PGE2-stimulated cAMP Production Research Articles

PGE2 -induced migration of human brain endothelial cell is mediated though protein kinase A in cooperation of EP receptors.

PGE2 plays a critical role in angiogenesis, ischemic, and neuro-inflammatory disorders of the brain, which breakdown the blood-brain barrier (BBB). However, the effects of PGE2 on human brain endothelial cell (HBECs) migration, a key process in the angiogenic response and BBB stability, are not well defined. In this study, we investigated the mechanism of PGE2 in HBECs migration in vitro. Here we showed that PGE2 stimulated migration of HBECs in a dose-time and matrix-dependent manner, evaluated by the Boyden chamber assay, but other prostanoids failed to do so. PGE2 receptor (EP2; butaprost), EP3 (sulprostone), and EP4 (PGE1 -OH) receptor agonists stimulated HBECs migration, but the silencing of EP significantly attenuated this effect. EP1 agonist (11-trinor PGE1 ) had no effect on HBECs migration on silencing of the EP1 receptor. We further showed that PGE2 stimulated cAMP production and activated protein kinase A (PKA), whereas pretreatment with the adenyl cyclase inhibitor (dideoxyadenosine; 1 μM) or PKA inhibitors, H89 (0.5 μM)/PKAI (1 μM), completely abrogated PGE2 -induced migration. Furthermore, silencing of the EP2/EP4 receptors significantly inhibited PGE2 -induced cAMP and PKA activation, whereas EP3 receptor silencing failed to do so. These results suggest that PGE2 regulates HBEC migration via cooperation of EP2, EP3, and EP4 receptors. Coupling of PGE2 to these receptors resulted in increased production of cAMP, which regulates HBEC migration via PKA pathway. The elucidation of molecular events involved is critical for the development of targeted strategies to treat cerebrovascular diseases associated with dysregulated angiogenesis.

Pharmacological characterisation of CR6086, a potent prostaglandin E2 receptor 4 antagonist, as a new potential disease-modifying anti-rheumatic drug

BackgroundProstaglandin E2 (PGE2) acts via its EP4 receptor as a cytokine amplifier (e.g., interleukin [IL]-6) and induces the differentiation and expansion of inflammatory T-helper (Th) lymphocytes. These mechanisms play a key role in the onset and progression of rheumatoid arthritis (RA). We present the pharmacological characterisation of CR6086, a novel EP4 receptor antagonist, and provide evidence for its potential as a disease-modifying anti-rheumatic drug (DMARD).MethodsCR6086 affinity and pharmacodynamics were studied in EP4-expressing HEK293 cells by radioligand binding and cyclic adenosine monophosphate (cAMP) production, respectively. In immune cells, IL-6 and vascular endothelial growth factor (VEGF) expression were analysed by RT-PCR, and IL-23 and IL-17 release were measured by enzyme-linked immunosorbent assay (ELISA). In collagen-induced arthritis (CIA) models, rats or mice were immunised with bovine collagen type II. Drugs were administered orally (etanercept and methotrexate intraperitoneally) starting at disease onset. Arthritis progression was evaluated by oedema, clinical score and histopathology. Anti-collagen II immunoglobulin G antibodies were measured by ELISA.ResultsCR6086 showed selectivity and high affinity for the human EP4 receptor (Ki = 16.6 nM) and functioned as a pure antagonist (half-maximal inhibitory concentration, 22 nM) on PGE2-stimulated cAMP production. In models of human immune cells in culture, CR6086 reduced key cytokine players of RA (IL-6 and VEGF expression in macrophages, IL-23 release from dendritic cells, IL-17 release from Th17 cells). In the CIA model of RA in rats and mice, CR6086 significantly improved all features of arthritis: severity, histology, inflammation and pain. In rats, CR6086 was better than the selective cyclooxygenase-2 inhibitor rofecoxib and at least as effective as the Janus kinase inhibitor tofacitinib. In mice, CR6086 and the biologic DMARD etanercept were highly effective, whereas the non-steroidal anti-inflammatory drug naproxen was ineffective. Importantly, in a study of CR6086/methotrexate, combined treatment greatly improved the effect of a fully immunosuppressive dose of methotrexate.ConclusionsCR6086 is a novel, potent EP4 antagonist showing favourable immunomodulatory properties, striking DMARD effects in rodents, and anti-inflammatory activity targeted to immune-mediated inflammatory diseases and distinct from the general effects of cyclooxygenase inhibitors. These results support the clinical development of CR6086, both as a stand-alone DMARD and as a combination therapy with methotrexate. The proof-of-concept trial in patients with RA is ongoing.

