Stimulation Of Prostaglandin Biosynthesis Research Articles

Synthesis of prostaglandins I2 and E2 by the canine gallbladder in vitro: Studies using a new incubation chamber

Using a specially designed incubation chamber, differential synthesis and the response to added arachidonic acid (25 mM) and L-alpha-phosphatidylcholine (LAP; 2 mM) was quantified in gallbladders from male and female dogs. Prostaglandin I2 (PGI2) was the predominant prostanoid synthesized, and tissues from females produced much more PGI2 under basal conditions than did gallbladders harvested from male dogs. Addition of arachidonic acid stimulated PGI2 synthesis by almost 100 per cent. Arachidonate-stimulated mucosal and serosal production of PGI2 were (mean(s.e.m.] comparable, 343(178) and 375(89) pg/cm2/min, respectively. Gallbladders from female animals synthesized significantly more PGI2 than did tissue from males. Indomethacin inhibited PGI2 synthesis in a dose-response manner; at 7 x 10(-5) M, prostanoid synthesis was inhibited by greater than 80 per cent. Arachidonic acid did not stimulate prostaglandin E2 (PGE2) production by gallbladder tissue. LAP similarly stimulated PGI2 biosynthesis, but in contrast to the effect of arachidonic acid, the effect was significantly greater in the serosa than the mucosa, 355(107) and 213(59) pg/cm2/min, respectively. LAP also stimulated PGE2 biosynthesis by the canine gallbladder in a pattern very similar to that of PGI2. Based on the differences in response to the two agents added, we conclude that arachidonic acid and L-alpha-phosphatidylcholine stimulate prostaglandin biosynthesis via independent pathways. We advocate the use of the incubation chamber for the assessment of prostanoid biosynthesis by the gallbladder in vitro.

Ovarian expression, cellular localization, and hormonal regulation of rat secretory phospholipase A2: increased expression by interleukin-1 and by gonadotropins.

It has been suggested that ovulation may constitute a cyclic inflammatory-like process and that gonadotropin-inducible intraovarian interleukin (IL)-1, an established mediator of inflammation, may play a central role in this regard. In support of this hypothesis, our group has been able to document the ability of IL-1 to potently stimulate prostaglandin biosynthesis by cultured rat ovarian cells. Herein we explore the possibility that the prostaglandin-stimulating action of IL-1 is due, in part, to the enhanced expression of ovarian secretory phospholipase-A2 (sPLA2). A single sPLA2 transcript of 1.4 kilobases was noted in all extraovarian tissues of immature rat origin subjected to Northern blot analysis. However, only a barely detectable signal was apparent in ovarian tissue. In contrast, the more sensitive RNase protection assay revealed the unequivocal presence of ovarian sPLA2 transcripts. Cellular localization studies by way of in situ hybridization documented sPLA2 transcripts primarily in the granulosa cell of the periovulatory ovary. Molecular probing of untreated cultured whole ovarian dispersates disclosed spontaneous elaboration of sPLA2 transcripts as early as 20 h after the introduction of cells into culture. Treatment of cultured whole ovarian dispersates with IL-1beta for 48 h produced a 1.7-fold increase (over the value in untreated controls) in the relative expression of sPLA2 transcripts (p < 0.01) along with a 1.7-fold increase in media PLA2 activity (p < 0.01). A more marked increase was documented for IL-1beta-treated cultured isolated granulosa cells (12.5-fold increase, p < 0.001). Treatment of whole ovarian dispersates with an IL-1 receptor antagonist (IL-1RA) produced a reduction in the basal expression of sPLA2 transcripts (55% at the 5 microg/ml dose level; p < 0.01) and PLA2 activity (40%; p < 0.01), thereby suggesting basal endogenous IL-1-like bioactivity. Treatment of cultured whole ovarian dispersates with either hCG or FSH led to 2.6-fold (p = 0.056) and 3-fold (p = 0.029) increases in the abundance of sPLA2 transcripts, respectively, effects blocked by the concurrent presence of IL-1RA. These observations 1) document the immature rat ovary as a site of sPLA2 gene expression, 2) localize the relevant transcripts to the postovulatory granulosa cell, 3) confirm the presence of functional secreted ovarian PLA2 activity, 4) reveal PLA2 expression to be IL-1- and gonadotropin-dependent, and 5) suggest the existence of endogenous PLA2-stimulating IL-1-like activity. These findings also suggest that the ability of hCG or FSH to up-regulate ovarian sPLA2 transcripts may be due, in part, to the endogenous elaboration of IL-like activity.

