Follicle size-dependent induction of prostaglandin G/H synthase-2 during superovulation in cattle

Under physiological conditions, prostaglandin G/H synthase-2 (PGHS-2) is induced in bovine preovulatory follicles by the endogenous surge of gonadotropins. To characterize the pattern of follicular PGHS-2 expression during superovulation in cattle, heifers were treated with exogenous FSH and ovulation was induced with hCG. Animals were ovariectomized 0, 18, and 24 h post-hCG, and extracts of follicles > or = 6 mm were analyzed by Western blotting. Follicular fluid concentrations of prostaglandin (PG) E2, PGF2alpha, progesterone, and estradiol-17beta were determined by RIAs, and the morphology of the cumulus oocyte complex was examined. Results showed that PGHS-2 protein was absent in all follicles isolated at 0 h post-hCG (n = 119) and in small follicles (6 to < 8 mm) isolated between 0 and 24 h post-hCG (n = 27 follicles). In contrast, 12.3% of medium (8 to < 10 mm) and 43.7% of large (> or = 10 mm) follicles were PGHS-2-positive at 18 h post-hCG, and these percentages rose at 24 h to 45.9% and 91.0% in medium and large follicles, respectively (p < 0.05). Follicular fluid concentrations of PGE2 and PGF2alpha were low in follicles isolated at 0 h and increased only in PGHS-2-positive follicles isolated 24 h post-hCG (p < 0.05). Concentrations of progesterone and estradiol-17beta at 0 h were 28.2 +/- 5.8 and 291.8 +/- 13.0 ng/ml, respectively, and a shift from estradiol-17beta to progesterone dominance (luteinization) occurred at 24 h post-hCG only in PGHS-2-positive follicles. Also, expansion of the cumulus oocyte complex was detected at 24 h post-hCG only in PGHS-2-positive follicles. Lack of PGHS-2 induction in follicles of ovulatory size (> 8 mm) was associated with an apparent failure to respond to hCG (absence of luteinization and cumulus expansion). Collectively, these results demonstrate the presence of a time- and follicle size-dependent induction of PGHS-2 in bovine follicles during superovulatory treatment and suggest that PGHS-2 expression can be used as a marker for follicular commitment to ovulation during ovarian hyperstimulation protocols.     

Dexamethasone induces an increase in intracellular and membrane-associated lipocortin-1 (annexin-1) in rat astrocyte primary cultures

1. The antiinflammatory actions of glucocorticoid steroids are thought to occur through induction of the protein lipocortin-1 (LC-1; annexin-1). The purpose of the current study was to investigate whether astrocytic LC-1 content was increased in the presence of a synthetic glucocorticoid, dexamethasone. 2. Steroid-induced changes in cellular levels of LC-1 in astrocytes were determined by electrotransfer and immunoblotting techniques. Separate cell fractions were investigated to study the influence of dexamethasone on astroglial LC-1 content. The effect of culture state on LC-1 expression was also examined. 3. Intracellular LC-1 content was found to decrease after initiation of culture, with a substantial rise in both cell proliferation and LC-1 expression occurring after the replenishment of medium containing steroid-free serum. A further increase in intracellular LC-1 occurred upon incubation with dexamethasone. The glucocorticoid-induced change in intracellular LC-1 was a time-dependent event and coincided with an increase in membrane-associated LC-1. 4. The findings in this study indicate that astrocytic LC-1 content is influenced by cell culture conditions and, in the presence of glucocorticoid steroids, the cellular localization of LC-1 is altered. This may indicate that LC-1 has functions at more than one cellular locality.     