Steroid hormones, prostanoids, and angiogenic systems during rescue of the corpus luteum in pigs.

In order to characterize the transition of the corpora lutea (CL) from acquisition of luteolytic sensitivity to rescue of luteal function: i) the expression of 38 factors associated with steroids, prostanoids, and angiogenic systems and ii) concentrations of the main hormones responsible for maintenance of CL function in cyclic and pregnant pigs were examined. Additionally, the effect of prostaglandin (PG) E2 and F2 α on luteal function during the estrous cycle and pregnancy was evaluated in vitro. Significantly up-regulated gene expression was revealed in CL collected on day 14 of the estrous cycle (CYP19A1, ESR2, PTGS2, HIF1A, and EDN1) and on days 12-14 of pregnancy (SCARB1, PGRMC1, STAR, HSD3B1, NR5A1, PTGFR, PTGER4, and VEGFA). Elevated concentrations of estradiol-17β and PGE2 occurred in CL on days 12 and 14 of pregnancy respectively, while an increased intraluteal PGF2 α content was noted on day 14 of the estrous cycle. Both PGs increased the synthesis of progesterone by cultured luteal slices obtained on day 14 of pregnancy, in contrast to the action of PGF2 α on the corresponding day of the estrous cycle. PGE2 stimulated cAMP production via PTGER2 and PTGER4, while PGF2 α elevated the content of CREB in cultured luteal slices from CL of pregnant pigs. In silico analysis showed that infiltration of lymphocytes and apoptosis of microvascular endothelium were activated in CL on day 12 of the estrous cycle vs pregnancy. Summarizing, an abundance of E2 and PGE2 during pregnancy regulates specific pathways responsible for steroidogenesis, the prostanoid signaling system and angiogenesis during rescue from luteolysis in porcine CL.

Abstract P3-12-06: Subpopulations of peripheral sensory neurons are differentially sensitive to the microtubule-targeting agent, paclitaxel

Abstract Paclitaxel (PTX), a microtubule-targeting anticancer agent, produces a debilitating peripheral neuropathy that is accompanied by neuropathic pain. Patients develop localized pain in their distal extremities, and more frequently complain of increased sensitivity to cold rather than hot temperatures. These clinical observations led us to hypothesize that PTX differentially damages subpopulations of peripheral sensory neurons. The purpose of this study was to evaluate the effects of PTX on bradykinin (BK) and eicosanoid (EP) receptor-expressing sensory neurons. PTX (11.7 mg/kg) or vehicle (Cremophor EL/EtOH/PBS; 1:1:6) was administered intraperitoneally to Sprague-Dawley rats (250-300 g) every other day for 5 days. Following treatment, PTX-treated rats exhibited increased sensitivity to cold and mechanical stimuli, but decreased sensitivity to heat stimulation as compared to vehicle-treated rats. Intraplantar (i.pl.) injection with BK (5 μg) produced a prolonged allodynia to cold, but a diminished allodynia to heat in PTX-treated rats. Interestingly, following treatment with PGE2 (0.3 μg, i.pl.), PTX-treated rats displayed no statistically significant differences in the allodynia to cold or heat compared to vehicle-treated rats. Efficacy of BK (1 pM - 1 μM) to stimulate inositol phosphate (IP) production in cultured primary sensory neurons from PTX-treated rats was significantly reduced. By contrast, stimulation of cAMP production by PGE2 (0.01 pM - 100 nM) was unaffected by PTX treatment. Preliminary mechanistic studies treating naïve cultures of rat primary sensory neurons directly with PTX (5 nM, 0 - 48 hr) yielded a time-dependent, tri-phasic effect on BK-stimulated IP accumulation and reduced PGE2-stimulated cAMP production, suggesting PTX differentially disrupts BK- and PGE2-mediated signaling. Collectively, these data provide evidence that subpopulations of peripheral sensory neurons are differentially sensitive to PTX. Understanding why particular subclasses are less sensitive, or resistant, to damage could reveal novel approaches to protect sensory neurons from chemotoxicity and ultimately prevent chemotherapy-induced peripheral neuropathy. Supported by USPS grant #8ULITR000149, Greehey Fellowship, and Translational Science Training Across Disciplines Program at UTHSCSA The project described was supported by Award Number 8UL1TR000149 from the National Center for Advancing Translational Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Advancing Translational Sciences. Citation Format: Peter M LoCoco, Teresa C Chavera, Raehannah J Jamshidi, Susan L Mooberry, Kelly A Berg, William P Clarke. Subpopulations of peripheral sensory neurons are differentially sensitive to the microtubule-targeting agent, paclitaxel [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-12-06.

