Prostaglandin production in cultured gastric mucosal cells: Role of cAMP on its modulation

The effect of cAMP on prostaglandin production may depend on cell types. To clarify the relationship between PG and cAMP, we examined arachidonate's effects on PG synthesis and intracellular cAMP accumulation in monolayers of rat gastric mucosal cells. These cells produced PGE₂, PGI₂ and thromboxaneA₂ (TXA₂) in amounts of 316±18, 100±7 and 30±5 pg per 10⁵ cells in 10 min, respectively, in response to 10μM arachidonic acid (AA). The production of these PG, however, leveled off subsequently. Cells initially exposed to AA responded poorly to a subsequent stimulation by AA. AA simultaneously stimulated intracellular cAMP accumulation; this stimulatory effect on cAMP production was abolished by the pretreatment with indomethacin. Nevertheless, the pretreatments with dibutyryl cAMP (0.1–5mM) did not alter the amount of subsequent AA-induced PGE₂ production. Furthermore, the preincubation with 1mM isobutyl methyl xanthine also failed to affect PGE₂ synthesis, while it increased intracellular cAMP accumulation. Our studies suggest (1) AA stimulates intracellular cAMP formation in cultured gastric mucosal cells, linked with conversion of AA to cyclooxygenase metabolites, (2) AA-induced PG production is limited in these cells, and (3) it seems, however, unlikely that intracellular cAMP modulates AA metabolism to PG.     

Regulation of prostaglandin production in cultured gastric mucosal cells

The aims of this study were to investigate whether exogenous prostaglandin modulates prostaglandin biosynthesis by cultured gastric mucosal cells, and to clarify the role of cyclic nucleotides in the possible modulation of prostaglandin production. After pretreatment for 30 min with buffer alone (control) or 1 to 100ng/ml PGE2, cells were incubated with 4 uM arachidonic acid for 30 min. Pretreatments with greater than 5ng/ml PGE2 inhibited arachidonate-induced PGE2 and PGI2 production in a dose-dependent fashion, as compared with control, with inhibition by 64 +/- 8% and 75 +/- 4% respectively, at 100ng/ml PGE2. PGE2, at 100ng/ml, significantly increased intracellular cAMP accumulation, but pretreatment with dibutyryl cAMP (0.01-mM) did not alter the amounts of arachidonate-induced PGE2 production. Furthermore, while greater than 10ng/ml PGE2 increased cGMP production dose-dependently, preincubation with dibutyryl cGMP (0.001-0.1mM) also failed to affect PGE2 synthesis significantly. In addition, pretreatment with isobutyl-methyl-xanthine, while increasing accumulation of cellular cyclic nucleotides, did not significantly change PGE2 production. Calcium ionophore A23187-induced PGE2 production was also inhibited by pretreatment with PGE2. These results indicate that exogenous PG inhibits subsequent arachidonate or A23187-induced PG biosynthesis in rat gastric mucosal cells, and suggest the possibility that PG regulates its own biosynthesis via feedback inhibition independent of cyclic nucleotides in these cells.     

Interrelationships among prostaglandins, vasopressin and cAMP in renal papillary collecting tubule cells in culture

To determine the influence of prostaglandins on cAMP metabolism in renal papillary collecting tubule (RPCT) cells, intracellular cAMP levels were measured after incubating cells with prostaglandins (PGs) alone or in combination with arginine vasopressin (AVP). PGE1, PGE2 and PGI2, but not PGD2 or PGF2 alpha, increased intracellular cAMP concentrations. At maximal concentrations (10(-5) M) the effects of PGE2 plus PGI2 (or PGE1), but not of PGI2 plus PGE1, were additive suggesting that at least two different PG receptors may be present in RPCT cell populations. Bradykinin treatment of RPCT cells caused an accumulation of intracellular cAMP which was blocked by aspirin and was quantitatively similar to that observed with 10(-5) M PGE2. PGs, when tested at concentrations (e.g. 10(-9) M) which had no independent effect on intracellular cAMP levels, did not inhibit the AVP-induced accumulation of intracellular cAMP in RPCT cells. These results indicate that PGs do not block AVP-induced accumulation of intracellular cAMP in RPCT cells at concentrations of PGs which have been shown to inhibit the hydroosmotic effect of AVP on perfused collecting tubule segments. However, at higher concentrations of PGs (e.g. 10(-5) M), the effects of AVP plus PGE1, PGE2, PGI2 or bradykinin on intracellular cAMP levels were not additive. Thus, under certain conditions, there is an interaction between PGs and AVP at the level of cAMP metabolism in RPCT cells.     