Induction by interleukin-1beta peptide of prostaglandin E2 formation via enhanced prostaglandin H synthase-2 expression in 3T6 fibroblasts

Several synthetic interleukin-1 (IL-1) peptides were tested in vivo for pyrogenic activity and in vivo for their ability to stimulate prostaglandin production. Only the IL-1beta fragment (208-240) enhanced body temperature, although both IL-1beta (208-240) and IL-1alpha (223-250) stimulated prostaglandin E2 (PGE2) production in vitro. We report here that the IL-1beta fragment (208-240) did not have the capacity to induce arachidonic acid (AA) mobilization by 3T6 fibroblasts. However, this peptide was able to increase the expression of the inducible prostaglandin H synthase isoform (PGHS-2; EC 1.14.99.1.), which is related to its ability to stimulate prostaglandin E2 synthesis.     

Comparison of structural stabilities of prostaglandin H synthase-1 and -2

There are two known isoforms of prostaglandin H synthase (PGHS), a key enzyme in the conversion of arachidonic acid to bioactive prostanoids. The "constitutive" isoform, PGHS-1, is thought to have housekeeping functions, and the "inducible" isoform, PGHS-2, has been implicated in cellular responses to cytokines. The two isoforms have high sequence conservation in the cyclooxygenase active site and quite similar crystallographic structures, but differ markedly in their interactions with many cyclooxygenase substrates and inhibitors. We have evaluated the stability of the overall folding, and of the active sites of ovine PGHS-1 and human PGHS-2 using denaturation with guanidinium hydrochloride (GdmHCl). Changes in hydrodynamic and cross-linking properties indicated a dimer --> monomer transition for both isoforms between 0.5 and 2 M GdmHCl; the monomers unfolded at higher GdmHCl levels. Changes in overall secondary and tertiary structure, measured by tryptophan fluorescence and circular dichroism, occurred in two phases for each isoform, with the transition between the phases at 0.2-0.5 M GdmHCl. Disruption of active site functions (cyclooxygenase, peroxidase, and cyclooxygenase inhibitor binding activities) began at GdmHCl levels below 0.2 M. The structural and functional changes were completely reversible up to about 2 M GdmHCl, they were more pronounced at lower protein levels, and they required lower GdmHCl levels for PGHS-2 than for PGHS-1. The results are consistent with a four-state denaturation process for both isoforms: native dimers --> inactive dimers --> compact monomers --> unfolded monomers. The first two steps are reversible for both isoforms; PGHS-2 undergoes the first and last steps more readily than PGHS-1. Thus, the structural stability of PGHS-2, both in the active site regions and in the subunits overall, is distinctly less than that of PGHS-1. These differences in structural stability may contribute to the isoforms' active site ligand selectivity.     

Lipopolysaccharide induces prostaglandin H synthase-2 protein and mRNA in human alveolar macrophages and blood monocytes

We and others have previously demonstrated that human alveolar macrophages produce more PGE2 in response to lipopolysaccharide (LPS) than do blood monocytes. We hypothesized that this observation was due to a greater increase in prostaglandin H synthase-2 (PGHS-2) enzyme mass in the macrophage compared to the monocyte. To evaluate this hypothesis, alveolar macrophages and blood monocytes were obtained from healthy nonsmoking volunteers. The cells were cultured in the presence of 0 to 10 micrograms/ml LPS. LPS induced the synthesis of large amounts of a new 75-kD protein in human alveolar macrophages, and a lesser amount in monocytes. Synthesis of this protein required more than 6 h and peaked in 24 to 48 h; the protein reacted with an anti-PGHS-2 antibody prepared against mouse PGHS-2. Associated with synthesis of the protein was a marked increase in LPS-stimulated and arachidonic acid-stimulated synthesis of PGE2 by alveolar macrophages compared to monocytes. Cells not exposed to LPS contained only PGHS-1 and synthesized very little PGE2 during culture or in response to exogenous arachidonic acid. An LPS-induced mRNA, which hybridized to a human cDNA probe for PGHS-2 mRNA, was produced in parallel with production of this new protein and was produced in much greater amounts by alveolar macrophages compared to blood monocytes. This mRNA was not detectable in cells not exposed to LPS. In contrast, both types of cells contain mRNA, which hybridizes to a cDNA probe for PGHS-1. This mRNA did not increase in response to LPS. LPS also had no effect on PGHS-1 protein. These data demonstrate that PGE2 synthesis in human alveolar macrophages and blood monocytes correlates to the mass of PGHS-2 in the cell. We conclude that the greater ability of the macrophage to synthesize PGE2 in response to LPS is due to greater synthesis of PGHS-2 by the macrophage.     

