Inhibition of prostaglandin E2-stimulated cAMP accumulation by lipopolysaccharide in murine peritoneal macrophages.

Treatment of murine peritoneal macrophages with 100 nM prostaglandin E2 (PGE2) produced a rapid biphasic increase in intracellular cAMP that was maximal at 1 min and sustained through 20 min. Pretreatment of macrophages with 100 ng/ml of lipopolysaccharide (LPS) for 60 min prior to PGE2 decreased the magnitude of cAMP elevation by 50%, accelerated the decrease of cAMP to basal levels, and abolished the sustained phase of cAMP elevation. The effect of LPS was concentration-dependent, with maximal effect at 10 ng/ml in cells incubated in the presence of 5% fetal calf serum and at 1 microgram/ml in the absence of fetal calf serum. LPS also inhibited cAMP accumulation in cells treated with 100 microM forskolin, but the decrease was about half that seen in cells treated with PGE2. LPS concentrations that inhibited cAMP accumulation produced a 30% increase in soluble low Km cAMP phosphodiesterase activity while having no effect on particulate phosphodiesterase activity. The nonspecific phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, as well as the more specific inhibitors rolipram and Ro-20-1724 were effective in inhibiting soluble phosphodiesterase activity in vitro, producing synergistic elevation of cAMP in PGE2-treated cells, and blocking the ability of LPS to inhibit accumulation of cAMP. Separation of the phosphodiesterase isoforms in the soluble fraction by DEAE chromatography indicated that LPS activated a low Km cAMP phosphodiesterase. The enzyme(s) present in this peak could be activated 6-fold by cGMP and were potently inhibited by low micromolar concentrations of Ro-20-1724 and rolipram. Using both membranes from LPS-treated cells and membranes incubated with LPS, no decrease in adenylylcyclase activity could be attributed to LPS. Although effects of LPS on the rate of synthesis of cAMP cannot be excluded, the present evidence is most consistent with a role for phosphodiesterase activation in the inhibitory effects of LPS on cAMP accumulation in murine peritoneal macrophages.     

Activation of the Ras-ERK pathway inhibits retinoic acid-induced stimulation of tissue transglutaminase expression in NIH3T3 cells

Retinoic acid (RA) is a potent activator of tissue transglutaminase (TGase) expression, and it was recently shown that phosphoinositide 3-kinase (PI3K) activity was required for RA to increase TGase protein levels. To better understand how RA-mediated TGase expression is regulated, we considered whether co-stimulation of NIH3T3 cells with RA and epidermal growth factor (EGF), a known activator of PI3K, would facilitate the induction or increase the levels of TGase expression. Instead of enhancing these parameters, EGF inhibited RA-induced TGase expression. Activation of the Ras-ERK pathway by EGF was sufficient to elicit this effect, since continuous Ras signaling mimicked the actions of EGF and inhibited RA-induced TGase expression, whereas blocking ERK activity in these same cells restored the ability of RA to up-regulate TGase expression. However, TGase activity is not antagonistic to EGF signaling. The mitogenic and anti-apoptotic effects of EGF were not compromised by TGase overexpression, and in fact, exogenous TGase expression promoted basal cell growth and resistance to serum deprivation-induced apoptosis. Moreover, analysis of TGase expression and GTP binding activity in a number of cell lines revealed high basal TGase GTP binding activity in tumor cell lines U87 and MDAMB231, indicating that constitutively active TGase may be a characteristic of certain cancer cells. These findings demonstrate that TGase may serve as a survival factor and RA-induced TGase expression requires the activation of PI3K but is antagonized by the Ras-ERK pathway.     

