Prevention of prostaglandin E2-induced desensitization of rat ovarian cyclic AMP response by concanavalin A

Prolonged exposure (> 6 h) of cultured granulosa cells to Prostaglandin E₂ (PGE₂; 1 μg/ml) led to a near-total loss of the cyclic AMP response to subsequent addition of fresh hormone. Pre-treatment of the cells with concanavalin A (ConA; 2.0 μg/ml) for 1 h blocked the desensitizing action of PGE₂, so that the decline in the response was reduced by 60% with the hormone at high concentration (1.0 μg/ml); a full response was preserved at submaximal concentration of PGE₂ (0.1 – 0.3μg/ml). Other lectins (succinyl Con a, peanut agglutinin and, to a lesser extent, phytohemagglutinin and wheat germ agglutinin) had a stabilizing effect similar to that of Con A. Addition of alpha-methyl-mannoside either with Con A or various times following the addition of Con A to the cells prevented the protective effect of Con A. Concomitant treatment with colchicine or cytochalasin B abolished the ability of Con A to prevent PGE₂-induced desensitization.     

The mechanism of action of soluble lymphocyte mediators. IV. Effect of migratio inhibitory factor (MIF) on macrophage cyclic AMP and on responsiveness to adenylate cyclase stimulators.

Intracellular levels of cyclic 3′,5′-adenosine monophosphate (cAMP) in purified guinea pig peritoneal macrophages were elevated following incubation with the adenylate cyclase stimulators prostaglandins E₁ and E₂ (PGE₁, PGE₂), isoproterenol, and cholera toxin. Exposure of macrophages to antigen-stimulated lymphocyte culture supernatants, containing migration inhibitory factor (MIF), resulted in a moderate but consistent decrease in the cAMP level, which was best expressed after 1–2 hr of incubation. Incubation of macrophages with MIF-containing supernatants or partially purified MIF for 1–2 hr resulted in reduced cAMP accumulation in response to PGE₁, PGE₂, isoproterenol, and cholera toxin (nonspecific refractoriness). These findings indicate that MIF-induced inhibition of macrophage migration is not due to an increase in the cellular level of cAMP and that the reduction in cAMP concentration, caused by MIF, is probably a secondary phenomenon unrelated to the inhibition of cellular motility.     

Competition for adenyl cyclase coupled (3H)-prostacyclin binding sites with prostaglandin E2 in rat peritoneal macrophages

Prostaglandins regulate macrophage function by their action on membrane-associated adenyl cyclase. In order to define more directly macrophage-prostaglandin interactions, a binding assay has been developed for macrophage receptors using (³H)-PGI₂ as ligand. (³H)-PGI₂ binding was specific, saturable and reversible. Moreover, specific binding showed to be enriched in a membrane-enriched fraction of the cells. The assay conditions ensured stability of (³H)-PGI₂ during incubations and should exclude intracellular accumulation of the ligand in macrophages. Unlabelled PGE₂ and PGI₂ competed for (³H)-PGI₂ specific binding in both macrophages and membrane preparations. PGE₂ showed to be more potent in this respect than PGI₂, a phenomena which was also observed for prostaglandin activation of cAMP production in macrophages.The data suggest an interaction at receptor level of endogenously released PGE₂ and PGI₂ by peritoneal macrophages in vivo and provide support for a previously proposed mechanism of action of low concentrations of PGE₂, counteracting stimulation of cAMP production by PGI₂ in macrophages.     

Cerebral endothelial cell culture II. Adenylate cyclase response to prostaglandins and their interaction with the adrenergic system

The response of endothelial adenylate cyclase (AC) to prostaglandins (PGE₁, PGE₂, PGF_1α, PGF_2α, PGD₂ and PGI₂) and the relationship of PGE₂ to adrenergic systems were investigated in cerebrovascular endothelial cultures. E-type prostaglandins and PGI₂ were more effective in stimulating endothelial AC (EC₅₀ = 3 × 10^?7M, and 3 × 10^?7M, respectively) than prostaglandins of the F-series and PGD₂ which activated AC at high doses only. A modulation of endothelial AC response to either PGE₂ or norepinephrine (NE) was observed in the presence of both agents in the system. It was manifested by a dose-dependent NE inhibition of the PGE₂-stimulated formation of cAMP, which was partially restored by phentolamine. Alpha and β-adrenergic agonists (α, clonidine and 6-fluoronorepinephrine; β, isoproterenol) also partly blocked while forskolin and PGE₂ synergistically stimulated the production of cAMP in the endothelial cultures. These findings strongly suggest that the interaction of prostaglandins and α- and β-adrenergic agonists with the AC system in cerebrovascular endothelium may play a role in the regulation of the cerebral microcirculation and/or blood pressure.     