Activation of heterocyclic amines by combinations of prostaglandin H synthase-1 and -2 with N-acetyltransferase 1 and 2

Gender asymmetry in touch in U.S. populations is related to the age of the participants in some studies and to the relationships between the participants in others. In the present study, researchers observed dyads in public settings in the United States frequented by couples and recorded the occurrence of touch, the touch initiator, and the body areas touched. The researchers then approached the couples and asked them to complete questionnaires indicating their ages, their relationship, and their level of agreement on major issues. Age and relationship were predictive of the gender of touch initiators. Although levels of agreement were less predictive of touch initiation, the women indicated higher levels of agreement than the men did. The results were generally consistent with a model of sex differences in reproductive strategies.     

Prostaglandin synthase-1 and prostaglandin synthase-2 are coupled to distinct phospholipases for the generation of prostaglandin D2 in activated mast cells

Aggregation of IgE cell surface receptors on MMC-34 cells, a murine mast cell line, induces the synthesis and secretion of prostaglandin D2 (PGD2). Synthesis and secretion of PGD2 in activated MMC-34 cells occurs in two stages, an early phase that is complete within 30 min after activation and a late phase that reaches a maximum about 6 h after activation. The early and late phases of PGD2 generation are mediated by prostaglandin synthase 1 (PGS1) and prostaglandin synthase 2 (PGS2), respectively. Arachidonic acid, the substrate for both PGS1 and PGS2, is released from membrane phospholipids by the activation of phospholipases. We now demonstrate that in activated mast cells (i) secretory phospholipase A2 (PLA2) mediates the release of arachidonic acid for early, PGS1-dependent synthesis of PGD2; (ii) secretory PLA2 does not play a role in the late, PGS2-dependent synthesis of PGD2; (iii) cytoplasmic PLA2 mediates the release of arachidonic acid for late, PGS2-dependent synthesis of PGD2; and (iv) a cytoplasmic PLA2-dependent step precedes secretory PLA2 activation and is necessary for optimal PGD2 production by the secretory PLA2/PGS1-dependent early pathway.     

The late induction of prostaglandin G/H synthase-2 in equine preovulatory follicles supports its role as a determinant of the ovulatory process

PGs are important mediators of the ovulatory process and prostaglandin G/H synthase-2 (PGHS-2) is a key rate-limiting enzyme in the PG biosynthetic pathway. To determine whether PGHS-2 is regulated in equine follicles before ovulation and, if so, to characterize its time course of induction, preovulatory follicles were isolated during estrus, 0, 12, 24, 30, 33, 36, and 39 h after an ovulatory dose of hCG (n = 5 follicles/time point). Cellular extracts were obtained from preparations of follicle wall (theca interna with attached granulosa cells), isolated granulosa cells, and theca interna and were analyzed by Western blot using specific anti-PGHS antibodies. Immunohistochemistry was used to characterize the in situ localization of PGHS-2 protein in preovulatory follicles, and follicular fluid concentrations of PGE2 and PGF were determined. The results showed the induction of PGHS-2, but not PGHS-1, in equine follicles before ovulation. The PGHS-2 protein (72,000 mol wt) was undetectable 0, 12, and 24 h post-hCG, first became apparent at 30 h, and reached maximal levels 39 h after hCG treatment. The induction of follicular PGHS-2 was localized exclusively in granulosa cells, and a pronounced staining was observed in the perinuclear region. Follicular fluid concentrations of PGE2 and PGF were low and not different between 0-33 h, but levels were increased at 36 and 39 h post-hCG (P < 0.01). Thus, the time course of PGHS-2 induction in equine follicles (30 h post-hCG) is clearly distinct from those previously observed in rat (4 h post-hCG) and bovine (18 h post-hCG) preovulatory follicles. Interestingly, in all three species, the interval from PGHS-2 induction to follicular rupture is highly conserved (approximately 10 h). Therefore, the progressively delayed expression of PGHS-2 in species with longer ovulatory processes supports its role as a molecular determinant of the species-specific length of the ovulatory process.     

