cAMP-mediated upregulation of gelatinases in primary cultures of isolated rat hepatocytes

Matrix metalloproteinases (MMPs) play a major role in tissue remodelling and repair in pathophysiological conditions, such as liver fibrosis and regeneration. Regulation of the MMPs produced by liver cells is important in maintaining cell-matrix ratio in liver, which is a major target site for hormones that mediate their intracellular effects through cAMP. The possibility of cAMP affecting the activity of MMPs and their endogenous inhibitors, tissue inhibitor of MMPs (TIMPs) was studied using isolated rat hepatocytes in culture. Zymographic analysis showed that treatment with hormones like epinephrine, thyroxine and dexamethasone and Bt2 cAMP increased 92 kDa MMP-9 activity. Bt2 cAMP caused upregulation of MMP-9 in a dose-dependent manner. The effect of hormones was less on MMP-2. ELISA using specific antibodies showed increase in levels of MMP-9 and TIMP-1 protein. Kinetic analysis of production of MMPs and TIMPs showed that the response to Bt2 cAMP was a delayed one, indicating its effect on de novo protein synthesis. These results suggest the possibility of cAMP dependent regulation of MMP-9 in the hepatocytes.     

Metabolism of cyclic AMP in isolated renal tubules: effects of prostaglandins and parathyroid hormone.

Concentrations of cyclic AMP (cAMP) were increased in isolated renal cortical tubules from hamsters by both parathyroid hormone (PTH) and prostaglandin E1 (PGE1) with maximal effects of PGE1 being 6-8 fold greater than those of PTH during a 10 min period. However, cAMP concentrations in cells treated with 1-methyl-3-isobutylxanthine (MIX) were increased with maximal concentrations of either hormone to the same degree. Similar effects of both hormones were observed on adenylate cyclase activity in renal homogenates. Simultaneous addition of hormones produced changes in both cAMP concentrations in intact tubules as well as adenylate cyclase activity of homogenates which were not completely additive. Degradation of cAMP, estimated in intact tubules as the difference in cAMP levels in the presence and absence of MIX, was increased by both hormones, however, changes were 2-3 fold greater in tubules exposed to PTH than to PGE1. Neither hormone directly altered cAMP phosphodiesterase (PDE) activity in either 30,000 x g supernatant or pellets from renal cortical homogenates. The results suggest that both hormones increase the production of cAMP in renal cortical tubules and may share a common target cell type in this response. Degradation of cAMP, however, is differentially effected by the two hormones, probably reflecting differences exerted on intracellular mechanisms regulating the enzymatic hydrolysis of cAMP.     

Cyclic AMP increases COX-2 expression via mitogen-activated kinase in human myometrial cells

Cyclic AMP (cAMP) is the archetypal smooth muscle relaxant, mediating the effects of many hormones and drugs. However, recently PGI(2) , acting via cAMP/PKA, was found to increase contraction-associated protein expression in myometrial cells and to promote oxytocin-driven myometrial contractility. Cyclo-oxygenase-2 (COX-2) is the rate-limiting enzyme in prostaglandin synthesis, which is critical to the onset and progression of human labour. We have investigated the impact of cAMP on myometrial COX-2 expression, synthesis and activity. Three cAMP agonists (8-bromo-cAMP, forskolin and rolipram) increased COX-2 mRNA expression and further studies confirmed that this was associated with COX-2 protein synthesis and activity (increased PGE(2) and PGI(2) in culture supernatant) in primary cultures of human myometrial cells. These effects were neither reproduced by specific agonists nor inhibited by specific inhibitors of known cAMP-effectors (PKA, EPAC and AMPK). We then used shRNA to knockdown the same effectors and another recently described cAMP-effector PDZ-GEF(1-2) , without changing the response to cAMP. We found that MAPK activation mediated the cAMP effects on COX-2 expression and that PGE(2) acts through EP-2 to activate MAPK and increase COX-2. These data provide further evidence in support of a dual role for cAMP in the regulation of myometrial function.     

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.     