Multifactorial regulation of prostaglandin synthesis in preovulatory goldfish ovarian follicles.

Goldfish preovulatory ovarian follicles (prior to germinal vesicle breakdown) were utilized for studies investigating the actions of activators of different signal transduction pathways on prostaglandin (PG) production. The protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA; 100-400 nM), 1-oleoyl-2-acetylglycerol (5 and 25 micrograms/ml), and 1,2-dioctanoylglycerol (10 and 50 micrograms/ml) stimulated PGE production; the inactive phorbol 4 alpha-phorbol didecanoate, which does not activate PKC, had no effect. Calcium ionophore A23187 (0.25-4.0 microM) stimulated PGE production and acted in a synergistic manner with activators of PKC. Although produced in lower amounts than PGE, PGF was stimulated by PMA and A23187. The direct activator of phospholipase A2, melittin (0.1-1.0 microM), stimulated a dose-related increase in PGE production, whereas chloroquine (100 microM), a putative inhibitor of phospholipase A2, blocked basal and PMA + A23187-stimulated PGE production. Several drugs known to elevate intracellular levels of cAMP including the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1-1.0 mM), forskolin (10 microM), and dibutyryl cAMP (dbcAMP; 5 mM) attenuate PMA + A23187-stimulated PGE production. Melittin-stimulated production of PGE was inhibited by dbcAMP, suggesting that the action of cAMP was distal to the activation of phospholipase A2. In summary, these studies demonstrate that activation of PKC and elevation of intracellular calcium levels stimulate PG production, in part, through activation of phospholipase A2. The adenylate cyclase/cAMP signalling pathway is inhibitory to PG production by goldfish ovarian follicles.     

Regulation of human natural killing. III. Mechanism for interferon induction of loss of susceptibility to suppression by cyclic AMP elevating agents

In this study we demonstrated that human NK cells activated by IFN or poly I:C were partially resistant to suppression by PGE2, PGD2, PGA2, PGI2, dibutyryl cAMP, isoproterenol, and theophylline. This partial loss of inhibition was not due to endogenous PG production because the addition of indomethacin to cultures stimulated with IFN or poly I:C did not prevent the partial loss of sensitivity to PGE2. NK cells incubated in the presence of PGE2 overnight, however, were not sensitive to inhibition. IFN or poly I:C did not stimulate PG synthesis nor elevate intracellular cAMP levels of NK cells. On the other hand, IFN or poly I:C diminished the accumulation of intracellular cAMP levels in NK cells in response to PGE2 stimulation. Dibutyryl cAMP and theophylline suppressed the cytolytic activity of the unstimulated cells more than that of the activated cells. A possible mechanism for the IFN-induced unresponsiveness to PGE2 may be a compartmentalized loss of cAMP responsiveness. Cycloheximide, puromycin, emetine, and actinomycin D blocked NK activation by IFN and poly I:C as well as the acquisition of resistance to PGE2-mediated suppression.     