Regulation of prostaglandin H synthase-2 in chorion and decidual cells

The regulation of chorio-decidual prostaglandin biosynthesis has important implications for our understanding of the mechanisms of human parturition. It has been known for many years that prostaglandin H synthase (PGHS) is a key enzyme in prostaglandin biosynthesis but recently an inducible form (PGHS-2) has been identified that is separate from the constitutive form (PGHS-1). We have evaluated whether substances known to stimulate prostaglandin biosynthesis in chorio-decidua act via PGHS-2. Human chorion and decidual cells were grown to confluence and treated for up to 16 h with interleukin 1β 10 ng/ml), epidermal growth factor (EGF) (10 ng/ml) or phorbol 12-myristate 13-acetate (PMA) (10 −7 M). Microsomal protein was isolated, separated by gel electrophoresis and PGHS-2 amounts determined using an antibody specific for PGHS-2. All three test agents caused an increase in PGHS-2 levels in chorion cells (189%–288%) and decidual cells (153%–541%). Hence, induction of PGHS-2 is one of the mechanisms by which several substances can elevate the rate of prostaglandin biosynthesis.

β-endorphin inhibits the production of prostaglandin E 2 by human chorio 2-decidual cells in carson

The regulation of chorio-decidual prostaglandin biosynthesis has important implications for our understanding of the mechanisms of human parturition. It has been known for many years that prostaglandin H synthase (PGHS) is a key enzyme in prostaglandin biosynthesis but recently an inducible form (PGHS-2) has been identified that is separate from the constitutive form (PGHS-1). We have evaluated whether substances known to stimulate prostaglandin biosynthesis in chorio-decidua act via PGHS-2. Human chorion and decidual cells were grown to confluence and treated for up to 16 h with interleukin 1β 10 ng/ml), epidermal growth factor (EGF) (10 ng/ml) or phorbol 12-myristate 13-acetate (PMA) (10−7 M). Microsomal protein was isolated, separated by gel electrophoresis and PGHS-2 amounts determined using an antibody specific for PGHS-2. All three test agents caused an increase in PGHS-2 levels in chorion cells (189%–288%) and decidual cells (153%–541%). Hence, induction of PGHS-2 is one of the mechanisms by which several substances can elevate the rate of prostaglandin biosynthesis.

Regulation of Prostaglandin H Synthase 1 and 2 in MyoD Transfected Cells

Exponentially growing DD-1 cells (differentiation-defective cells) synthesize prostaglandins, while exponentially growing MyoDD-1 cells (differentiation-competent cells; DD-1 cells transfected with the myogenic regulatory gene MyoD) synthesize little or no prostaglandins and have reduced levels of prostaglandin H synthase-1 RNA [J. R. Wolf et al., 1993, Exp. Cell Res. 207, 439]. However, MyoDD-1 cells have the capability of synthesizing prostaglandins in response to serum stimulation of serum-deprived cells. The serum stimulation of prostaglandin biosynthesis is associated with transient increases in both prostaglandin H synthase-2 RNA and protein. DD-1 cells and MyoDD-1 cells are similar with respect to prostaglandin biosynthesis and expression of prostaglandin H synthase-2 following serum stimulation of serum-deprived cells. In contrast, the expression of prostaglandin H synthase-1 is higher in DD-1 cells as compared to MyoDD-1 cells under all culture conditions. Thus, expression of myogenic regulatory genes is associated with the differential regulation of prostaglandin H synthase-1 and prostaglandin H synthase-2.