Activation of protein kinase C modulates the adenylate cyclase effector system of B-lymphocytes

Antibodies to surface immunoglobulins activate inositol phospholipid hydrolysis in B-lymphocytes, but very little is known concerning their effects on cAMP levels. In other cells, products from the hydrolysis of phosphatidylinositol 4,5-bisphosphate can increase and/or potentiate cAMP accumulation. In this study we have examined whether goat anti-mouse IgM (mu-chain-specific) stimulates and/or potentiates increases in the cAMP levels of splenocytes from athymic nude mice. Goat anti-mouse IgM, by itself, stimulated a 60% increase in cAMP within 2 min. Pretreating the cell suspensions at 37 degrees C with anti-IgM produced opposite effects on the forskolin- and prostaglandin E1 (PGE1)-induced increase in cAMP. Anti-IgM (25 micrograms/ml) potentiated the rise in cAMP induced by 100 microM forskolin 76%, but it decreased the response to 50 nM PGE1 by 30%. Direct activation of protein kinase C (Ca2+/phospholipid-dependent enzyme) by 12-O-tetradecanoylphorbol 13-acetate and/or sn-1,2-dioctanoylglycerol resulted in a similar pattern of responses. A 3-min preincubation with 97 nM 12-O-tetradecanoylphorbol 13-acetate potentiated the forskolin-induced response from 1.7 +/- 0.1 to 4.3 +/- 0.6 pmol of cAMP/10(6) cells but reduced the PGE1 response from 0.98 +/- 0.06 to 0.51 +/- 0.03 pmol of cAMP/10(6) cells. Similarly, preincubating the cells for 3 min with 5 microM sn-1,2-dioctanoylglycerol increased the forskolin response from 1.7 +/- 0.1 to 5.1 +/- 0.2 pmol of cAMP/10(6) cells but reduced the response to PGE1 from 1.15 +/- 0.03 to 0.75 +/- 0.04 pmol of cAMP/10(6) cells. Thus, activation of protein kinase C by hydrolysis products of inositol phospholipids, 12-O-tetradecanoylphorbol 13-acetate, or exogenous diacylglycerols modified adenylate cyclase itself and sites upstream of adenylate cyclase such as the receptor or G proteins coupling the receptor to the cyclase. Furthermore, modification of the PGE1 response by anti-IgM provides a mechanism by which antigen can differentially regulate T- and B-cells responding to macrophage-produced prostaglandins during an immune response.     

Hormone-sensitive particulate cAMP phosphodiesterase activity in 3T3-L1 adipocytes. Regulation of responsiveness by dexamethasone

Confluent 3T3-L1 fibroblasts incubated for 72 h with methylisobutylxanthine, dexamethasone, and insulin differentiate and acquire phenotypic characteristics of mature adipocytes, including hormone-sensitive cAMP phosphodiesterase activity located in a particulate fraction of homogenates. About 10 days after initiating differentiation, a maximally effective concentration of insulin (100 pM) increased particulate cAMP phosphodiesterase activity 40 to 60% in 8 min; activation persisted for at least 30 min in the presence of insulin. Incubation of adipocytes for 6-8 min with agents that increased cAMP, e.g. 1 microM epinephrine, 0.1 microM isoproterenol, corticotropin (2 mu units/ml), or thyroid-stimulating hormone (15 ng/ml), also increased particulate phosphodiesterase activity 40-60%. Changes in phosphodiesterase activity produced by epinephrine tended to lag behind changes in cAMP. Insulin, epinephrine, and corticotropin increased Vmax, not Km (0.5 microM), for cAMP. Particulate phosphodiesterase activity, solubilized with detergent, eluted in a single peak from DEAE-Bio-Gel. Insulin and epinephrine increased the activity eluted in this peak. Neither insulin nor lipolytic hormones increased activity in soluble fractions from differentiated cells or particulate or soluble fractions from undifferentiated cells. Incubation of adipocytes for 48 h with 1 microM dexamethasone prevented insulin-induced activation of the particulate phosphodiesterase and did not alter basal activity. After incubation for 72 h with 0.1 microM dexamethasone, insulin and epinephrine activation were abolished. These effects of dexamethasone on hormonal regulation of particulate phosphodiesterase activity could account for some of the so-called permissive effects of glucocorticoids on cAMP-mediated processes as well as the "anti-insulin" effects of glucocorticoids.     