Prostaglandin E2 promotes IL-4-induced IgE and IgG1 synthesis

PG of the E series are generally known to suppress immune responses, however, we have found that PGE synergizes with IL-4 to induce IgE and IgG1 production in LPS-stimulated murine B lymphocytes. PGE2 and PGE1 (10(-6) to 10(-8) M) significantly increase IgE and IgG1 production (up to 26-fold) at all concentrations of IL-4 tested. In addition to its effects on IgE and IgG1, PGE also causes a significant decrease in IgM and IgG3 synthesis, suggesting that PGE may promote IL-4-induced class switching. The specificity of the E series PG effect is demonstrated by the fact that PGF2 alpha (10(-6) M) does not alter production of any of these isotypes. Because PGE can mediate its effects through cAMP in some cases, we investigated the importance of cAMP levels in regulation of isotype expression. Other agents that increase intracellular cAMP levels (cholera toxin and dibutyryl cAMP) were assessed for their ability to regulate isotype differentiation. Cholera toxin (100 pg/ml) and dibutyryl cAMP (100 microM) significantly enhanced IgE and IgG1 production and diminished IgM and IgG3 synthesis. We also show that PGE and cholera toxin elevate intracellular cAMP in B lymphocytes in a dose-dependent manner. In contrast, PGF2 alpha (10(-6) M) and the B subunit of cholera toxin (100 pg/ml) did not increase cAMP and did not regulate the isotype of Ig produced, reiterating the importance of cAMP in enhancing isotype differentiation. Although PGE is known to inhibit a number of immune responses, our data show that it is not always inhibitory. PGE may play a role in atopy in vivo where PGE-secreting cells such as macrophages, follicular dendritic cells, and fibroblasts can promote IgE synthesis. This research emphasizes the importance of PGE in regulation of the humoral immune response and adds a new stimulatory action to the repertoire of known PGE effects.     

Prostaglandin E2 stimulates the formation of mineralized bone nodules by a cAMP-independent mechanism in the culture of adult rat calvarial osteoblasts

The effects of prostaglandin E2 (PGE2) on the proliferation and differentiation of osteoblastic cells were studied in osteoblast-like cells isolated from adult rat calvaria. Treatment of the cells with PGE2 within the concentration range 10(-8)-10(-5) M resulted in a dose-dependent increase in alkaline phosphatase (ALP) activity, [3H]proline incorporation into collagenase-digestible protein, and mineralized bone nodule (BN) formation, as well as a dose-dependent decrease in [3H]thymidine incorporation into the cells. PGE2 also caused a dose-dependent increase in the intracellular cyclic adenosine monophosphate (cAMP) content, with a maximal effective concentration of 10(-5) M; this effect of PGE2 was mimicked by forskolin, an adenylate cyclase activator. The treatment of adult calvarial cells with forskolin decreased BN formation, ALP activity, and collagen synthesis. These results suggested that cAMP does not have a stimulatory, but rather a suppressive, effect on the differentiation of adult rat calvarial cells. A time-course study of cAMP accumulation showed that both PGE2- and forskolin-induced cAMP reached a maximum at 5 min after the treatment, but the former rapidly returned to the basal level by 40 min, while the latter declined slowly and was still at 70% of the maximal level at 60 min, suggesting that PGE2 activates phosphodiesterase as well as adenylate cyclase. The presence of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, reduced the rate of degradation of cAMP formed after PGE2 treatment, suggesting the involvement of calmodulin in the activation of phosphodiesterase. However, PGE2 also caused the production of inositol 1,4,5-triphosphate (IP3) and an elevation of the intracellular Ca2+ concentration ([Ca2+]i), both of which peaked at 15 s and returned to the basal level within 1 min. Submaximal responses of the IP3 production and the [Ca2+]i elevation to PGE2 were obtained at 10(-5) M. W-7 decreased both basal and PGE2-induced ALP activity, collagen synthesis and BN formation, indicating the involvement of Ca2+/calmodulin-dependent protein kinase in the PGE2-induced differentiation of calvarial cells. From these results, we concluded that PGE2 inhibits the proliferation and stimulates the differentiation of calvarial osteoblasts by elevating the [Ca2+]i through the activation of a phosphoinositide turnover, but not via an activation of adenylate cyclase. We also found that BN formation varies, depending on the time of PGE2 addition, suggesting that responsiveness of the cells to PGE2 may change during the culture period.     