Modulation of T-cell functions: I. Effect of 2-mercaptoethanol and macrophages on T-cell proliferation

The in vitro effects of 2-mercaptoethanol (2-ME), macrophages (MØ), and concanavalin A (Con A) on the proliferation of normal spleen cells (NSC), MØ-depleted spleen cells (DSC), T cells, T-cell subpopulations, and B cells were assessed by [³H]thymidine incorporation. 2-ME alone was consistently shown not to be mitogenic for purified T cells; however, 2-ME enhanced the early (Days 1 and 2) Con A (2 μg/ml)-induced response of NSC, DSC, and T-cell preparations, but depressed the late response (Days 4 and 5). 2-ME alone was mitogenic for purified B-cells, as reported previously; and the 2-ME-induced B-cell response was inhibited by Con A. Preincubation of T cells with 2-ME was sufficient for enhanced Con A responsiveness; however, if 2-ME was added 24 hr after the initiation of culture, no alteration of the Con A-induced response was observed. Ly-2,3⁺ T cells were unresponsive to Con A (0.3–20 μg/ml), but the addition of 2-ME or peritoneal cells enhanced the Con A responsiveness of Ly-2,3⁺ T cells over 200-fold. Ly-1⁺ T cells responded with a similar doseresponse and kinetic profile as unselected T cells. Although Ly-1⁺ T cells responded to Con A, unlike Ly-2, 3⁺ T cells, extensive removal of MØ significantly reduced the Con A-induced responsiveness of the Ly-1⁺ T cells. The reactivities of Ly-1⁺ and Ly-2,3⁺ DSC could be reconstituted by the addition of MØ or 2-ME; however, the kinetic response of Ly-1⁺ T cells peaked on Day 2–3, and Ly-2,3⁺ T cells had a delayed response which peaked on Day 4–5. The results indicated that (i) 2-ME and/or MØ accelerate the response kinetics of T-cells to Con A; (ii) T-cell subpopulations have differential requirements for MØ and/or 2-ME in the response to Con A; (iii) T-cell subpopulations exhibit differential dose responsiveness to Con A; and (iiii) 2-ME alters Con A responsiveness by a direct effect on T cells.     

Cytosolic phospholipase C activity: II. Relationship to concanavalin A-induced phosphatidylinositol-turnover in splenocytes

We have described in the first paper the coupling betweencytosolic Giα and cytosolic PLC activity in a cell free preparation. In order to establish the functional significance of the cytosolic Giα coupled soluble PLC, we examined the effects of Dex, NaF, and trifluopeerizine (TEP) on concanavalin A(Con A)-induced PI-turnover in intact slenocytes and, in parallel, on soluble PLC activity in cytosol preparations. Vytosolic PLC activity was measured with [³H]PIP and [³H]PIP₂ as substrates. (1) The con A-induced increase (2–4 fold) in Pl-turnover in intact splenocytes was paralleled by an 1.2–5-fold increase in soluble PLC activity in vitro. Con A administration also increased cytosolic Giα immunoreactivity 3–6-fold as expected if cytosolic Giα was coupled to soluble PLC activation. (2) DEX (10^?7 M), administered 6 h prior to Con A administration inbited the Con A-induced increase in Pl-turnover in intact splenocytes. This was paralleled by DEX inhibition of the Con A-induced increase in soluble PLC activity measured in vitro and cytosolic Giα imunoreactivity. (3) We have demonstrated in the first paper that NaF and TEP inhibited soluble PLC activity. Here we show that NaF and TFP inhibited the Con A-induced increase in PI-turnover extending the similarities between soluble PLC activity and Con A- Stimulated PLC Activity in intact splenocytes. (4) In order to examine Whether or not the Con A-induced PLC activity and Con A-stimulated PLC activity in intact splenocytes. (4) In order to examine Whether or not the Con A-induced PLC was similar to PLCγ, we measured PI-turnover induced by Con A or BaVO₃ in combination with DEX and PMA. Whereas the Con A-induced PI-turnover was significantly inhibited (40–60%) by DEX, the NaVO₃ -induced PI-turnover was not affected by DEX. The Con A-induced PI-turnover was not affected by PMA (50nM), But the NaVO₃-induced Pi-turnover was increased over 2-fold PMA (50nM), suggesting that the Con A-induced PLC in intact splenocytes is different from NaVO₃-induced PLC. Based on these results a model for the sequential activation of substrate-specific PLCs in splenocyte by mitogen is presented.     