Mitogenic effect of prostaglandin E1 in Swiss 3T3 cells: role of cyclic AMP

Addition of prostaglandin E1 (PGE1) to quiescent cultures of Swiss 3T3 cells rapidly elevates the intracellular levels of cAMP and increases the activity of adenylate cyclase in particulate fractions of these cells. In the presence of insulin, PGE1 stimulates the reinitiation of DNA synthesis. Both effects (increase in cellular cAMP and stimulation of DNA synthesis) are markedly potentiated by 1-methyl-3-isobutyl xanthine (IBMX) or by 4-(3-butoxy-4 methoxy benzyl)-2-imidazolidine (Ro 20-1724), both of which are potent inhibitors of cyclic nucleotide phosphodiesterase activity. In the presence of 50 microM IBMX, PGE1 caused a dose-dependent increase in cAMP levels and in [3H]thymidine incorporation into acid-insoluble material at concentrations (5-50 ng/ml) that are orders of magnitude lower than those used in previous studies (50 micrograms/ml) to demonstrate growth-inhibitory effects. Thus, the inhibitory effects produced by adding high concentrations of PGE1 on the initiation of DNA synthesis in Swiss 3T3 cells are not mediated by cAMP and should be regarded as nonspecific. In contrast, the mitogenic activity of PGE1 parallels its ability to increase the intracellular levels of cAMP. The findings support the proposition that a sustained increase in the level of this cyclic nucleotide acts as a mitogenic signal for confluent and quiescent Swiss 3T3 cells.     

Effects of dimaprit on growth and differentiation of human promyelocytic cell line, HL-60

The human promyelocytic cell line HL-60, differentiates in response to a variety of agents including dibutyryl cAMP and agents which increase intracellular cAMP concentrations (phosphodiesterase inhibitors, PGE2, and cholera toxin). HL-60 is also known to be rich in H2 -histamine sensitive adenylate cyclase activity. The present study was therefore designed to test the effects of H2-stimulation on growth and differentiation of HL-60 using the potent H2 agonist dimaprit. Dimaprit markedly increased cAMP production in a dose-dependent manner reaching maximal levels after 30-60 minutes. Intracellular cAMP levels decreased thereafter and by 24 hours were approximately 2-3 fold increased above control. Intracellular cAMP levels were not altered by dimaprit (10(-7)M to 10(-4)M) at 4 days in culture compared to either untreated HL-60 cells or dimethylsulfoxide (DMSO) (1.3%) treated cells. While exponential growth was unaltered by dimaprit (10(-7)M to 10(-4)M) as compared to control, dimaprit induced i) morphologic maturation to the myelocyte and metamyelocyte form with no differentiation seen beyond the metamyelocyte even after 6 days in culture, ii) increased NBT reductase activity and iii) dose-dependent increase in lysozyme activity which could be completely blocked by cimetidine, a specific H2 antagonist. Dimaprit-induced differentiation of HL-60 cells was associated with an initial but transient increase in intracellular cAMP production. Maturation beyond the metamyelocyte stage was not observed. Acquisition of NBT reductase and lysozyme activity correlated with morphologic maturation.     

Effects of tissue transglutaminase on retinoic acid-induced cellular differentiation and protection against apoptosis.

Retinoic acid (RA) and its various synthetic analogs affect mammalian cell growth, differentiation, and apoptosis. Whereas treatment of the human leukemia cell line HL60 with RA results in cellular differentiation, addition of the synthetic retinoid, N-(4-hydroxyphenyl) retinamide (HPR), induces HL60 cells to undergo apoptosis. Moreover, pretreatment of HL60 cells as well as other cell lines (i.e. NIH3T3 cells) with RA blocks HPR-induced cell death. In attempting to discover the underlying biochemical activities that might account for these cellular effects, we found that monodansylcadaverine (MDC), which binds to the enzyme (transamidase) active site of tissue transglutaminase (TGase), eliminated RA protection against cell death and in fact caused RA to become an apoptotic factor, suggesting that the ability of RA to protect against apoptosis is linked to the expression of active TGase. Furthermore, it was determined that expression of exogenous TGase in cells exhibited enhanced GTP binding and transamidation activities and mimicked the survival advantage imparted by RA. We tested whether the ability of this dual function enzyme to limit HPR-mediated apoptosis was a result of the ability of TGase to bind GTP and/or catalyze transamidation and found that GTP binding was sufficient for the protective effect. Moreover, excessive transamidation activity did not appear to be detrimental to cell viability. These findings, taken together with observations that the TGase is frequently up-regulated by environmental stresses, suggest that TGase may function to ensure cell survival under conditions of differentiation and cell stress.     