Metabolism of cyclic AMP in isolated renal tubules: Effects of prostaglandins and parathyroid hormone

Concentrations of cyclic AMP (cAMP) were increased in isolated renal cortical tubules from hamsters by both parathyroid hormone (PTH) and prostaglandin E₁ (PGE₁) with maximal effects of PGE₁ being 6–8 fold greater than those of PTH during a 10 min period. However, cAMP concentrations in cells treated with 1-methyl-3-isobutylxanthine (MIX) were increased with maximal concentrations of either hormone to the same degree. Similar effects of both hormones were observed on adenylate cyclase activity in renal homogenates. Simultaneous addition of hormones produced changes in both cAMP concentrations in intact tubules as well as adenylate cyclase activity of homogenates which were not completely additive. Degradation of cAMP, estimated in intact tubules as the difference in cAMP levels in the presence and absence of MIX, was increased by both hormones, however, changes were 2–3 fold greater in tubules exposed to PTH than to PGE₁. Neither hormone directly altered cAMP phosphodiesterase (PDE) activity in either 30,000 x g supernatant or pellets from renal cortical homogenates.The results suggest that both hormones increase the production of cAMP in renal cortical tubules and may share a common target cell type in this response. Degradation of cAMP, however, is differentially effected by the two hormones, probably reflecting differences exerted on intracellular mechanisms regulating the enzymatic hydrolysis of cAMP.     

Multiple cyclic AMP receptors are linked to adenylyl cyclase in Dictyostelium.

cAMP receptor 1 and G-protein alpha-subunit 2 null cell lines (car1- and g alpha 2-) were examined to assess the roles that these two proteins play in cAMP stimulated adenylyl cyclase activation in Dictyostelium. In intact wild-type cells, cAMP stimulation elicited a rapid activation of adenylyl cyclase that peaked in 1-2 min and subsided within 5 min; in g alpha 2- cells, this activation did not occur; in car1- cells an activation occurred but it rose and subsided more slowly. cAMP also induced a persistent activation of adenylyl cyclase in growth stage cells that contain only low levels of cAMP receptor 1 (cAR1). In lysates of untreated wild-type, car1-, or g alpha 2- cells, guanosine 5'-O-'(3-thiotriphosphate) (GTP gamma S) produced a similar 20-fold increase in adenylyl cyclase activity. Brief treatment of intact cells with cAMP reduced this activity by 75% in control and g alpha 2- cells but by only 8% in the car1- cells. These observations suggest several conclusions regarding the cAMP signal transduction system. 1) cAR1 and another cAMP receptor are linked to activation of adenylyl cyclase in intact cells. Both excitation signals require G alpha 2. 2) cAR1 is required for normal adaptation of adenylyl cyclase. The adaptation reaction caused by cAR1 is not mediated via G alpha 2. 3) Neither cAR1 nor G alpha 2 is required for GTP gamma S-stimulation of adenylyl cyclase in cell lysates. The adenylyl cyclase is directly coupled to an as yet unidentified G-protein.     

cAMP-dependent protein kinase activation lowers hepatocyte cAMP

Rat hepatocyte protein kinase was activated by incubating the cells with various cAMP analogs. Boiled extracts were then prepared and Sephadex G-25 chromatography was carried out. The G-25 procedure separated the analogs from cAMP since the resin had the unexpected property of binding cyclic nucleotides with differing affinities. Separation was necessary because the analogs would otherwise interfere with the sensitive protein kinase activation method developed for assay of cAMP. The cAMP analogs, but not 5'-AMP, lowered basal cAMP by 50-70%. The effect was rapid, analog concentration-dependent, and occurred parallel with phosphorylase activation, suggesting that the cAMP analogs act through cAMP-dependent protein kinase activation. A cAMP analog completely blocked the cAMP elevation produced by relatively low concentrations of glucagon, but did not block the phosphorylase response, indicating that the cAMP analog substitutes for cAMP as the intracellular activator of protein kinase. One implication of the results is that elevation of cAMP and protein kinase activity by hormones has a negative feedback effect on the cellular cAMP level.     

The role of insulin, glucagon, and cAMP in the regulation of hepatocyte guanylate cyclase activity

Rat liver regeneration is regulated by a humoral signal that includes insulin and a sustained elevation in glucagon. The intracellular response is characterized by a rise in cAMP as well as altered cGMP metabolism, i.e. increased particulate guanylate cyclase activity. To evaluate the role of hormones in the regulation of guanylate cyclase during liver regeneration, the enzyme activity of primary cultures of rat hepatocytes was examined. Hepatocytes were maintained for 22 h in medium containing various combinations of insulin, glucagon, and cAMP. The cells were then harvested and homogenized and the guanylate cyclase activity was assessed in vitro. Hepatocytes maintained in 100 nM insulin exhibited a 42% (p < 0.001) increase in guanylate cyclase activity when compared to cells cultured in medium alone. Incubation with glucagon (100 nM) produced a 52% (p < 0.01) rise. In the presence of insulin (100 nM), culturing with as little as 5 nM glucagon resulted in increased activity, and 100 nM glucagon produced a 161% (p < 0.001) rise above cultures maintained in insulin alone. Thus, the combination of the two hormones produced an effect that was significantly (p < 0.01) greater than additive. Dibutyryl cAMP and 8-bromoadenosine 3':5'-monophosphoric acid were at least as effective as glucagon; the enzyme activity of cells maintained in 5 microM N6,02'-dibutyryl adenosine 3':5'-monophosphoric acid and 100 nM insulin was 243% (p < 0.001) above those in insulin alone. The findings suggest that the activity of hepatocyte guanylate cyclase is regulated by a synergistic action of insulin and glucagon and that positive interactions between the two cyclic nucleotide second messenger systems exist.     