Arachidonic acid regulation of prostanoid synthesis in macrophages

The dynamics of prostaglandin (PG) E2 synthesis by mouse peritoneal macrophages during the delivery of the basic substrate, arachidonic acid (AA), from different sources to the enzyme system of the cells was investigated. The dynamics of PGE2 synthesis in these cells was studied both after addition of exogenous AA and after stimulating the liberation of AA from intracellular pools with the calcium ionophore A23187. The kinetics of PGE2 synthesis when AA was supplied from intracellular and extracellular sources were absolutely different. PGE2 metabolism and the inactivation of the key enzyme of PG synthesis (PGH-synthase) during the reaction may be the regulating factors in the kinetics of PGE2 synthesis in the cells. For the different sources of AA in the cells, the rate constants of PGE2 consumption (k2) and PGH-synthase inactivation in the course of the reaction (kin) were calculated. The experimentally determined value of the apparent rate constant kin was identical to the theoretically calculated kin value for the case when AA was provided from an intracellular source. An observed deceleration in the PGE2 synthesis kinetics from exogenous AA is characterized by a 10-fold drop in the apparent kin and k2 values. The possibility of prostanoid synthesis regulation at the level of the traditional, constitutive isoenzyme PGH-synthase-1 is discussed.     

Up-regulation of prostaglandin E2 binding and of prostanoid release in rabbits producing antibodies against prostaglandin E2

German Giant rabbits successfully immunized against prostaglandin (PG) E2 as shown by a rise in antibody titers developed gastric mucosal lesions. Enzymatically dispersed gastric mucosal cells of these animals had a significantly enhanced production of PG E2 and PG I2 as measured by specific radioimmunoassays. This may be explained by an increased supply with endogenous arachidonic acid (as indicated by an enhanced phospholipase A2/LAT ratio) and by a higher activity of the subsequent PG forming enzymes (as indicated by a more effective stimulation of PG production by exogenous arachidonic acid). Gastric mucosal plasma membranes of immunized rabbits had significantly higher PG E2 binding capacity (108 +/- 9 fmol/mg protein) than those of nonimmunized rabbits (72 +/- 5 fmol/mg protein). The ligand affinity was not affected by immunization. Neither histamine-stimulated 14C-amino-pyrine uptake of isolated parietal cells as a marker for acid production nor its inhibition by PG E2 were influenced by receptor up-regulation. The increased eicosanoid release can be regarded as an endogenous defense mechanism against increased mucosal vulnerability caused by PG E2 scavenging. The potential role of PG E2 receptor up-regulation in support of this process remains to be established.     

Gonadotropic regulation of prostaglandin production by ovarian follicular cells of the pig

Prepubertal gilts were treated with 750 IU pregnant mare's serum gonadotropin (PMSG) and 72 h later with 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Dispersed granulosa cells (GC) and theca interna cells (TC) from follicles of gilts 72 h (GC-72 and TC-72, respectively) and 108 h (GC-108 and TC-108 h, respectively) after PMSG treatment were cultured for 0, 12, 24, and 36 h in medium with or without luteinizing hormone (LH), dibutyryl cyclic adenosine 3',5'-monophosphate [Bu)2cAMP), calcium ionophore (A23187), and/or arachidonic acid (AA), and the production of prostaglandin E2 (PGE) and prostaglandin F2 alpha (PGF) was measured by radioimmunoassay. TC-72 was the principal source of PGs 72 h after PMSG. At 108 h, the production of PGE and PGF by GC was increased 10- and 30-fold, respectively, whereas corresponding increases by TC were 2-fold. LH and A23187 significantly stimulated PGE and PGF production by both GC-72 and TC-72, but only thecal PG production was stimulated by (Bu)2cAMP. LH had minimal or no effect on PG production by GC-108 and TC-108, but A23187 (GC-108, TC-108) and (Bu)2cAMP (TC-108) were stimulatory. Basal PG production by GC-72, GC-108, and TC-108 was stimulated by AA. However, production by GC and TC cultured in medium containing AA and LH, A23187, or (Bu)2cAMP was not different from that produced by AA alone. These findings suggested that GC and TC can synthesize PGs in vitro, but AA availability is rate-limiting in GC. After exposure to hCG in vivo, the capacity of both cell types to produce PGs is increased but is limited by AA availability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycerylprostaglandin synthesis by resident peritoneal macrophages in response to a zymosan stimulus