The role of cyclic-3′,5′-adenosine monophosphate in prostaglandin-mediated inhibition of basic calcium phosphate crystal-induced mitogenesis and collagenase induction in cultured human fibroblasts

Synovial fluid basic calcium phosphate (BCP) crystals are associated with severe destructive arthropathies characterised by synovial proliferation and non-inflammatory degradation of intra-articular collagenous structures. BCP crystals stimulate fibroblast and chondrocyte mitogenesis, metalloprotease secretion and prostaglandin production. As a tissue protective effect of prostaglandins has been suggested, we recently studied the effect of PGE₁ on BCP crystal-induced mitogenesis and collagenase mRNA accumulation in human fibroblasts (HF). We demonstrated a dose-dependent inhibition of BCP crystal-induced mitogenesis and collagenase mRNA accumulation. The mechanism of PGE₁ inhibition of BCP crystal-induced mitogenesis and collagenase mRNA accumulation was therefore explored. PGE₁ (100 ng/ml) increased HF intracellular cAMP 40-fold over control. BCP alone caused no such change but inhibited the PGE₁-induced increase in intracellular cAMP by at least 60%. The PGE₁-induced increase in intracellular cAMP was also blocked by the adenyl cyclase inhibitor, 2′,5′-dideoxyadenosine (ddA) (10 μM) and ddA reversed the PGE₁-mediated inhibition of BCP crystal-induced mitogenesis. Dibutyrul cAMP also inhibited BCP crystal-induced mitogenesis in a concentration-dependent manner. Agents which increase intracellular cAMP levels such as the adenyl cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) mimicked the effect of PGE₁ on HF collagenase mRNA levels. PGE₁ inhibits the biologic effects of BCP crystals through the cAMP signal transduction pathway and such inhibition may have significant therapeutic implications.     

Further evidence for lack of specific receptors for PGE2 in the rat ovary

Prostaglandin E₂ (PGE₂) seems to stimulate cAMP accumulation in ovaries of all mammals. While it acts through specific receptors in some species, our earlier observations (1) suggest absence of PGE₂ receptors in the rat ovary. In order to further substantiate this assumption we digested ovarian membranes from the bovine and the rat with various enzymes and measured cAMP after stimulation with PGE₂, NaF, and hCG. Pronase, trypsin, and phospholipase C abolished cAMP accumulation completely. Neuraminidase, β-galactosidase and phospholipase D did not interfere with cAMP formation. After treatment with phospholipase A₂ PGE₂-mediated cAMP accumulation was abolished in the bovine but not in the rat ovary. Formation of cAMP disappeared after hCG but not after NaF in both species. Furthermore specific binding of PGE₂ could not be demonstrated in phospholipase A₂-treated bovine ovaries. These findings are consistent with presence of specific PGE₂ receptors in the bovine and their absence in the rat ovary.     

Interrelationships among prostaglandins, vasopressin and cAMP in renal papillary collecting tubile 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 PGF_2α, increased intracellular cAMP concentrations. At maximal concentrations (10⁻⁵ tthe 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 PGE₂. PGs, when tested at concentrations (e.g. 10⁻⁹ ) 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 hydroosmatic effect of AVP on perfused collecting tubule segments. However, at higher concentrations of PGs (e.g. 10⁻⁵ ), 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.     