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.     

Cyclic AMP response to recombinant human relaxin by cultured human endometrial cells--a specific and high throughput in vitro bioassay.

A specific and high throughput 96-well format bioassay for recombinant human relaxin (rhRLX) has been developed using human endometrial cells (NHE cells). rhRLX caused a time- and dose-dependent stimulation of cyclic AMP (cAMP) with 1/2 maximal activity of 3.56 +/- 0.65 ng/ml (n = 30). The range of the standard curve was 0.39 to 25 ng/ml with interplate precision of 17 and 22% CV for high and low controls respectively. The cAMP response requires forskolin and 3-isobutyl-1-methylxanthine, and is enhanced by prostaglandin E2 and F2 alpha. The NHE cells do not respond to A or B chains of rhRLX, or a whole array of hormones. Preincubation of rhRLX with specific monoclonal antibody completely abolished the cAMP response. This bioassay has been used to determine the biological activity of several manufactured lots of recombinant human relaxin.     

Aromatase activity of human adipose tissue stromal cells: effects of thyroid hormones and progestogens

In order to determine the direct effects of thyroid hormones and progestogens on extraglandular aromatization, human adipose stromal cells in monolayer culture were used as a model system for this study on the regulation of aromatase enzyme activity. It was found that 1-thyroxine at 2- and 4-fold normal concentrations (220 and 440 nM, respectively) and triiodothyronine at 4-fold normal concentration (7.4 nM) had no effect on basal, dibutyryl cyclic AMP ((Bu)2 cAMP)-induced, or dexamethasone-induced aromatization. Medroxyprogesterone acetate at a concentration of 25.9 nM, but not progesterone, 47.7 nM, stimulated basal aromatization slightly but not significantly. In contrast, both medroxyprogesterone acetate and progesterone potentiated the effect of (Bu)2 cAMP on aromatase activity (P less than 0.05 and P less than 0.01, respectively) but had no effect on dexamethasone-stimulated aromatase activity. We concluded that (i) the increased peripheral aromatization associated with hyperthyroidism is not due to the direct effect of thyroid hormones on aromatase activity, and (ii) neither progesterone nor medroxyprogesterone acetate inhibit aromatase activity of adipose tissue stromal cells, but may stimulate this activity under certain conditions.     

Pleiotropic drug resistance in cystic fibrosis fibroblasts: increased resistance to cyclic AMP.

In previous studies, cystic fibrosis (CF) fibroblasts were demonstrated to be resistant to the cytotoxic effects of ouabain, dexamethasone, and the sex hormones, dihydrotestosterone, 17beta-estradiol, and progesterone. We now show that CF fibroblasts also exhibit greatly increased resistance to the cytotoxic effects of exogenous dibutyryl cyclic AMP (cAMP), as well as to isoproterenol and theophylline, drugs which are known to increase endogenous levels of cAMP. CF cells were also shown to have normal amounts of (3H)cAMP binding to protein kinase as well as normal amounts of cAMP-stimulated protein kinase activity. Phosphodiesterase in CF cells was also found to be stimulated by cAMP to the same degree as in normal cells. These findings suggest that there is no detectable protein kinase deficiency in CF cells. cf cells thus appear to be unlike some cAMP-resistant mutants described by others which are defective in protein kinase activity and cAMP regulation of phosphodiesterase levels. The cross-resistance of CF fibroblasts to ouabain, steroid hormones, and cAMP may provide a unique opportunity to study the biochemical events involved in the metabolism of these drugs as well as the basic biochemical defect in a common human genetic disease.     

Elevation of cyclic 3'5' adenosine monophosphate levels by cholera toxin inhibits the generation of interleukin 2 activity

Several molecules can interact with membrane receptors on mononuclear cells to increase intracellular levels of cyclic adenosine monophosphate (cAMP). We used the cholera toxin (CT), a cAMP elevating agent, to study the influence of this nucleotide on the production of interleukin 2 (IL-2) by human peripheral blood mononuclear cells stimulated by phytohemagglutinin and phorbol myristate acetate. Stimulated generation of IL-2 activity was inhibited by CT but not by its B subunit. The inhibition was potentiated by addition of theophylline. Therefore the synthesis and/or release of IL-2 is controlled by intracellular cAMP levels and may be modulated by agents active on this nucleotide system, such as bacterial toxins, glycoprotein hormones, or neurotransmitters.     