Failure of tetrodotoxin to inhibit the prostaglandin-induced secretory response of rat small intestine in-vitro.

Tetrodotoxin (TTX, 10 microM) did not inhibit prostaglandin E2 (PGE2)-stimulated increases in electrical activity in intact or stripped sheets of rat small intestine, although it reduced basal electrical activity in the intact preparation. Basal and PGE2-stimulated cAMP production by enterocytes isolated from the small intestine was unaffected by TTX. Thus its reported ability to inhibit prostaglandin-induced fluid secretion in-vivo does not appear to represent a direct interaction of the neurotoxin with the mechanism of prostaglandin action at the enterocyte.

Bradykinin enhancement of PGE 2 signalling in bovine trabecular meshwork cells

Kinins and prostaglandins activate signalling pathways in cells of the trabecular meshwork and have opposing effects on outflow resistance to aqueous humor. Consequently, interactions between these pathways may be important in the regulation of intraocular pressure. In the present study, the influence of bradykinin on PGE 2 signalling was examined in primary cultures of bovine trabecular meshwork cells. Incubation of cells with bradykinin produced a concentration-dependent (EC 50=3·6±0·7 n m) elevation of intracellular free Ca 2+. At a maximal concentration of 100 n m, the increase in Ca 2+ was rapid, peaking in 30 sec, and then slowly returned to baseline. This effect was completely inhibited in cells pretreated with the selective B 2 kinin receptor antagonist, Hoe-140. Treatment of trabecular meshwork cells with PGE 2, in comparison, had no effect on cellular Ca 2+ but produced a concentration-dependent increase in adenosine 3′, 5′-cyclic monophosphate (cAMP) formation. Bradykinin had no effect on basal cAMP. However, incubation of cells with PGE 2 in combination with bradykinin resulted in a 3- to 5-fold enhancement of PGE 2-stimulated cAMP production. Bradykinin enhancement of cAMP stimulation was concentration-dependent with an EC 50 of 3·6±1·8 n m. Treatment of cells with bradykinin increased the response maximum for PGE 2 signalling, while the EC 50 for PGE 2 was not changed. This action of bradykinin was again blocked in cells pretreated with Hoe-140. Bradykinin also produced a 2- to 3-fold increase in isoproterenol and cholera toxin-stimulated cAMP accumulation. However, when adenylyl cyclase was stimulated directly with forskolin, bradykinin failed to alter cAMP production. These results indicate that bradykinin activates B 2 kinin receptors in trabecular meshwork cells to amplify PGE 2-stimulated cAMP formation by facilitating the interaction between activated G s and the catalytic unit of adenylyl cyclase.