Interleukin-1 induces slow-wave sleep at the prostaglandin D2-sensitive sleep-promoting zone in the rat brain

To determine the site of action of the sleep-promoting effect of interleukin-1 (IL-1), we continuously infused (between 11 P.M. and 5 A.M.) murine recombinant IL-1beta into seven different locations in the ventricular and subarachnoid systems of the brain in freely moving rats. When IL-1 was infused at 10 ng/6 hr into the subarachnoid space underlying the ventral surface of the rostral basal forebrain, which previously was defined as the "prostaglandin (PG) D2-sensitive sleep-promoting zone" (PGD2-SZ), the total amount of slow-wave sleep (SWS) increased by 110.7 min (IL-1 was 208.1 +/- 14.3 min vs control at 97.4 +/- 9.3 min; n = 8; p < 0.01 by paired Student's t test) from the baseline control level obtained under continuous infusion of saline vehicle. The hourly SWS during the infusion period reached the level of daytime SWS, the physiological maximum, whereas paradoxical sleep (PS) was decreased transiently. This site of action for the SWS promotion was dissociated from the site in the third ventricle sensitive to the IL-1-mediated PS suppression, fever, and anorexia. The SWS increase caused by IL-1 infusion into the PGD2-SZ was blocked completely by coadministered diclofenac, a nonselective cyclooxygenase (COX) inhibitor. Pretreatment of rats with NS-398 or piroxicam (3 mg/kg of body weight, i.p.), which are said, respectively, to possess high and relative specificity for the COX-2 enzyme, also blocked the SWS-promoting effect of IL-1. We present a hypothesis that IL-1 induces SWS, at least in part, via COX-2-mediated PG production in the PGD2-SZ.     

Interleukin-1 beta modulates prostaglandin and progesterone production by primate luteal cells in vitro

Increasing evidence suggests that cytokine products of the immune system may play a regulatory role in corpus luteum regulation in several species. The role of cytokines in primate luteal function, however, remains unclear. In the present study we examined the effects of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and interferon-gamma (IFN-gamma) on progesterone and prostaglandin (PGE2, PGF2 alpha) production by primate luteal cells in vitro. Specifically, corpora lutea were removed from normally cycling cynomolgus monkeys (n = 30 corpora lutea) during either the early (Days 3-5 after the estimated LH surge), mid (Days 8-10), or late (Days 12-14) luteal phase of the menstrual cycle. The corpora lutea were dispersed into individual cells using collagenase, DNase, and hyaluronidase. Approximately 50,000 viable luteal cells per tube were incubated in Ham's F-10 medium with increasing concentrations of IL-1 beta (0.1-10 ng/ml), TNF alpha (1-100 ng/ml), or IFN-gamma (10-1000 U/ml) in the presence and absence of hCG for 8 h at 37 degrees C. TNF alpha and IFN-gamma had no effect on progesterone PGE2, or PGF2 alpha production during any phase of the cycle at the doses tested. In contrast, IL-1 beta significantly stimulated PGF2 alpha production in a dose-dependent manner during the mid and late luteal phases (p < 0.05). Human CG alone had no effect on PGE2 or PGF2 alpha production by dispersed luteal cells in vitro but inhibited IL-1 beta-stimulated PGF2 alpha production. As expected, hCG stimulated progesterone production by primate luteal cells in vitro. Interestingly, IL-1 beta inhibited this hCG stimulation of progesterone production. In summary, these date suggest that IL-1 beta is a potentially important modulator of prostaglandin production by the primate corpus luteum. In view of this, cytokine-mediated changes in prostaglandin production by the primate corpus luteum may participate in the physiological regulation of luteal function.     