Cyclooxygenase (COX)-2 oxygenates arachidonic acid (AA) and 2-arachidonylglycerol (2-AG) to endoperoxides, which are subsequently transformed to prostaglandins (PGs) and glycerylprostaglandins (PG-Gs). PG-G formation has not been demonstrated in intact cells treated with a physiological agonist. Resident peritoneal macrophages, which express COX-1, were pretreated with lipopolysaccharide to induce COX-2. Addition of zymosan caused release of 2-AG and production of the glyceryl esters of PGE2 and PGI2 over 60 min. The total quantity of PG-Gs (16 +/- 6 pmol/10(7) cells) was much lower than that of the corresponding PGs produced from AA (21,000 +/- 7,000 pmol/10(7) cells). The differences in PG-G and PG production were partially explained by differences in the amounts of 2-AG and AA released in response to zymosan. The selective COX-2 inhibitor, SC236, reduced PG-G and PG production by 49 and 17%, respectively, indicating a significant role for COX-1 in PG-G and especially PG synthesis. Time course studies indicated that COX-2-dependent oxygenation rapidly declined 20 min after zymosan addition. When exogenous 2-AG was added to macrophages, a substantial portion was hydrolyzed to AA and converted to PGs; 1 microm 2-AG yielded 820 +/- 200 pmol of PGs/10(7) cells and 78 +/- 41 pmol of PG-Gs/10(7) cells. SC236 reduced PG-G and PG production from exogenous 2-AG by 88 and 76%, respectively, indicating a more significant role for COX-2 in the utilization of exogenous substrate. In conclusion, lipopolysaccharide-pretreated macrophages produce PG-Gs from endogenous 2-AG during zymosan phagocytosis, but PG-G formation is limited by substrate hydrolysis and inactivation of COX-2.     

Prostaglandin inhibition of testosterone production induced by luteinizing hormone, dibutyryl cyclic AMP or 3-isobutyl-1-methyl xanthine in dispersed rat testicular interstitial cells.

Testicular interstitial cells were utilized to study the effects of prostaglandins (PG) on in vitro testosterone production and to examine the role of cyclic adenosine-3',5'-monophosphate (cAMP) in this process. Testosterone production was assessed after 3 hour incubations while cAMP accumulation was examined after a 0.5 hour incubation period. Testosterone and cAMP were measured by radioimmunoassay. None of the PGs tested (PGA, PGA2, PGB1, PGE1, PGE2, PGF1alpha PGF2alpha) altered basal testosterone production when present in incubates at concentrations of 1.3 X 10(-8) M to 1.3 X 10(-4). However, at concentrations of 1.3 X 10(-4) M all of these PGs were capable of decreasing Luteinizing Hormone (LH; 100ng)-induced testosterone production. The inhibition of LH-induced testosterone production by the B, E and F series PGs was less pronounced than that for the A series. PGA1 and PGA2 exhibited 80% and 95% inhibition, respectively, at 1.3 X 10(4) M. The inhibitory action of 4 X 10(5) M PGA1 or PGA2 was evident within 30 minutes. Preincubation of interstitial cells with indomethacin (10(-5) or 10(-6) M) for 30 minutes did not alter subsequent basal or LH (100ng)-induced testosterone production. Accumulation of cAMP was stimulated by LH (10 microgram) or by PGs (1.3 X 10(-4) M PGA1, PGA2, PGB1, PGE1 or PGF2alpha). The PG-induced cAMP accumulation thus occurred at concentrations where LH-stimulated testosterone production was inhibited. Furthermore, PGA1 and PGA2 (1.3 X 10(-4) M) inhibited testosterone production induced by either 3-isobutyl-1-methyl xanthine (MIX; 10(-4) M or 10(-3) M) or dibutyryl cAMP (dbcAMP; 10(-4) M or 10(-3) M). These results indicate that PGs can block testosterone production by a direct effect on testicular interstitial cells and suggest that PGs exert their inhibitory action distal to stimulation of cAMP formation. PGs do not appear to play a role in the mechanism of LH action.     