Cell-to-substratum adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus

Summary Some plant lectins, Concanavalin agglutinin (Con A), succinyl Con A and wheat germ agglutinin (WGA) increased the adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus, to the substratum (plastic and glass surface) in vitro. Other plant lectins,Ulex europeus agglutinin (UEA) andDolichos biflorus agglutinin (DBA) had no effect on the cell-to-substratum interaction. A specific monocarbohydrate inhibitor of lectins, -methyl-d-mannoside, inhibited the Con A-induced cell-to-substratum adhesion of dissociated embryonic cells. This observation suggests that the Con A-induced cell-to-substratum adhesion may be attributed to the Con A-carbohydrate interaction. In Millipore-filtered sea water (MPFSW) containing Con A (0.1 mg/ml), dissociated embryonic cells adhered to the substratum for more than 6 h at 18°C, while in MPFSW as control, almost all the dissociated cells were released from the substratum after 1 h. A scanning electron microscopic study showed that dissociated embryonic cells adhered to the substratum were surrounded by an extracellular fibrous material, when the cells were cultured in MPFSW containing Con A. The induction of the extracellular fibrous material by Con A was inhibited by -methyl-d-mannoside. The appearance of this material may be related to the cell-to-substratum adhesion of dissociated cells. Sequential extractions of Con A-treated dissociated cells with Triton X 100 and urea solubilized most of the cellular components, leaving the fibrous material on the surface. Biochemical conponents of the isolated fibrous material included sea urchin fibronectin, Con A and minor components (88 and 140 kilodalton proteins). Fibronectin preformed in the cells was excreted after the dissociation, while the 88 and 140 kilodalton proteins were synthesized and released to the extracellular space.     

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

Sublethaladministration of lipopolysaccharide (LPS) renders rats tolerant tomultiple lethal stimuli. Tolerant macrophages exhibit differentialalterations in LPS-stimulated cytokine and inflammatory mediatorrelease. Increased cAMP levels stimulated byPGE₂ or prostacyclin(PGI₂) result in differentialeffects on LPS-induced cytokine release and protect against thepathophysiological changes of endotoxemia. In the present studies, wesought to determine whether PGE₂-and PGI₂-stimulated cAMP levelsare altered in tolerant macrophages. Incubation of macrophages withcicaprost or 11-deoxy-PGE₁ in thepresence 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 resultin significantly elevated cAMP levels. Prostacyclin (IP) receptor mRNAlevels were significantly increased in tolerant cells compared withcontrols, whereas[³H]PGE₂binding and PGE₂ EP4 receptor mRNAlevels were not significantly changed. These studies suggest that LPStolerance induces selective alterations in eicosanoid regulation ofcAMP formation.

     

Comparison between the effect of luteinizing hormone and prostaglandin E1 on ovarian cyclic AMP

Isolated whole ovaries from 23–24 day-old rats were studied in order to compare the effects of prostaglandin E₁ (PGE₁) and luteinizing hormone (LH) on ovarian cyclic adenosine 3′,5′-monophosphate (cAMP) production. Both substances produced a dose-dependent accumulation of cAMP in the ovarian tissue as well as in the incubation medium. The release of cAMP to the incubation medium was considerable after long periods of incubation (60–120 min). Time-relationships for LH- and PGE₁-effects were different. Maximal cAMP content in the tissue after addition of PGE₁ was seen already after 5–15 min of incubation whereas LH gave a maximal response after around 60 min. Accumulation of cAMP in the medium was approximately linear with time for both LH and PGE₁. Addition of theophylline potentiated the action of PGE₁ and LH but did not change the time-courses of the effects. It is concluded that the accumulation of cAMP in the medium should be considered in studies with various in vitro types of ovarian preparations. It is also pointed out that the different time-courses of the LH- and PGE₁-effects make the interpretation of additivity experiments difficult.     

Suppression of prostaglandin synthesis by analogues of cyclic AMP and forskolin in chondrocyte monolayer cultures

Recent experimental studies indicated that prostaglandin E₂ (PGE₂) is the most abundant prostanoid synthesized by rabbit articular chondrocytes. Exogenous PGE₂ stimulates cyclic AMP (cAMP) synthesis in these cells. Analogues of cAMP and forskolin have now been shown to suppress the biosynthesis of PGE₂ in the presence of serum in a time-dependent manner. The most abundant prostanoid, PGE₂ was most markedly affected. PGF_2α was unaffected. These results indicated that intracellular accumulation of cAMP in chondrocytes and relative resistance of cAMP to phosphodiesterases control prostanoid synthesis in a negative feedback loop.     