Distinct Roles for Gαi2, Gαi3, and Gβγ in Modulation of Forskolin- or Gs-mediated cAMP Accumulation and Calcium Mobilization by Dopamine D2S Receptors

Previous studies have shown that a single G protein-coupled receptor can regulate different effector systems by signaling through multiple subtypes of heterotrimeric G proteins. In LD2S fibroblast cells, the dopamine D2S receptor couples to pertussis toxin (PTX)-sensitive Gi/Go proteins to inhibit forskolin- or prostaglandin E1-stimulated cAMP production and to stimulate calcium mobilization. To analyze the role of distinct Galphai/o protein subtypes, LD2S cells were stably transfected with a series of PTX-insensitive Galphai/o protein Cys --> Ser point mutants and assayed for D2S receptor signaling after PTX treatment. The level of expression of the transfected Galpha mutant subunits was similar to the endogenous level of the most abundant Galphai/o proteins (Galphao, Galphai3). D2S receptor-mediated inhibition of forskolin-stimulated cAMP production was retained only in clones expressing mutant Galphai2. In contrast, the D2S receptor utilized Galphai3 to inhibit PGE1-induced (Gs-coupled) enhancement of cAMP production. Following stable or transient transfection, no single or pair set of mutant Galphai/o subtypes rescued the D2S-mediated calcium response following PTX pretreatment. On the other hand, in LD2S cells stably transfected with GRK-CT, a receptor kinase fragment that specifically antagonizes Gbeta gamma subunit activity, D2S receptor-mediated calcium mobilization was blocked. The observed specificity of Galphai2 and Galphai3 for different states of adenylyl cyclase activation suggests a higher level of specificity for interaction of Galphai subunits with forskolin- versus Gs-activated states of adenylyl cyclase than has been previously appreciated.

Increased prostacyclin and PGE2 stimulated cAMP production by macrophages from endotoxin-tolerant rats.

Sublethal administration of lipopolysaccharide (LPS) renders rats tolerant to multiple lethal stimuli. Tolerant macrophages exhibit differential alterations in LPS-stimulated cytokine and inflammatory mediator release. Increased cAMP levels stimulated by PGE2 or prostacyclin (PGI2) result in differential effects on LPS-induced cytokine release and protect against the pathophysiological changes of endotoxemia. In the present studies, we sought to determine whether PGE2- and PGI2-stimulated cAMP levels are altered in tolerant macrophages. Incubation of macrophages with cicaprost or 11-deoxy-PGE1 in the presence of phosphodiesterase inhibitors resulted in significantly higher (2.5- to 6.5-fold) cAMP concentrations in tolerant macrophages compared with control. In contrast, isoproterenol-stimulated cAMP levels were not significantly different between control and tolerant cells. Also, incubation of tolerant macrophages with LPS did not result in significantly elevated cAMP levels. Prostacyclin (IP) receptor mRNA levels were significantly increased in tolerant cells compared with controls, whereas [3H]PGE2 binding and PGE2 EP4 receptor mRNA levels were not significantly changed. These studies suggest that LPS tolerance induces selective alterations in eicosanoid regulation of cAMP formation.

Stimulation of adenylyl cyclase mediated by phospholipase C-linked M3 muscarinic receptor in human neuroblastoma SK-N-BE (2) C cells.

Muscarinic receptor in human neuroblastoma SK-N-BE(2)C cells was identified and characterized. Treatment of the cells with carbachol evoked the generation of inositol 1,4,5-trisphosphate (IP3) with a peak level reached at 1 min after stimulation. Carbachol increased intracellular Ca2+ ([Ca2+]i) with an EC50 value of 35 microM. In addition, carbachol produced a 1.3-3-fold increase in the cyclic AMP (cAMP) level compared with untreated control and elevated synergistically the cAMP level in the treatment with prostaglandin E2 (PGE2). The M3 antagonist p-fluorohexahydrosiladifenidol (IC50 = 0.5-0.8 microM) inhibited the increases in [Ca2+]i, IP3, and cAMP more effectively than the M1 antagonist pirenzepine (IC50 = 5-9 microM) and the M2 antagonist methoctramine (IC50 = 20-30 microM). The involvements of [Ca2+]i elevation and protein kinase C activation induced by phospholipase C activation were tested in the carbachol-induced cAMP production. The calcium chelator BAPTA/AM (75 microM) inhibited significantly the synergistic effects of carbachol and PGE2 on the production of cAMP, whereas the Ca2+ ionophore ionomycin (1 microM) clearly enhanced PGE2-induced cAMP production. However, phorbol 12-myristate 13-acetate did not enhance PGE2-stimulated cAMP production. These data suggest that phospholipase C-linked M3 receptors are present and that stimulation of the receptors activates adenylyl cyclase, at least in part, by the Ca(2+)-dependent system in the neuronal cells.