Regulation of bone metabolism by the kallikrein-kinin system, the coagulation cascade, and the acute-phase reactants

Inflammation-induced localized bone resorption in diseases such as marginal and apical periodontitis, rheumatoid arthritis, and osteomyelitis is due to activation and recruitment of osteoclasts by locally produced cytokines and inflammatory mediators. Thus several interleukins (1, 3, 4, 6, and 11), tumor necrosis factors (α, β), colony-stimulating factors (M and GM), leukemia inhibitory factor, γ-interferon, and transforming growth factor-β have effects on bone resorption and bone formation in vivo and in vitro. The kallikrein-kinin system and the coagulation cascade are also activated in inflammation. We have found that peptides produced in the kallikrein-kinin system (bradykinin, kallidin) and thrombin, the end product in the coagulation cascade, can stimulate bone resorption in vitro. The stimulatory effect of bradykinin is linked both to B1 and B2 bradykinin receptors. Both kinins and thrombin stimulate prostaglandin biosynthesis in bone parallel with the bone resorptive effect. The stimulatory effect of bradykinin on bone resorption is completely lost when the prostaglandin response is abolished, whereas thrombin can stimulate bone resorption both via prostaglandin-dependent and independent mechanisms. In addition, bradykinin and thrombin act in concert with interleukin-1 to synergistically stimulate bone resorption and prostaglandin biosynthesis. We also have found that one of the acute-phase reactants, haptoglobin, can stimulate bone resorption in vitro, indicating the possibility of generalized bone loss in chronic inflammatory diseases. Moreover, haptoglobin synergistically potentiates bradykinin-induced and thrombin-induced prostanoid biosynthesis in osteoblasts. These observations indicate that the rate of bone resorption in inflammation-induced bone loss may not be due to a single factor but to the concerted action of several local or systemic factors.

Protein kinase A interactions with prostaglandin biosynthesis at the chorio-decidual interface

Activation of the enzyme adenylate cyclase or treatment with dibutyryl cyclic AMP (dbcAMP) stimulates prostaglandin biosynthesis in vitro and in vivo. Prior activation of adenylate cyclase has been shown to inhibit the stimulation of amnion cell prostaglandin E 2 (PGE 2) biosynthesis by substances such as epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA). Little is known, however, concerning the effects of activation of the adenylate cyclase system on basal and stimulated prostaglandin biosynthesis in human chorion and decidual cells. Interleukin-1β (IL-1β), EGF, ionomycin (iono), and PMA are known to stimulate prostaglandin E 2 production in human chorion and decidual cells. Hence, we have evaluated the effects of co-treatment of chorion and decidual cells with dbcAMP in the presence and absence of EGF, PMA, IL-1β, and iono on prostaglandin production. dbcAMP alone stimulated chorion and decidual PGE 2 production. Coincubation of dbcAMP with all four test stimulants resulted in a further enhancement of decidual PGE 2 production that was often more than additive. Thus activation of adenylate cyclase can have effects on prostaglandin production that have specificity with respect to tissue source, presence of other stimulants and relative time of exposure of the tissues to each of the substances involved.

Regulation of chorion laeve prostaglandin E 2 production by epidermal growth factor, protein kinase C activation and calcium

The effects of epidermal growth factor (EGF), phorbol 12-myristate 13-acetate (PMM), A23187, and ionomycin on prostaglandin production by chorion laeve cells in culture for 3 days and 10 days were tested. Experiments were conducted at day 3 because at this time the cultures became confluent and again at day 10 because changes have been observed in the biochemical properties of these cells with time in culture. At 3 days of culture the cells did not respond to EGF but at 10 days EGF (10 ng/ml) induced a significant increase in prostaglandin E2 production. PMIA (10 −9 to 10 −6M) induced a significant increase in PGEZ production at both times in culture. The calcium ionophores, A23187 and ionomycin, were less effective in eliciting a response at either time in culture. Only A23187 (1μM) induced a significant increase in PGE 2 production at day 10 of culture. These data suggest that the presence of functional EGF receptors may increase with time in culture. Furthermore, activation of the protein kinase C pathway in the chorion laeve stimulates prostaglandin biosynthesis. On the other hand, chorion laeve cell prostaglandin biosynthesis is not responsive to increases in intracellular calcium induced by mobile ion carriers such as the ionophores used in this study.