Binding and second messengers of prostaglandins F2 alpha and E1 in primary cultures of rabbit endometrial cells

Several factors and hormones are thought to play a role in the growth control of endometrial cells. We have shown that prostaglandin F2 alpha (PGF2 alpha) is a growth factor for primary cultures of rabbit endometrial cells grown in serum-free, chemically defined medium and that prostaglandin E1 (PGE1) antagonizes the PGF2 alpha induction of growth (Orlicky et al., 1986). [3H]PGF2 alpha binds to whole cells in a time (optimal approximately 30 min)- and temperature-dependent (optimal 37 degrees C), disassociable (90% disassociable within 30 min), saturable (Kd1 = 4.9 X 10(-8) M, n1 = 1.2 X 10(5) molecules/cell; Kd2 = 2.6 X 10(-7) M, n2 = 3.0 X 10(5) molecules/cell), and specific manner. [3H]PGE1 binds in a time-dependent (optimal 25 min), disassociable (90% disassociable within 10 min), saturable (Kd = 6.4 X 10(-8) M, n = 1.2 X 10(5) molecules/cell), and specific manner. This specific binding of [3H]PGF2 alpha and [3H]PGE1 is down-regulatable by prior treatment of the cultures with unlabeled ligand, and up-regulatable by prior treatment of the cultures with indomethacin to inhibit endogenous PG synthesis. Proteolytic enzyme treatment for 2 min reduces the specific binding of PGF2 alpha by 75%. PGE1 stimulates intracellular cAMP synthesis and accumulation in a time (optimal 10 min)- and concentration (half-maximal stimulation at 10(-6) M)-dependent manner but has no effect on intracellular cGMP. PGF2 alpha has no effect on either intracellular cAMP or cGMP in this system. We describe here for the first time the analysis at a biochemical level of the interaction between two prostaglandins, antagonistic to each other in terms of growth regulation.     

Differential effects of prostaglandin F2 alpha and of prostaglandins E1 and E2 on cyclic 3',5'-monophosphate production and intracellular calcium mobilization in avian uterine smooth muscle cells

The effects of prostaglandins (PGs) E1 (PGE1), E2 (PGE2) and F2 alpha (PGF2 alpha) on cyclic 3',5'-adenosine monophosphate (cAMP) production and intracellular Ca mobilization were examined in smooth muscle cells of chicken uterus grown in primary culture. At subnanomolar concentrations, both PGE1 and PGE2 significantly suppressed cAMP levels. However, at higher concentrations (0.1-100 microM), both agonists caused a dose-related increase in cAMP production. PGF2 alpha, on the other hand, had no effect on cAMP production. Forskolin (1-100 microM), which also stimulated cAMP production in a dose-dependent fashion, potentiated the effects of both PGE1 and PGE2. In digitonin-permeabilized uterine cells preloaded with 45Ca2+, the addition of PGF2 alpha caused a biphasic 45Ca2+ efflux. There was a small but significant 45Ca2+ release (10.0 +/- 1.5%) within 30 s (rapid phase), followed by a larger one (32.0 +/- 2.0%) within 5 min (slow phase). PGE2, at doses above 1 nM (which significantly increased cAMP accumulation), promoted 45Ca2+ sequestration. This action of PGE2 was observed as early as 1 min and was complete by 5 min. In addition, 0.001 nM PGE2 (a dose that was ineffective on 45Ca2+ mobilization) enhanced PGF2 alpha-induced 45Ca2+ mobilization from 22.5 +/- 5% to 57.0 +/- 3.5%. These results show that PGs of the E series have distinctly different effects on cAMP production and intracellular Ca mobilization. PGF2 alpha action may be linked directly to intracellular Ca mobilization, whereas the effects of PGE may be exerted at multiple sites depending on its local concentration. At low concentrations, its action may be mediated by the suppression of cAMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for 6-keto-PGF1 alpha in adrenal cortex of the rat and effects of 6-keto-PGF1 alpha and PGI2 on adrenal cAMP levels and steroidogenesis.