Inhibition of prostaglandin E 2-responsive adenylyl cyclase in embryonal human kidney 293 cells by phorbol esters

A clonal primary embryonal human kidney cell line, 293, increased cAMP production in response to prostaglandin E 2 (PGE 2) (0.02–2 μM). The purpose of this study was to show the effects of tumor-promoting phorbol esters (e.g., 4β-phorbol 12-myristate 13-acetate, PMA) on PGE 2-stimulated cAMP production. Pretreatment with PMA (0.2–200 nM) for 30 min markedly reduced PGE 2-Stimulated CAMP production in the presence of 0.5 mM isobutylmethylxanthine. The reduction by PMA was dose- and time-dependent. PMA seems to attenuate the increase in cAMP accumulation elicited by PGE 2 primarily, if not entirely, by inhibiting adenylyl cyclase activity, since we were unable to demonstrate an effect of PMA on the degradation half-life of cAMP in intact 293 cells. The action of PMA had some specificity for the agonist used; thus, PMA inhibited PGE 2 activated adenylyl cyclase but had no effect on the forskolin-activated enzyme. Co-pretreatment with PMA and H-7, an inhibitor of protein kinase C (PKC), partially prevented the PMA-induced attenuation of the PGE 2-stimulated cAMP accumulation, and 1-oleoyl-2-acetylglycerol, a synthetic diacylglycerol analog, partially mimicked the PMA action. Thus, PMA appeared to decrease cAMP production by a PKC-mediated mechanism, inhibiting adenylyl cyclase activity at a point other than the catalytic subunit of the enzyme in the kidney 293 cell line.

PAF stimulates cAMP formation in P388D 1 macrophage-like cells via the formation and secretion of prostaglandin E 2 in an autocrine fashion

The role of cAMP in the formation of prostaglandin E 2 (PGE 2) was investigated in bacterial lipopolysaccharide (LPS)-primed P388D 1 macrophage-like cells stimulated with platelet activating factor (PAF). cAMP levels and PGE 2 secretion were correlated with stimulation by PAF or ionomycin. Indomethacin inhibited cAMP formation induced by PAF, but not PGE 2-stimulated cAMP production. Inositol (1,4,5)-trisphosphate levels were strongly reduced by exogenous PGE 2 and increased by H-89, an inhibitor of PKA. However, exogenous PGE 2 did not affect PAF-stimulated PGE 2 formation. These results suggest that cAMP levels in P388D 1 cells are regulated by PGE 2 in an autocrine fashion. Evidence is presented that this feedback mechanism regulates inositol (1,4,5)-triphosphate levels in these cells, while PGE 2 formation is not affected.

PGE2 regulates cAMP production in cultured rabbit CCD cells: evidence for dual inhibitory mechanisms.

In cultured cortical collecting duct (CCD) cells, exogenous prostaglandin E2 (PGE2) inhibited arginine vasopressin (AVP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production in a concentration-dependent manner. Although pertussis toxin (PT, 500 ng/ml) alone did not reverse the PGE2-dependent inhibition, PT and staurosporine, a protein kinase C inhibitor, together partially reversed the effect of exogenous PGE2. In contrast, PT completely reversed the inhibition of AVP-dependent cAMP production by sulprostone. These data suggest that exogenous PGE2 can inhibit AVP-stimulated cAMP production and that the inhibitory effects of PGE2 are mediated by staurosporine- and PT-sensitive component(s). Short-term (15-240 min) incubation with phorbol 12-myristate 13-acetate (PMA, 10(-7) M) inhibited PGE2-stimulated cAMP production. Long-term (20 h) incubation with PMA augmented PGE2-stimulated cAMP production. These data provide evidence for the maintenance of a PT-sensitive PGE2-dependent inhibitory pathway of cAMP production in cultured CCD cells. In addition, data are presented that support an inhibitory role for protein kinase C in the effects of PGE2 on the metabolism of cAMP in these cells.