Pharmacological profile of JTE-522, a novel prostaglandin H synthase-2 inhibitor, in rats

OBJECTIVE AND DESIGN: The antinociceptive, antipyretic, and anti-inflammatory effects of JTE-522, a novel selective prostaglandin H synthase (PGHS)-2 inhibitor, were examined in rats. MATERIALS: Sheep seminal vesicle PGHS-1 and placenta PGHS-2 were used for in vitro assay, while for in vivo experiments, male rats (4-8 weeks old) were used. TREATMENT: JTE-522 and reference compounds (0.01-100 microM) were subjected to enzyme assay. JTE-522 (0.3-30 mg/kg) and indomethacin (0.3-10 mg/kg) were administered orally. RESULTS: JTE-522 inhibited PGHS-2 (IC50: 0.64 microM) without affecting PGHS-1 activity at 100 microM. In rats with yeast-induced hyperalgesia, JTE-522 showed a dose-dependent antinociceptive effect (ED50: 4.4 mg/kg). In rats with yeast-induced pyrexia, JTE-522 significantly reversed the pyrexic response (ED50: 3.9 mg/kg). Orally administered JTE-522 dose-dependently inhibited carrageenin-induced rat paw edema (ED30: 4.7 mg/kg). In rats with adjuvant-induced arthritis, JTE-522 showed a significant inhibitory effect at daily doses of 0.3-3 mg/kg. JTE-522 did not cause severe gastric lesions at oral doses up to 300 mg/kg. CONCLUSIONS: Our results indicate that the selective PGHS-2 inhibitor JTE-522 may represent a novel type of anti-inflammatory drug without adverse effects on the gastrointestinal tract. JTE-522 may thus be a promising agent for treating both acute inflammatory disease and chronic inflammatory diseases such as rheumatoid arthritis.     

Interleukin-1 beta enhances interleukin-1 receptor antagonist content in human somatotroph adenoma cell cultures

In addition to the well-known modulation of immune and inflammatory responses, the interleukin-1 (IL-1) system has been shown to be involved in the regulation of anterior pituitary hormone secretion and growth. We previously demonstrated that IL-1 receptor antagonist (IL-1ra) is expressed in human pituitary adenomas cultured in vitro. In the present study, we investigated the regulation of IL-1ra protein by IL-1 beta (1-100 U/mL) in human somatotroph adenomas (n = 9) cultured for 12-48 h. IL-1 beta significantly enhanced the concentration of IL-1ra dose dependently in the somatotroph adenoma cell lysates, whereas IL-1ra concentrations remained unchanged in the culture supernatants. Furthermore, basal IL-1ra concentrations were significantly higher in the cell lysates compared with the corresponding culture supernatants. The regulation of IL-1ra in somatotroph adenoma cells is different from human cultured monocytes, in which IL-1 beta significantly stimulated IL-1ra secretion into the culture supernatants, and no change of intracellular IL-1ra content was observed. Incubation of the somatotroph adenoma cells with 100 U/mL IL-1 beta did not result in a change of GH concentrations in the culture supernatants. Enhancement of intracellular IL-1ra protein by IL-1 beta may represent a mechanism intrinsic to somatotroph adenoma cells to counterregulate the response to IL-1 beta on hormone secretion or cellular growth.     

The interaction of arginine 106 of human prostaglandin G/H synthase-2 with inhibitors is not a universal component of inhibition mediated by nonsteroidal anti-inflammatory drugs