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.     

Correlation between cyclic AMP levels and calcium efflux in isolated renal cortical tubules.

Isolated renal cortical tubules from male hamsters were utilized to examine the possible relationship between cyclic AMP (cAMP) and efflux of calcium. Both parathyroid hormone (PTH) and prostaglandin E1 (PGE1) produced dose-related increases in cAMP levels and calcium efflux from isolated tubules. Maximal concentrations of both hormones resulted in changes in cAMP which were 6 fold greater and changes in calcium efflux which were 2 fold greater with PGE1 than with PTH. Effects of sub-maximal amounts of either hormone on both cAMP and calcium efflux were potentiated to tubule incubations resulted in increases in tissue-associated cAMP over the same degree by inclusion of methyl-isobutylxanthine (MIX). Addition of either exogenous cAMP or dibutyryl cAMP (db-cAMP) produced dose-related increases in calcium efflux which occurred more rapidly with db-cAMP than with cAMP. Increasing amounts of cAMP added to the same concentration range resulting in increases in calcium efflux. Addition of 2', 3' cyclic AMP, 5'AMP or db-cyclic GMP had no significant effect on calcium efflux while 3', 5' cyclic CMP significantly reduced this response. The results indicate that cAMP increases efflux of calcium from renal tubules and may play a central role in hormone-dependent transport of this ion.     

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.     

Tissue transglutaminase mediates activation of RhoA and MAP kinase pathways during retinoic acid-induced neuronal differentiation of SH-SY5Y cells

All-trans-retinoic acid (RA) plays a crucial role in survival and differentiation of neurons. For elucidating signaling mechanisms involved in RA-induced neuronal differentiation, we have selected SH-SY5Y cells, which are an established in vitro cell model for studying RA signaling. Here we report that RA-induced neuronal differentiation of SH-SY5Y cells is coupled with increased expression/activation of TGase and in vivo transamidation and activation of RhoA. In addition, RA promotes formation of stress fibers and focal adhesion complexes, and activation of ERK1/2, JNK1, and p38alpha/beta/gamma MAP kinases. Using C-3 exoenzyme (RhoA inhibitor) or monodansylcadaverine (TGase inhibitor), we show that transamidated RhoA regulates cytoskeletal rearrangement and activation of ERK1/2 and p38gamma MAP kinases. Further, by using stable SH-SY5Y cell lines (overexpressing wild-type, C277S mutant, and antisense TGase), we demonstrate that transglutaminase activity is required for activation of RhoA, ERK1/2, JNK1, and p38gamma MAP kinases. Activated MAP kinases differentially regulate RA-induced neurite outgrowth and neuronal marker expression. The results of our studies suggest a novel mechanism of RA signaling, which involves activation of TGase and transamidation of RhoA. RA-induced activation of TGase is proposed to induce multiple signaling pathways that regulate neuronal differentiation.     

Hormonal regulation of a plasma membrane phosphodiesterase in differentiating granulosa cells. Reciprocal actions of follicle-stimulating hormone and a gonadotropin-releasing hormone agonist on cAMP degradation