Phorbol ester-like action of staurosporine on the cAMP response to prostaglandin E 2 in two macrophage-like cell lines at distinct differentiation stages

The purpose of this study is to investigate the involvement of protein inase C (PKC) in prostaglandin E 2 (PGE 2)-stimulated cAMP production of two macrophage-like cell lines (G3 and XC). XC cells are thought to be placed at a more differentiated stage than G3 cells [Orikasa et al. (1990) Cell Immunol. 132, 350–365]. In RPMI 1640 containing 10% (v/v) heat-inactivated foetal calf serum (FCS), in which the cAMP response of both cells to PGE 2 increased with duration of culture, XC cells showed greater response than G3 cells at 2 days of culture. In α-minimum essential medium (α-MEM) containing 20% (v/v) heat-inactivated horse serum (HS), the cAMP response of both cells was apparently greater than that in RPMI 1640 containing 10% (v/v) FCS. These cells increased cAMP production also in response to PGE 1 and PGF 2α, and the order of potency in increase was PGE 1 > PGE 2 ⪢ PGF 2α. Interestingly, a short-term (20 min) treatment with phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC or staurosporine, a relatively specific inhibitor of PKC, augmented the PGE 2-stimulated cAMP production in these cells cultured in α-MEM containing 20% (v/v) HS. However, a long-term (24 h) treatment with these compounds did not alter the cAMP response. In G3 cells, PMA appeared more potent than staurosporine in terms of augmentation, whereas in XC cells, the former appeared less potent than the latter. Although the mechanism of this augmentation seems complex and is still unclear, our findings suggest that PKC modulates the regulation of the PGE 2-cAMP signalling system, and that the macrophage differentiation may decrease the involvement of PMA-sensitive PKC, but not that of staurosporine-sensitive PKC, in this cAMP system.

Characterization of prostaglandin and thromboxane receptors expressed on a megakaryoblastic leukemia cell line, MEG-01s

MEG-01s, an established human megakaryoblastic leukemia cell line, exhibited specific high-affinity binding sites for [3H]iloprost, a stable prostaglandin (PG) I2 analogue, for [3H]SQ-29548, a stable thromboxane (TX) A2 antagonist and, for [3H]PGE2/PGE1, but not for [3H]PGD2. In the MEG-01s cells, iloprost/PGI2, or PGE1 stimulated cAMP production with ED50 values practically identical to the IC50 values for the [3H] iloprost binding. STA2 and U46619, TXA2/PGH2 agonists, PGE2/PGE1, iloprost/PGI2, and thrombin elevated the intracellular concentrations of Ca2+ ([Ca2+]i), as determined by Fura-2 fluorescence signals. Elevation of [Ca2+]i by PGE2/PGE1 and iloprost, but not that by TX-agonists or thrombin, was totally dependent on the presence of extracellular Ca2+. This effect by PGE2/PGE1 was partially inhibited by prior treatment of the cells with islet-activating protein (IAP), while that by TX-agonists or by PGI2/iloprost was not affected. We tentatively conclude from these results that: (1) MEG-01s cells express (a) PGI2/PGE1 receptor(s) coupled to adenylate cyclase and Ca2+ influx, a TXA2/PGH2 receptor coupled to the phosphatidylinositol-turnover-Ca2+ system, and the PGE2/PGE1 receptor coupled to Ca2+ influx; (2) the receptors for TXA2/PGH2 and iloprost and those for PGE2/PGE1 and thrombin are coupled to IAP-insensitive and IAP-sensitive GTP-binding proteins, respectively, and function in a different manner to elevate [Ca2+]i. Thus, the MEG-01s cell line is a pertinent model for studying eicosanoid receptor-mediated signal transduction in platelet/megakaryocyte systems.