Epidermal growth factor and the regulation of amnion prostaglandin biosynthesis

The production of prostaglandins by amnion is a key factor in the mechanism of human parturition yet the regulation of prostaglandin biosynthesis in amnion is poorly understood. Hence, we have investigated the regulation of epidermal growth factor (EGF) stimulation of prostaglandin biosynthesis in human amnion cells. This stimulatory action is inhibited by cycloheximide or actinomycin D at high concentrations, but enhanced at much lower concentrations of these protein synthesis inhibitors. An amnion-produced prostaglandin inhibitor or immediate early gene action may explain these effects. Pretreatment with phorbol esters (inhibition of protein kinase C activity) reduces basal prostaglandin production and attenualtes the stimulatory action of EGF on prostaglandin biosynthesis. Hence, amnion prostaglandin biosyn-thesis is dependent partly on protein kinase C activity.

Interleukin-1 alpha stimulates prostaglandin biosynthesis in serum-activated mesangial cells by induction of a non-pancreatic (type II) phospholipase A2

Enhanced prostaglandin (PG) biosynthesis is a hallmark of inflammation, and interleukin-1 (IL), a proinflammatory cytokine, is a potent stimulus of PG production. We investigated the mechanisms of IL-1 alpha-enhanced PG synthesis in serum-stimulated mesangial cells. The rIL-1-stimulated increase in PGE2 synthesis was dose- and time-dependent and inhibited by both cycloheximide and actinomycin D. Phospholipase (PL) activity was increased 5- to 10-fold in acid extracts of rIL-1-treated cells as measured by arachidonate release from exogenous [14C]arachidonyl-phosphatidyl-ethanolamine. This induced phospholipase activity was Ca(2+)-dependent and inhibited by the PLA2 inhibitors, aristocholic acid, 7,7-dimethyl-5,8-eicosadienoic acid, and p-bromophenacylbromide, but not by the 1,2-diacylglycerol lipase inhibitor RHC 80267. The rIL-1-stimulated PLA2 had an alkaline pH optimum, and phosphatidylethanolamine was preferred over phosphatidylcholine as substrate. The PLA2 activity increased by rIL-1 was inhibited in cells coincubated with cycloheximide and was measurable after 6 h. A sensitive and specific solution hybridization assay demonstrated a coordinate time-dependent induction of non-pancreatic PLA2 mRNA expression which was increased at least 6-fold by 24 h. In whole cells, IL-1 had no effect on basal [3H]arachidonic acid release but vasopressin (1 microM)-stimulated release was potentiated 2- to 3-fold, suggesting that IL-1 may prime cells for increased PG synthesis via increased PLA2 activity. Thus IL-1 directly stimulates, as well as primes cells for, enhanced PG synthesis, in part, by increasing PLA2 activity through new synthesis of a non-pancreatic (Type II) PLA2.

Regulation of prostaglandin biosynthesis by luteinizing hormone and bradykinin in rat preovulatory follicles in vitro

Luteinizing hormone (LH) stimulates prostaglandin biosynthesis and steroidogenesis in preovulatory (PO) follicles prior to ovulation. Since the ovulatory process shares many similarities with an inflammatory reaction, mediators of the inflammatory response, such as bradkinin (BK) have been suggested to modulate the effects of LH. In the present study the effect of BK (5μM) on: 1) prostaglandin biosynthesis (PGE 2, PGF 2α and 6-keto-PGF 1α), 2) the levels of two enzymes in the cyclo-oxygenase pathway, prostaglandin endoperoxide synthase (PGS) and prostacyclin synthase (PCS), and 3) cyclic adenosine 3'5'-monophosphate (cAMP) and progesterone response of PO follicles incubated in vitro were examined. LH (0.1μg/ml) stimulated the accumulation of cAMP and progesterone in the medium, while BK had no effect on these parameters. BK exerted a slight stimulatory effect on PGE 2, and PGF 2α, (p ≤ 0.01) but not on 6-keto-PGF 1α synthesis, but no changes in PGS or PCS levels could be detected. The effect of LH on prostaglandin biosynthesis was much more pronounced, with an increase of PGE 2, PGE 2α and 6-keto-PGF 1α. LH also induced PGS. The combination of LH and BK did not alter these responses compared to that of LH alone. This study demonstrates that BK stimulates prostaglandin biosynthesis in PO follicles. In contrast ti LH, this effect of BK does not seem to involve the adenylate cyclase system, since BK did not stimulate cAMP production. BK did not affect the levels of PGS or PCS, and the stimulatory effect of BK is suggested to involve an increase in the availability of substrate for the cyclo-oxygenase pathway.