The activity of a plasma membrane cAMP-phosphodiesterase in cultured ovarian granulosa cells was regulated by follicle-stimulating hormone (FSH) and the gonadotropin-releasing hormone (GnRH) agonist [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa). Degradation of cAMP was similar in cultures treated with FSH alone or FSH plus GnRHa when the labeled cyclic nucleotide was added from 24 to 42 h of culture. However, at 48 h and subsequent times of incubation, cAMP phosphodiesterase activity was significantly higher in cells incubated with FSH plus GnRHa. Phosphodiesterase activity was progressively increased by GnRHa concentrations between 10(-13) and 10(-10) M, and was maximally stimulated by 10(-9) M GnRHa. In comparison with control cells, FSH lowered the Vmax of cAMP catabolism by the high (1 microM cAMP substrate) and the low (50 microM) affinity phosphodiesterase, while GnRHa raised enzyme activity toward control levels. These actions of FSH and GnRHa were specific for a plasma membrane phosphodiesterase that was accessible to extracellular cAMP, since extracellular substrate was hydrolyzed, no intracellular uptake of [3H]cAMP was observed, and only a small fraction (10%) of cAMP was catabolized in the incubation medium in the absence of cells. Further, the actions of FSH and GnRHa on the membrane enzyme were the opposite of those observed when total phosphodiesterase activity was measured in cellular sonicates. Hormonal changes in phosphodiesterase activity were not due to leakage of the enzyme from damaged cells since a constant percentage of cAMP hydrolysis in the medium was observed during culture. Analysis of cAMP catabolites in granulosa cells indicated that the phosphodiesterase reaction product, 5'-AMP, was rapidly converted to adenosine by a plasma membrane 5'-nucleotidase, independent of the cellular hormonal status. These results indicate that the opposing actions of FSH and GnRHa upon granulosa cell differentiation include modulation of cAMP degradation at the plasma membrane level.     

Phosphodiesterase 4 inhibitors and db-cAMP inhibit TNF-alpha release from human mononuclear cells. Effects of cAMP and cGMP-dependent protein kinase inhibitors

We investigated the effects of specific inhibitors of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) on the inhibitory activity of phosphodiesterase (PDE) type 4 inhibitors and of the cell permeable analogue of cAMP, db-cAMP on LPS-induced TNF-alpha release from human mononuclear cells. Incubation from 30 min of mononuclear cells with dbcAMP (10(-5) to 10(-3) M), rolipram (10(-9) M to 10(-5) M) or Ro 20-1724 (10(-9) M to 10(-5) M) significantly inhibited LPS-induced TNF-alpha release. When mononuclear cells were preincubated for 30 min with the selective PKA inhibitor, H89 (10(-4) M), but not with the selective PKG inhibitor, Rp-8-pCPT-cGMPs (10(-4) M), a significant reduction of the inhibitory effect of db-cAMP was noted. Thirty min incubation of mononuclear cells with Rp-8-pCPT-cGMPs induced a significant reduction of the inhibitory activities of both rolipram and Ro 20-1724 (10(-9) to 10(-5) M) on LPS-induced TNF-alpha release, whereas H89 elicited a moderate, but significant inhibition. The present data indicate that db-cAMP inhibits TNF-alpha release from human mononuclear cells through a PKA-dependent mechanism. In contrast, PDE 4 inhibitors elicit their in vitro anti-inflammatory activities via a PKG-dependent rather than PKA-dependent activation.     

Cyclic AMP-mediated modulation of immunoglobulin production in B cells by prostaglandin E1.

We had previously demonstrated in a transformed human B cell line, LA350, the existence of an inverse relationship between cyclic adenosine monophosphate (cAMP) content and immunoglobulin secretion using the cAMP-elevating agents such as cholera toxin and forskolin. In this paper we report that cAMP acting as a second messenger for prostaglandin exerts a similar effect on the antibody response of B lymphocytes. Incubation of the cells with PGE1 in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) produced a concentration- and time-dependent elevation of intracellular cAMP. Significant increases of cAMP production were observed at physiologically relevant levels of PGE1 (10(-7) and 10(-8) M). Immunoglobulin production, whether measured as the total number of immunoglobulin-secreting cells by a reverse hemolytic plaque assay or as specific immunoglobulin production (IgM) by an enzyme-linked immunoadsorbent assay, was suppressed in a dose-dependent fashion by the presence of IBMX. This suppression of immunoglobulin production was significantly enhanced by the presence of PGE1. Phorbol myristate acetate-induced IgM production was also inhibited by the presence of PGE1. These results imply that prostaglandins regulate B cell activation and immunoglobulin production by signal transduction mechanisms involving cyclic nucleotides.     