The three-dimensional cocrystal structures of ovine prostaglandin G/H synthase-1 (PGHS-1) with S-flurbiprofen and murine PGHS-2 with S-flurbiprofen and indomethacin reveal that the carboxylate acid groups of these nonsteroidal anti-inflammatory drugs (NSAIDs) form a salt bridge with the guanidinium group of Arg120 in PGHS-1 and Arg106 in PGHS-2. Mutagenesis studies confirmed that the Arg120 residue of PGHS-1 is critical for binding of substrate and inhibitors through ionic interactions of its guanidinium group with the carboxylate moieties of arachidonic acid and certain NSAIDs. We report here that the analogous R106E substitution in human PGHS-2 results in a catalytically active enzyme with a 30-fold higher Km value for arachidonic acid. Comparison of the inhibition of hPGHS-2(R106E) with wild-type hPGHS-2 by 11 structurally diverse selective and nonselective PGHS inhibitors revealed a 0-1000-fold decrease in inhibitory potency on the mutant enzyme. The loss of inhibitory potency of NSAIDs on hPGHS-2(R106E) could not be correlated with the presence or absence of a carboxylate functional group in the inhibitor, as was demonstrated previously for the PGHS-1(R120E) mutant, or with the selective or nonselective nature of the PGHS inhibitor. The decreases in the inhibitory potencies on hPGHS-2(R106E) by the carboxylate-containing NSAIDs flurbiprofen, indomethacin, meclofenamic acid, and diclofenac on hPGHS-2(R106E) were 965-, 48-, 5.5-, and 4.5-fold, respectively. The nonuniversal requirement for interaction of the carboxylate group of certain NSAIDs with the Arg106 residue in hPGHS-2 is supported by the observation that the methyl ester derivative of indomethacin was a more potent inhibitor than indomethacin on both hPGHS-2 and hPGHS-2(R106E). The greatest loss of potency for inhibition of hPGHS-2(R106E) was observed with the hPGHS-2-selective sulfonamide-containing inhibitors NS-398 and flosulide. The PGHS-2-selective inhibitor DuP697 and a desbromo-sulfonamide analogue of DuP697 displayed equivalent potency on hPGHS-2(R106E) and hPGHS-2. The change in inhibitory potency of NS-398 on hPGHS-2(R106E) was due to a difference in the kinetics of inhibition, with NS-398 displaying time-dependent inhibition of hPGHS-2 but time-independent inhibition of PGHS-2(R106E). The time-dependent inhibition of hPGHS-2 by DuP697 was not affected by the presence of the R106E mutation. We conclude that the Arg106 residue of hPGHS-2 is involved in binding arachidonic acid and certain NSAIDs, but interactions with Arg106 are not a universal requirement for inhibition by either carboxylate-containing NSAIDs or PGHS-2-selective inhibitors.     

Regulation of prostaglandin H synthase-2 expression in cerebromicrovascular smooth muscle by serum and epidermal growth factor

Growth factors may play a role in the formation of prostaglandins (PG) by cerebral blood vessels during development or reaction to injury. In smooth muscle cultures isolated from murine cerebral microvessels PG production was induced with either serum or epidermal growth factor (EGF). Prostaglandin H synthase (PGHS) activity peaked at 6 h after the addition of 10% serum or 50 ng/ml EGF. Increases in expression of PGHS-1 mRNA were small (7- to 10-fold) compared with PGHS-2 (30- to 120-fold), and the induction patterns were different for serum and EGF. An increase in PGHS-2 message was detected by 0.5 h of adding either agent, but peak induction occurred earlier for EGF than for serum, 1 h vs. 3 h, respectively. The response to either stimulus had returned to prestimulation levels by 12 h. The induction of PGHS-2 protein was also transient, but followed a more delayed time course (peak levels at 6 h). Induction of activity, message, and protein by either agent was blocked by 1 microM dexamethasone and attenuated by genistein (100 microM), a nonspecific tyrosine kinase inhibitor. Tyrphostin 47, a more selective EGF receptor tyrosine kinase inhibitor, dose-dependently inhibited EGF-stimulated PGHS activity, completely abolishing PG production at 100 microM. However, this inhibitor had no effect on serum-stimulated PG production. Curiously, 100 microM tyrphostin 47 enhanced EGF-induced PGHS-2 mRNA and protein expression. These data suggest that EGF induces the expression of PGHS-2 in cerebromicrovascular smooth muscle by a mechanism that requires tyrosine kinase activity and that is distinct from serum.