRNA levels were quantified by real-time RT-PCR. RESULTS: Exposure of MIN6 cells to AA (3.1-12.5 microM) caused concentration-dependent reductions in apoptosis, and similar results were obtained when endogenous AA levels were elevated in cytosolic phospholipase A(2)-overexpressing MIN6 cells. 25mM glucose caused both a significant up-regulation of MIN6 cell COX2 mRNA levels and a decrease in apoptosis. Inhibition of MIN6 cell COX2 activity with a selective inhibitor, NS-398 (10-100 microM), increased apoptosis and exogenous PGE(2) (0.2-5 microM) reduced NS-398-induced apoptosis in a concentration-dependent manner. The protective effects of AA and PGE(2) were also observed in primary mouse islets. CONCLUSION: These data show that AA and its COX2-generated metabolite, PGE(2), can protect beta-cells from apoptosis.     

Triacylglycerol lipase mediated release of arachidonic acid for prostaglandin synthesis in rabbit kidney medulla microsomes

The effect of triarachidonin on the synthesis of prostaglandins in rabbit kidney medulla microsomes was examined. Medulla microsomes were incubated with triarachidonin in 0.1 M--Tris/HCl buffer (pH 7.0) containing reduced glutathione and hydroquinone and the formed prostaglandin E2, prostaglandin F2 alpha and prostaglandin D2 were measured by high-pressure liquid chromatography using 9-anthryldiazomethane for derivatization. The addition of triarachidonin (1-10 microM) stimulated prostaglandin formation in a dose-dependent manner. Under our incubation conditions rabbit kidney medulla was found to produce prostaglandin E2 mainly. When arachidonic acid, instead of triarachidonin, was added to the incubation mixture of microsomes, the identical profile of prostaglandin products was obtained. When the pH of the reaction mixture was changed from 7.0 to 8.0, the rate of triarachidonin-induced prostaglandin E2 formation was approximately 60% of that observed at pH 7.0. Studies utilizing Ca2+ and EGTA revealed that triacylglycerol lipase of kidney medulla is independent of Ca2+. The addition of epinephrine made the stimulatory effect of triarachidonin on prostaglandin E2 formation more pronounced. These results suggest that epinephrine-activated triacylglycerol lipase is present in the renomedullary microsomes, and this enzyme activity is a potential mediator of release of arachidonic acid for prostaglandin synthesis in the kidney medulla.