Augmentation of tissue transglutaminase expression and activation by epidermal growth factor inhibit doxorubicin-induced apoptosis in human breast cancer cells

Tissue transglutaminase (TGase) exhibits both a GTP binding/hydrolytic capability and an enzymatic transamidation activity. Increases in TGase expression and activation often occur in response to stimuli that promote cellular differentiation and apoptosis, yet the signaling mechanisms used by these stimuli to regulate TGase expression and activation and the role of TGase in these cellular processes are not well understood. Retinoic acid (RA) consistently induces TGase expression and activation, and it was shown recently that RA-induced TGase expression was inhibited in NIH3T3 mouse fibroblasts co-stimulated with epidermal growth factor (EGF). Here we investigate whether EGF also antagonized RA-induced TGase expression in breast cancer cells. We found that EGF stimulation affected TGase expression and activation very differently in these cancer cells. Not only did EGF fail to block RA-induced TGase expression, but also EGF alone was sufficient to potently up-regulate TGase expression and activation in SKBR3 cells, as well as MDAMB468 and BT-20 cells. Inhibiting phosphoinositide 3-kinase activity severely diminished the ability of EGF and RA to increase TGase protein levels, whereas a constitutively active form of phosphoinositide 3-kinase potentiated the induction of TGase expression by EGF in SKBR3 cells. Because EGF is an established antiapoptotic factor, we examined whether the protection afforded by EGF was dependent on its ability to up-regulate TGase activity in SKBR3 and BT-20 cells. Exposure of cells to a TGase inhibitor or expression of a dominant-negative form of TGase potently inhibited EGF-mediated protection from doxorubicin-induced apoptosis. Moreover, expression of exogenous TGase in SKBR3 cells mimicked the survival advantage of EGF, suggesting that TGase activation is necessary and sufficient for the antiapoptotic properties of EGF. These findings indicate for the first time that EGF can induce TGase expression and activation in human breast cancer cells and that this contributes to their oncogenic potential by promoting chemoresistance.     

Acute in vivo stimulation of low-Km cyclic AMP phosphodiesterase activity by insulin in rat-liver Golgi fractions

A low-Km phosphodiesterase activity, which is acutely stimulated by insulin in vivo, has been identified in plasma membranes and Golgi fractions prepared from rat liver homogenates in isotonic sucrose. Within seconds after insulin injection (25 micrograms/100 g body weight) cAMP phosphodiesterase activity increases by 30-60% in Golgi fractions and by 25% in plasma membranes; activity in crude particulate and microsomal fractions is unaffected. The increase in activity is short-lived in the light and intermediate Golgi fractions, but persists for at least 10 min in the heavy Golgi fraction. It precedes the translocation of insulin and insulin receptors to these fractions, which is maximal at 5 min. The doses of insulin required for half-maximal and maximal activation are, respectively, 7.5 micrograms/100 g and 25 micrograms/100 g body weight. Golgi-associated cAMP phosphodiesterase activity shows non-linear kinetics; a high-affinity component (Vmax, 13 pmol min-1 mg protein-1; Km, 0.35 microM) is detectable. Insulin treatment increases the Vmax 60-70%, but does not affect the Km. Unlike the low-Km cAMP phosphodiesterase associated with crude particulate fractions, the Golgi-associated activity is not easily extractable by solutions of low or high ionic strength. On analytical sucrose density gradients, low-Km cAMP phosphodiesterase associated with the total particulate fraction equilibrates at lower densities than endoplasmic reticulum and lysosomal markers, but at a higher densities than plasma membrane, Golgi markers and insulin receptors. Insulin treatment increases the specific activity of the enzyme by 20-60% at densities below 1.12 g cm-3, and by 20-40% in the density interval 1.23-1.25 g cm-3. Such treatment also causes a slight, but significant shift in the distribution of phosphodiesterase towards lower densities. It is suggested that Golgi elements or physically similar subcellular structures are a major site of localization of insulin-sensitive cAMP phosphodiesterase in rat liver. However, internalization of the insulin-receptor complex is probably not required for enzyme activation.     