Both intact cortical tissue and isolated cortical cells from the adrenal gland of the rat were analyzed for 6-keto-PGF1 alpha, the hydrolysis metabolite of PGI2, using high-performance liquid chromatography and gas chromatography-mass spectrometry. 6-Keto-PGF1 alpha was present in both incubations of intact tissue and isolated cells of the adrenal cortex, at higher concentrations than either PGF2 alpha or PGE2. Thus, the cortex does not depend upon vascular components for the synthesis of the PGI2 metabolite. Studies in vitro, using isolated cortical cells exposed to 6-keto-PGF1 alpha (10(-6)-10(-4)M), show that this PG does not alter cAMP levels or steroidogenesis. Cells exposed to PGI2 (10(-6)-10(-4)M), however, show a concentration-dependent increase of up to 4-fold in the levels of cAMP without altering cortico-sterone production, ACTH (5-200 microU/ml) increased cAMP levels up to 14-fold, and corticosterone levels up to 6-fold, in isolated cells. ACTH plus PGI2 produced an additive increase in levels of cAMP, however, the steroidogenic response was equal to that elicited by ACTH alone. Adrenal glands of the rat perfused in situ with PGI2 showed a small decrease in corticosterone production, whereas ACTH greatly stimulated steroid release. Thus, while 6-keto-PGF1 alpha is present in the rat adrenal cortex, its precursor, PGI2, is not a steroidogenic agent in this tissue although it does stimulate the accumulation of cAMP.     

Prostaglandin E2 potentiates granulocyte-macrophage colony stimulating factor-induced histamine synthesis in bone marrow cells: role of cAMP.

Histamine synthesis in response to Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) by murine hematopoietic cells is strikingly potentiated by prostaglandin E2 (PGE2). This synergy is mediated by an increase in intracellular adenosine 3':5'-cyclic monophosphate (cAMP), since: (a) exogeneous and endogeneous cAMP generated either by forskolin or IBMX potentiate GM-CSF-induced histamine synthesis, (b) the maximal potentiating effects of PGE2 and cAMP are not cumulative, and (c) GM-CSF together with PGE2 enhances intracellular cAMP content in a bone marrow population enriched for GM-CSF target cells. cAMP and PGE2 enhance histidine decarboxylase activity induced by GM-CSF showing that both factors act on histamine synthesis rather than on its release. Conversely, histamine synthesis promoted by Interleukin 3 (IL-3), the unique cytokine sharing this property with GM-CSF, is not modulated by PGE2 or cAMP, suggesting two distinct mechanisms for the induction of this biological activity in hematopoietic cells.     

Effects of prostaglandin E2 and F2 alpha on cytoplasmic pH in a clonal osteoblast-like cell line, MOB 3-4.

Prostaglandin E2 (PGE2, 5 ng/ml to 5 micrograms/ml) induced a dose-dependent increase in cAMP accumulation, inositol phosphates (IPs) accumulation, and cytoplasmic free Ca2+ ([Ca2+]i) in a clonal osteoblast-like cell line, MOB 3-4. In contrast, prostaglandin F2 alpha (PGF2 alpha, 5 ng/ml to 5 micrograms/ml) stimulated increases in IPs accumulation and [Ca2+]i without stimulating an increase in cAMP accumulation. Both PGE2 (greater than 0.5 micrograms/ml) and PGF2 alpha (greater than or equal to 5 micrograms/ml) increased cytoplasmic pH (pHi) from approximately 7.15 to 7.35 in BCECF-loaded cells. A tumor promotor, phorbol 12-myristate 13-acetate (PMA, 0.1-100 nM) also increased pHi without effect on phosphoinositide hydrolysis. Both PGE2-(5 micrograms/ml) and PMA- (100 nM) induced cytoplasmic alkalinization was inhibited by removal of extracellular Na+, or by pretreatment of the cells with amiloride (0.5 mM), an inhibitor of Na+/H+ exchange, or H-7 (100 microM), a nonspecific inhibitor of protein kinase C. Thus, MOB 3-4 cells appeared to possess PGE2 receptors and PGF2 alpha receptors: the former are coupled to adenylate cyclase and phospholipase C, and the latter are predominantly coupled to phospholipase C. Also the cells appeared to possess an amiloride-sensitive Na+/H+ exchange activity, which increases pHi in response to PGE2 and PGF2 alpha, as well as to PMA. Long-term (48 hr) exposure of the cells to PGE2 at a high concentration (5 micrograms/ml), but not to PGF2 alpha and PMA, decreased DNA synthesis in the serum-deficient medium. Thus, cytoplasmic alkalinization appeared insufficient for cell replication. At least in MOB 3-4 cells, the inhibitory effect of PGE2 on DNA synthesis may be due to the cAMP messenger system.     