Prostaglandin E2 inhibits the proliferation of human gingival fibroblasts via the EP2 receptor and Epac

Elevated levels of prostaglandins such as PGE₂ in inflamed gingiva play a significant role in the tissue destruction caused by periodontitis, partly by targeting local fibroblasts. Only very few studies have shown that PGE₂ inhibits the proliferation of a gingival fibroblast (GF) cell line, and we expanded this research by using primary human GFs (hGFs) and looking into the mechanisms of the PGE₂ effect. GFs derived from healthy human gingiva were treated with PGE₂ and proliferation was assessed by measuring cell number and DNA synthesis and potential signaling pathways were investigated using selective activators or inhibitors. PGE₂ inhibited the proliferation of hGFs dose‐dependently. The effect was mimicked by forskolin (adenylate cyclase stimulator) and augmented by IBMX (a cAMP‐breakdown inhibitor), pointing to involvement of cAMP. Indeed, PGE₂ and forskolin induced cAMP generation in these cells. Using selective EP receptor agonists we found that the anti‐proliferative effect of PGE₂ is mediated via the EP₂ receptor (which is coupled to adenylate cyclase activation). We also found that the effect of PGE₂ involved activation of Epac (exchange protein directly activated by cAMP), an intracellular cAMP sensor, and not PKA. While serum increased the amount of phospho‐ERK in hGFs by ～300%, PGE₂ decreased it by ～50%. Finally, the PGE₂ effect does not require endogenous production of prostaglandins since it was not abrogated by two COX‐inhibitors. In conclusion, in human gingival fibroblasts PGE₂ activates the EP₂—cAMP—Epac pathway, reducing ERK phosphorylation and inhibiting proliferation. This effect could hamper periodontal healing and provide further insights into the pathogenesis of inflammatory periodontal disease. J. Cell. Biochem. 108: 207–215, 2009. © 2009 Wiley‐Liss, Inc.     

Regulation of human peripheral blood monocyte collagenase by prostaglandins and anti-inflammatory drugs

Macrophages, which produce the collagenolytic enzyme collagenase, are commonly found at sites of connective tissue destruction in chronic inflammatory lesions. Since tissue macrophages are derived from circulating peripheral blood monocytes, we used these less-differentiated, more readily available cells to examine the production and regulation of collagenase. Human monocytes, isolated in large quantities by counterflow centrifugal elutriation, were shown to produce substantial amounts of collagenase when stimulated by concanavalin A (Con A) and to a lesser extent with lipopolysaccharide, while unstimulated monocyte cultures produced negligible collagenase. Collagenase was detected in the culture media within the first 24 hr of culture after activation with peak production at 48 hr. Analysis of the intracellular regulation of collagenase revealed that synthesis of this enzyme required a prostaglandin (PGE₂)-dependent step since indomethacin-inhibited enzyme synthesis was reversed by PGE₂. Additionally, dibutyryladenosine cyclic monophosphate (dBcAMP) restored collagenase synthesis in indomethacinblocked cultures, indicating a PGE₂-dependent generation of cAMP requirement for collagenase production similar to that demonstrated in experimental animals systems. In additional studies, anti-inflammatory drugs which are known to modulate connective tissue destruction were analyzed for their influence on monocyte-derived collagenase. Dexamethasone, colchicine or retinoic acid all inhibited collagenase synthesis by monocytes in a dose-dependent manner although the effect of these drugs on monocyte PGE₂ synthesis differed. Dexamethasone inhibited PGE₂ synthesis, which resulted in the suppression of collagenase. However, PGE₂ production was unaffected by colchicine whereas retinoic acid caused a significant increase in PGE₂ levels. Inhibition of collagenase synthesis by dexamethasone, but not colchicine or retinoic acid, could be reversed by PGE₂ or phospholipase A₂. These findings provide insight into the intracellular events regulating monocyte collagenase synthesis and also implicate monocytes as a target of anti-inflammatory agents which ameliorate connective tissue degradation associated with chronic inflammatory lesions.     