Inhibition of prostaglandin E 2 synthesis by a blocker of epithelial chloride channels

Arginine-vasopressin (AVP) elicits a variety of responses in cultured rat mesangial cells, among them stimulation of prostaglandin biosynthesis and activation of Cl- channels. AVP produced an 11-fold increase over basal levels in prostaglandin E2 release from cultured mesangial cells. This response was completely inhibited by 25 microM indomethacin and 82 +/- 5% inhibited by 25 microM 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) which is a potent blocker of epithelial Cl- channels. The IC50 for NPPB inhibition of prostaglandin E2 release was 8 microM. Indomethacin and NPPB at 25 microM also inhibited AVP-stimulated cellular accumulation of prostaglandin E2 by 98% and 79 +/- 7% respectively. The inhibitory effect of NPPB was not due to interference with the cellular response to AVP since at 50 microM it did not block AVP-stimulated release of arachidonate metabolites from cells metabolically labeled with [3H]-arachidonic acid. It is suggested that NPPB inhibition of prostaglandin E2 synthesis is at the cyclooxygenase level on the basis of its structural similarity to the fenamic acid type of cyclooxygenase inhibitors.

Interlukin-1 stimulates prostaglandin biosynthesis by human amnion

The purpose of these studies was to determine if Interleukin-1 (IL-1) alters the rate of prostaglandin biosynthesis by human amnion. Primary monolayer cultures of amnion cells were established from women undergoing elective cesarean section before the onset of labor. Natural purified and recombinant human IL-1α and IL-1α were incubated with amnion cells in culture, and prostaglandin E 2 (PGE 2) biosynthesis was measured by radioimmunoassay in cell-free media. A concentration-dependent increase in PGE 2 production by amnion cells occurred in response to natural purified and recombinant IL-1 preparations. No differences in the parameters of the dose-response curves between the two IL-1 gene products could be determined (p > 0.05). Indomethacin blocked the effectof IL-1 in prostaglandin biosynthesis by human amnion. Interleukin-1, a fever mediator, could serve as a signal for the initiation of labor in cases of intrauterine or systemic infection.

Effects of some phenolics on the prostaglandin synthesizing enzyme system.

N-cis- and N-t__-r__-a__-n__-s__--feruloyltyramines were isolated as the inhibitors of prostaglandin (PG) biosynthesis from Ipomoea aquatica. Aromatic amides consisting of naturally occurring cinnamic acids and β-phenethylamines were synthesized and tested for their effects on PG biosynthesis. Of those caffeoyl-β-phenethylamine(CaP) stimulated PG biosynthesis at a lower concentration and inhibited at a higher concentration. It has been shown that CaP inhibits cyclooxygenase and stimulates peroxidase in PG biosynthesis. o__-- and m__--Hydroxycinnamic acids were found to act as "tryptophan-like cofactors" even at a very high concentration where p__--coumaric acid significantly inhibit PG biosynthesis.

Gossypol-hypokalaemia interrelationships.

Around 1% of 8806 volunteers taking gossypol as a male contraceptive had hypokalaemic paralysis and more had simple hypokalaemia, the direct cause being renal potassium loss. In gossypol takers not showing hypokalaemia, serum potassium levels were within the normal range but were significantly lower than levels in controls. In the majority of patients suffering from gossypol-induced hypokalaemia, recovery was prompt and complete following potassium repletion, but in some men there were recurrent attacks of hypokalaemia during a period of several months to years after cessation of gossypol treatment. The incidence of hypokalaemic paralysis in gossypol takers showed distinct regional differences, being much higher in Nanjing, where the dietary potassium level of the inhabitants was low, than in Taian, where the dietary potassium level was high. In rats fed a low-K fodder, gossypol reduced the intracellular Mg and K concentrations of the skeletal muscle, while in regularly fed rats, this effect of gossypol was not observed. A potassium deficient diet could thus be considered a contributing factor in the development of gossypol-induced hypokalaemia. Potassium deficiency has also been shown to enhance the anti-spermatogenic effect of gossypol. Suggested mechanisms for the development of gossypol-induced hypokalaemia include inhibition of Na-K-ATPase activity, stimulation of prostaglandin biosynthesis, damage to the renal tubule, and modification of membrane transport.