Role of Ca(2+)-independent phospholipase A(2) and cyclooxygenase/lipoxygenase pathways in the nitric oxide production by murine macrophages stimulated by lipopolysaccharides.

There is evidence of molecular cross talk between inflammatory mediators such as nitric oxide (NO) and prostaglandins (PG), which may regulate tissue homeostasis and contribute to pathophysiological processes. Here we examine the role of endogenous arachidonic acid (AA) and its AA metabolites in the regulation of NO release by lipopolysaccharide (LPS)-stimulated macrophages RAW 264.7. Our results suggest that bromoenol lactone-sensitive phospholipase A(2) is involved in AA release and the subsequent PG and leukotriene (LT) production. The cyclooxygenase inhibitor, indomethacin, and lipoxygenase inhibitors such as baicalein and zileuton blocked the dose-dependent PGE(2) or LTB(4) and nitrite (NO(2)(-)) production induced by LPS. Furthermore, the effects of indomethacin were reverted by exogenous PGE(2) and forskolin, whereas AH23848B, an EP(4) PGE(2) subtype receptor antagonist, decreased NO(2)(-) release. On the other hand, the effect of baicalein on NO(-)(2) production was reverted by exogenous LTB(4) and the fibrate WY 14,643, a natural and a synthetic peroxisome proliferator-activated receptor alpha (PPAR alpha), respectively. Thus, PGE(2) via EP(4) receptor/cAMP and LTB(4) via PPAR alpha may be involved in the control of NO synthesis by LPS in macrophage RAW 264.7 cultures.     

The adenylate cyclase of rheumatoid synovial fluid macrophages is more sensitive for dl-5E, 19,14-di dehydro-carbo-prostacyclin, a stable prostacyclin analogue, than for prostaglandin E2

dl-5E, 19,14-di dehydro-carbo-prostacyclin (DDH-carbo PGI2), a stable prostacyclin (PGI2) derivative, but not prostaglandin (PG) E2, stimulated the adenylate cyclase of synovial fluid macrophages, isolated from rheumatoid patients with an active synovitis, in a dose dependent manner (10-1000 ng/ml). DDH-carbo PGI2 also stimulated synovial macrophage cAMP synthesis when injected into the knee joint. Exogenous arachidonic acid (AA) had little effect on cyclic-AMP (cAMP) formation or PGI2 release (assayed as 6ketoPGF1 alpha). It stimulated, however, the release of PGE2 and, to a lesser extent, thromboxane (Tx) A2 (measured as TxB2).     

Interactions between parathyroid hormone and prostaglandins on renal cortical cyclic AMP

Effects of parathyroid hormone (PTH) and several prostaglandins (PGs) on cyclic AMP (cAMP) metabolism were studied and compared in isolated renal cortical tubules from male hamsters. Both production and intracellular degradation of cAMP were increased by PTH and each of the PGs tested (PGE2, PGE1, PGI2). Production of cAMP was increased to similar levels by maximal concentrations of PTH and each PG, however, degradation of cAMP was significantly higher in response to PTH than with any of the PGs. This difference in intracellular degradation of cAMP was responsible for the much higher concentrations of cAMP in renal cortical tubules exposed to PGs (PGE1, PGE2, PGI2) than to PTH. Submaximal amounts of each PG produced additive increases in cAMP concentrations in the presence of maximal amounts of PTH. Additivity of the combined responses was lost, however, as the PGs concentrations reached their maxima. The results suggest that renal PGs (PGE2 and PGI2) may modulate the effects of PTH on cAMP concentrations in renal cortical tubules.