Production of arachidonic acid metabolites by operationally defined macrophage subsets

The antitumor activity and arachidonic acid metabolism of operationally defined macrophage populations was examined. Macrophages from mice injected with (strain BCG) or with pyran-copolymer were cytotoxic for tumor cells. The major arachidonic acid metabolite of these cells was PGE₂. Neither resident nor elicited macrophages were cytotoxic. However, elicited macrophages as well as macrophages from BCG injected mice inhibited tumor cell growth. The production of arachidonic acid metabolites by elicited cells, while low initially, was followed by a rapid increase in PGE₂. The major metabolites of resident cells were PGE₂ and prostacyclin. The cAMP:cGMP ratio correlated with the metabolic activity of the cells.     

Purinergic modulation of T-lymphocyte activation: differential susceptibility of distinct activation steps and correlation with intracellular 3',5'-cyclic adenosine monophosphate accumulation

The mechanism by which purinergic agonists modulate murine T-lymphocyte activation and proliferation was investigated. Adenosine and other compounds such as ATP and 2-chloroadenosine (ClAdo) were found to block T-cell mitogenesis induced by concanavalin A (Con A) in a dose-dependent fashion. The nonmetabolizable adenosine analog ClAdo was the most potent agent capable of inhibiting T-cell mitogenesis. Extracellular addition of the permeable cAMP analog dibutyryl cyclic AMP (dbcAMP) also led to a dose-dependent blockade of T-cell mitogenesis, although with less efficiency when compared to ClAdo. Addition of IL-2-enriched fluids failed to reverse blockade of T-cell mitogenesis by ClAdo or dbcAMP. ClAdo blocked T-cell enlargement induced after 20 hr of culture with Con A. We analyzed the effect of micromolar concentrations of ClAdo on interleukin-2 (IL-2) production, expression of IL-2 receptors (7D4 and 3C7 surface antigens), and induction of IL-2 responsiveness after in vitro cultivation with Con A. ClAdo inhibited both IL-2 secretion and induction of IL-2 responsiveness up to control levels in the same dose range it inhibited T-cell mitogenesis. However, cell surface expression of IL-2 receptors was not affected. Short incubations of resting splenic T cells with ClAdo led to a dose-dependent accumulation of cyclic AMP in responding cells. This effect was markedly reduced by the purinergic antagonist 3-isobutyl-1-methylxanthine (IBMX) but was not prevented by the adenosine uptake blocker dipyridamole. ClAdo elicited cAMP accumulation in the same dose range it inhibited T-cell activation events. Extracellular administration of dbcAMP to splenic T cells stimulated by Con A mimicked the effects of ClAdo on T-cell activation parameters, as revealed by a dose-dependent blockade of both IL-2 secretion and IL-2 responsiveness induction, without affecting IL-2 receptor expression. Short incubations of Con A-activated T-cell blasts with ClAdo also led to a dose-dependent accumulation of cAMP. We then analyzed the effect of purines and dbcAMP on IL-2-mediated activated T-cell growth. Purines caused a dose-dependent inhibition of IL-2-mediated T-cell proliferation and ClAdo was the most potent purinergic agonist tested. The effect of ClAdo on Con A-induced T blasts was shifted to the right, if compared to earlier T-cell activation steps.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Differential Effects of Prostaglandin E1 and Prostaglandin E2 on Growth and Differentiation of Murine Myeloid Leukemic Cell Line,M1

Effects of prostaglandins (PGs) of the E series on growth and differentiation of murine myeloid leukemic cell line M1 were studied. PGE₁, but not PGE₂, inhibited the growth of M1 cells. PGE₂ neither inhibited nor augmented the antiproliferative effect of PGE₁. PGE₁ augmented the differentiation of M1 cells into macrophage-like cells induced by interleukin 6. PGE₂, however, did not exhibit any effect on the differentiation. PGE₁ caused a marked increase in intracellular cAMP level in M1 cells, whereas PGE₂ had no effect. These results indicate that M1 cells are able to respond only to PGE₁. Radiolabeled PGE₁ binding experiments, however, revealed that there was no specific binding in M1 cells, suggesting that the cells express low numbers of receptors or very low affinity receptors specific for PGE₁. Stable agonists of PGI₂, iloprost, cicaprost or carbacyclin, also potently inhibited the growth of M1 cells. These findings suggest that PGE₁ as well as PGI₂ may play a role in the differentiation of monocyte-macrophage lineage cells.