Phorbol ester (TPA) reduces prostaglandin E2-stimulated cAMP production by desensitization of prostaglandin E2 receptors in a clonal osteoblast-like cell line, MOB 3-4

A clonal osteoblast-like cell line, MOB 3-4, increased cAMP production in response to prostaglandin E2 (PGE2) (5–500 ng/ml). The purpose of this study was to show the effects of tumor-promoting phorbol ester (e.g., 12-O-tetradecanoylphorbol 13-acetate, TPA) on basal and PGE2-stimulated cAMP production and the affinity of PGE2 receptors in the cells. Pretreatment with TPA (1 nM–10 μM) for 30 minutes increased basal cAMP production, whereas it markedly reduced the PGE2-stimulated cAMP production in the presence of 0.1 mM isobuthylmethyl xanthine. Both the TPA increase and reduction were dose- and time-dependent. However, TPA exerted no effect on forskolinor cholera toxin-stimulated cAMP production. Copretreatment with TPA and H-7, an inhibitor of protein kinase C (PKC), prevented the TPA-induced increase in basal cAMP production, whereas it did not prevent the reduction of the PGE2-stimulated cAMP production. On the other hand, TPA (0.1–10 μM) decreased3H-PGE2 binding in a dose- and time-dependent manner. Scatchard analysis revealed that TPA decreased the apparent affinity of PGE2 receptors without effect on their apparent number. In addition, 1-oleoyl-2-acetylglycerol (12.6 μM), a synthetic diacylglycerol analog, did not mimic the TPA action on3H-PGE2 binding. Thus, TPA at relatively high concentrations appeared to increase basal cAMP production by a PKC-mediated mechanism, and it appeared to directly act on PGE2 receptors to decrease their apparent affinity and thereby reduce the PGE2-stimulated cAMP production in the clonal osteoblast-like MOB 3-4 cell line.

骨芽細胞ＭＣ３Ｔ３‐Ｅ１におけるＰＧＥ２のａｄｅｎｙｌａｔｅ ｃｙｃｌａｓｅおよびｐｈｏｓｈｏｌｉｐａｓｅＣに対する作用

We have reported previously that PGE2 evoked an increase in intracellular calcium level ( [Ca2+] i) in mouse osteoblastic cells (Eicosanoids, 3, 157-160) . Here, we investigated the effects of PGE1 and PGF2a on cAMP production and [Ca2+] i in comparison with those of PGE2. In osteoblastic clone, MC3T3-E1 cells, PGE1 stimulated cAMP production, but has no effect on [Ca2+] i whereas PGF2a evoked only [Ca2+] i increase. In contrast, PGE2 not only stimulated cAMP production, but also increased [Ca2+] i. From the Scatchard plot analysis of PGE2 it is confirmed that there are two classes of PGE2 binding sites (Kd value, 9.2 nM; binding site, 29 fmole/mg protein, and Kd value, 134 nM; binding site, 148 fmole/mg protein) .These data indicate the possibility that the dual action of PGE2 is mediated by distinct receptor systems. As increase in [Ca2+] i was caused by PGF2a and PGE2, but not by PGE1, we investigated the displacement study of [3H] -PGF2a binding. The displacement capacity of unlabeled PGE2 was about 1/10 of that of PGF2a, while that of PGE1 was very low even at 500-fold excess.These data suggest that the dual effect of PGE2 is mediated by distinct receptor systems.

Differential effects of calcium on progesterone production in small and large bovine luteal cells