Endogenous stimulant of prostaglandin endoperoxide synthase activity in human amniotic fluid

In this study we describe the discovery and characterization of a substance in human amniotic fluid that stimulates prostaglandin biosynthesis by a microsome-enriched preparation of bovine seminal vesicles. The stimulatory activity is not retained substantially upon anisotropic ultrafiltration through a filter with a molecular weight exclusion limit of 500. Stimulation of prostaglandin biosynthesis by this substance is time- and concentration-dependent; maximal stimulation of approx. 200% being observed within 20 min of commencing incubation with 1 mi-equivalent of stimulant fraction. Stimulatory activity is demonstrable both in the presence of reduced glutathione (1.3 mM) and l-tryptophan (20 mM), either separately or combined, and in the presence of exogenous arachidonic acid (5–120 μM). In the absence of added cofactors, the stimulatory substance increases the rates of biosynthesis of prostaglandin E 2 and prostaglandin F 2α to equal extents. The amount of stimulatory substance added to incubations is correlated positively with increased oxygen consumption during incubations. The stimulatory substance is stable to heating at 100°C for 10 min but is inactivated substantially (to less than 20% of original activity) by treatment with pronase. It is concluded that human amniotic fluid contains a substance of relatively low molecular weight, which is proteinaceous in character, that stimulates prostaglandin endoperoxide synthase activity.

Stimulation of Prostaglandin Biosynthesis by Urine or the Human Fetus May Serve as a Trigger for Parturition

Stimulation of Prostaglandin Biosynthesis by Urine or the Human Fetus May Serve as a Trigger for Parturition

Stimulation of prostaglandin biosynthesis by urine of the human fetus may serve as a trigger for parturition.

Urine of the human fetus stimulated prostaglandin biosynthesis in vitro by increasing the conversion of arachidonic acid into prostaglandins. The stimulatory activity in urine from fetuses delivered at term after labor of spontaneous onset was greater than that in urine from fetuses delivered by cesarean section at term before the onset of labor. Such stimulation of prostaglandin biosynthesis by the fetal membranes, by way of a substance released into the urine and thence into amniotic fluid, could serve as a signal for the initiation of parturition.

Stimulants of prostaglandin biosynthesis in human fetal membranes, uterine decidua vera and placenta

Modulation of prostaglandin (PG) biosynthesis by cytosolic fractions derived from homogenates of human amnion, chorion laeve, decidua vera and placenta was examined. PGF 2α and 6-oxo-PGF 1α synthesis by bovine seminal vesicle (BSV) PG synthase was stimulated by the cytosolic fractions of each tissue in a dose-dependent manner. The cytosols from decidua vera and placenta were the most effective in stimulating synthesis and also stimulated PGE 2 biosynthesis. Reduced glutathione (GSH) acted to increase the biosynthesis of PGE 2 at the expense of other PGs both in the presence and absence of various cytosols. These data are indicative that the mode of action of cytosolic fractions on the stimulation of PG biosynthesis is unlike that of GSH. Indomethacin and aspirin, inhibitors of fatty acid cyclooxygenase activity, strongly inhibited the cytosol-induced stimulation of BSV PG synthase. The cytosolic factors that stimulated PG biosynthesis exhibited differential behavior towards boiling and dialysis. The stimulatory effect of all cytosolic fractions was sensitive to boiling except in the case of chorion leave effects toward 6-oxo-PGF 1α production. In dialysis studies we found that the cytosolic components that stimulated the production of PGF 2α were not removed by dialysis except in the case of cytosol of placenta whereas the stimulatory effects of various cytosols toward the biosynthesis of PGE 2 and 6-oxo-PGF 1α were removed by dialysis. These results are indicative of the presence of endogenous factors in human intrauterine tissues that preferentially stimulate the biosynthesis of PGF 2α and 6-oxo-PGF 1α and are further suggestive that PC biosynthesis in intrauterine tissues is, at least in part, regulated by cytosolic factors.

Stimulation of prostaglandin biosynthesis by cytoplasmic factors in human intra-uterine tissues

Stimulation of prostaglandin biosynthesis by cytoplasmic factors in human intra-uterine tissues