Role of calcium and cAMP in the action of adrenocorticotropin on aldosterone secretion

When the dose-response curve of adrenocorticotropin (ACTH)-induced aldosterone secretion is compared to that of ACTH-induced intracellular cAMP, the ED50 for intracellular cAMP is more than 10 times as high as that for aldosterone production. In contrast, the dose-response curve of forskolin-induced aldosterone secretion correlates well with that for forskolin-induced intracellular cAMP. ACTH, but not forskolin, increases calcium influx into glomerulosa cells without inducing the mobilization of calcium from an intracellular pool. The effect of ACTH on calcium influx is dose-dependent and ED50 is 3.5 X 10(-11) M. In a perifusion system, the effect of 1 nM ACTH on aldosterone secretion is much greater than that of 1 microM forskolin, even though these two stimulators induce identical increases in the intracellular cAMP. Perifusion with combined A23187 (50 nM) and forskolin (1 microM) stimulates aldosterone secretion to a value comparable to that induced by 1 nM ACTH. Likewise, BAY K 8644 (1 nM), which induces a comparable increase in calcium influx, potentiates the effect of 1 microM forskolin. When the intracellular [Ca2+] is fixed at either 100 or 300 nM, forskolin-stimulated intracellular cAMP content is identical, but ACTH-stimulated intracellular cAMP content at 100 nM [Ca2+]i is 60% of that at 300 nM [Ca2+]i. Both the ACTH- and forskolin-induced aldosterone secretion rate is higher at 300 nM than at 100 nM [Ca2+]i. These results indicate that ACTH stimulates calcium influx, that calcium potentiates ACTH-induced but not forskolin-induced cAMP generation, and that Ca2+ and cAMP act as synarchic messengers in ACTH-mediated aldosterone secretion.     

Functional compartmentalization of arginine-vasopressin-activated cyclic AMP in anterior pituitary gland: the presence of a compartment activated by prostaglandin E2

AVP(10(-8)-10(-6)M) increased ACTH as well as PGE2 release from rat anterior pituitary quarters in vitro in a concentration dependent manner. IBMX (0.1 mM), a phosphodiesterase inhibitor, increased the ACTH response to AVP. The cAMP content in pituitary tissue was increased by AVP. Cyclooxygenase inhibition by indomethacin(1.4 X 10(-5) M) or diclofenac (1.8 X 10(-5)M) led to a potentiation of AVP-evoked ACTH secretion and to a decrease in AVP-stimulated cAMP formation. PGE2(10(-6)M) significantly increased pituitary cAMP content and indomethacin did not affect cAMP levels activated by PGE2. PGE2 attenuated the AVP-induced ACTH release. These results indicate that at least two functional compartments of AVP-activated cAMP responses are involved in the AVP-induced ACTH release. One compartment is directly activated by AVP and participates in the propagation of AVP-induced ACTH release. The second compartment is activated by PGE2. The contribution of the second compartment to the regulation of ACTH secretion is not well understood since PGE2 shows an inhibitory effect on AVP-induced ACTH secretion.     

Effect of cyclic nucleotides on the mitotic activity of dental anlage and alveolar bone cells in tissue culture

A study was made of the effects of cAMP, db-cGMP and db-cAMP on the mitotic activity of the cells of the tooth anlage and alveolar bone in tissue culture of mouse embryo aged 15 days. The data indicate that db-cGMP and db-cAMP at concentrations 10(-6) M and 10(-8) M do not have any essential effect on the mitotic activity of the cells of the tooth anlage whereas cAMP inhibits the mitotic activity in these cells as compared with control. A definite relationship was established between the character of differentiation of the osseous and dental tissues in tissue culture and the concentration of cyclic nucleotides. db-cAMP, db-cGMP and cAMP raise the mitotic activity of the osteogenic cells, inhibit resorption of the alveolar bone, and stimulate formation of a new osseous tissue.