We studied the effects of calcium (Ca2+) ions in progesterone (P) production by separated small and large luteal cells. Corpora lutea were collected from 31 heifers between days 10 and 12 of the estrous cycle. Purified small and large cells were obtained by unit gravity sedimentation and flow cytometry. P accumulation in cells plus media was determined after incubating 1 x 10(5) small and 5 x 10(3) large cells for 2 and 4 h respectively. Removal of Ca2+ from the medium did not influence basal P production in the small cells (P greater than 0.05). However, stimulation of P by luteinizing hormone (LH), prostaglandin E2 (PGE2), 8-bromo-cyclic 3',5' adenosine monophosphate (8-Br-cAMP) and prostaglandin F2 alpha (PGF2 alpha) was impaired (P less than 0.05) by low Ca2+ concentrations. LH and PGE2-stimulated cAMP production was not altered by low extracellular Ca2+ concentrations, and PGF2 alpha had no effect on cAMP. In contrast, basal as well as LH and forskolin-stimulated P production were attenuated (P less than 0.05) in Ca2(+)-deficient medium in the large cells. However, P production stimulated by 8-Br-cAMP was not altered in Ca2(+)-deficient medium. Steroidogenesis in large cells was also dependent on intracellular Ca2+, since 8-N, N-diethylamineocytyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular Ca2+ release and/or action, suppressed (P less than 0.05) basal, LH and 8-Br-cAMP stimulated P. In contrast, basal P in small cells was not altered by TMB-8; whereas LH-stimulated P was reduced 2-fold (P less than 0.05). The calcium ionophore, A23187, inhibited LH-stimulated P in small cells and both basal and agonist-stimulated P in large cells. These studies show that basal P production in small cells does not require Ca2+ ions, while hormone-stimulated P production in small cells and both basal and hormone-stimulated P in large cells do require Ca2+. The inhibitory effect of Ca2+ ion removal was exerted prior to the generation of cAMP in the large cells, but distal to cAMP generation in hormone-stimulated small cells. The calmodulin/protein kinase C antagonist, W-7, also inhibited both basal and hormone-stimulated P production in both small and large luteal cells, indicating that P production in luteal cells also involves Ca2(+)-calmodulin/protein kinase C-dependent mechanisms.

Effects Of Atrial Natriuretic Factor on Cyclic Nucleotides, Bone Resorption, Collagen and Deoxyribonucleic Acid Synthesis, and Prostaglandin E2Production in Fetal Rat Bone Cultures*

We examined the effects of synthetic human atrial natriuretic factor (human ANF 99-126) on adenylate cyclase activity, cAMP and cyclic GMP (cGMP) levels, bone resorption, collagen and DNA synthesis, and prostaglandin E2 (PGE2) production in fetal rat bone organ cultures. ANF (100 nM) inhibited PTH- and PGE2-stimulated cAMP production but had no effect on basal cAMP production in 21-day fetal rat calvaria. ANF increased cGMP levels, and this was not affected by PTH. ANF (10 nM) partially inhibited bone resorption stimulated by PGE2 but had no effect on control or PTH-stimulated resorption in 19-day fetal rat long bones. ANF had no effect on collagen and DNA synthesis or PGE2 production and did not alter responses to PTH or PGE2 in the fetal rat calvaria. Thus, ANF has no major direct effect on bone resorption or formation, but it is possible that ANF modulates the local regulatory function of PGE2 in bone.

PGE2-induced cyclic AMP accumulation in human CD4+ T cells is strongly enhanced during the CD2-mediated activatory process

The effect of a mitogenic combination of two different anti-CD2 monoclonal antibodies on the PGE2-stimulated and basal cAMP production in human CD4+ T cell clones was investigated. The anti-CD2 stimulation strongly potentiates the PGE2-induced cAMP production while both PMA and A23187 produced a less potent effect. On the opposite the anti-CD2 treatment is without any effect on the basal cAMP level contrasting with a marked increase of intracellular cAMP concentrations with A23187 or the combination of A23187 and PMA. These results suggest that activation of CD4+ human T cells via the CD2 molecule significantly influences the cAMP-related transduction pathway. Although PMA and A23187 also modulate the activity of this pathway, their effect in this model is more likely mediated through an amplification of basal cAMP production.

Agonist but not phorbol ester induced desensitization of human lymphocyte prostaglandin receptor is dependent on tubulin polymerization

The regulation of prostaglandin stimulated cAMP accumulation in cells of the human T-cell leukemia line Jurkat was examined. Pretreatment with PGE2 (0.1-10 nM) for 2 hour caused a concentration dependent desensitization of the prostaglandin receptor. Tumor promoting phorbol esters (1-1000 nM) could also inhibit PGE2 stimulated cAMP production dose dependently. Inhibition of tubulin polymerization with colchicine or nocodazole (1 microM) eliminated prostaglandin but not phorbol ester induced desensitization of the receptor. It is concluded that agonist and phorbol ester induced desensitization are mediated by two distinct mechanisms and that tubulin polymerization appear to be required only for agonist induced desensitization of the prostaglandin receptor.