Spatial organisation of AKAP18 and PDE4 isoforms in renal collecting duct principal cells

A plethora of stimuli including hormones and neurotransmitters mediate a rise of the cellular level of cAMP and thereby activation of protein kinase A (PKA). PKA phosphorylates and thereby modulates the activity of a wide range of cellular targets. It is now appreciated that different stimuli induce the activation of PKA at specific sites where the kinase phosphorylates particular substrates in close proximity. The tethering of PKA to cellular compartments is facilitated by A kinase-anchoring proteins (AKAPs). The incorporation of phosphodiesterases (PDEs) into AKAP-based signalling complexes provides gradients of cAMP that regulate PKA activity locally. An example for a process depending on compartmentalised cAMP/PKA signalling is the arginine-vasopressin (AVP)-mediated water reabsorption in renal collecting duct principal cells. Upon activation through AVP, PKA phosphorylates the water channel aquaporin-2 (AQP-2) located on intracellular vesicles. The phosphorylation triggers the redistribution of AQP2 to the plasma membrane. AKAP-anchored PKA has been shown to be involved in AQP2 shuttling. Here, AKAP18 isoforms and members of the PDE4 family of PDEs are shown to be differentially localised in renal principal cells.     

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.     

Transiently elevated levels of c-fos and c-myc oncogene messenger ribonucleic acids in cultured murine Leydig tumor cells after addition of human chorionic gonadotropin

The gonadotropic hormones LH and human CG (hCG) normally function to stimulate steroidogenesis in testicular and ovarian cells through receptor-mediated activation of adenylate cyclase. These hormones are also important in regulating the development and growth of responsive cells. Such regulation requires tightly controlled gene expression. Herein we demonstrate that hCG induces increases in mRNAs encoding the competence oncogenes c-fos and c-myc in a murine Leydig cell tumor line (MA-10). When stimulated by hCG (40 ng/ml), the mRNA levels of both genes increase rapidly, peaking at 30 min for c-fos and 1 h for c-myc. Both mRNAs fall to near control levels by 3-6 h. This response to hCG is dose-dependent with half-maximal stimulation of these genes occurring at a concentration of 3 ng/ml, approximating the level required for 50% occupancy of the LH/hCG receptors and the ED50 for steroidogenesis. (Bu)2 cAMP (2 mM) elicits responses similar to those produced by hCG. The observation of oncogene control by the gonadotropin hCG provides further insight regarding the pathways by which such hormones may regulate steroidogenesis, growth, and differentiation of endocrine and neoplastic cells.     

Hormonal activation of adenylate cyclase in macrophage membranes is regulated by guanine nucleotides

Many macrophage functions such as chemotaxis, phagocytosis, enzyme secretion, and cytotoxicity are influenced by intracellular cyclic nucleotide levels, but the regulatory mechanisms involved are poorly defined. We have developed methods that allowed us to study the activation of AC in isolated guinea pig (g.p.) macrophage membranes. AC in these membrane preparations could be stimulated approximately twofold by guanine nucleotides. We could not obtain any hormonal activation of membrane-bound AC in the absence of guanine nucleotides. In the presence of GTP, however, the hormones isoproterenol and PGE1 elicited an additional threefold rise in AC activity, which subsided after approximately 15 min. As little as 10(-8) M concentrations of these two hormones induced significant elevations of AC activity. Replacement of GTP by its nonhydrolyzable analogue Gpp(NH)p resulted in a persistent hormone-independent activation of AC, and addition of hormones enhanced this level of activation. Thus, GTP-ase activity is present in macrophage membrane preparations and serves to regulate AC activation. Hormonal stimulation of AC was receptor mediated, because the effect of the beta-adrenergic agonist isoproterenol, but not PGE1, was inhibited by the beta-adrenergic blocker propranolol. In addition, the potency series of PG corresponded to that observed for stimulation of cAMP production in intact g.p. macrophages, i.e., PGE1 = PGE2 greater than PGA1 greater than PGF2 alpha. AC activation by PG in the membrane preparation was inhibited by an alpha-adrenergic agonist, thus demonstrating one means for down regulating cAMP production in g.p. macrophages. Our studies also showed that certain hormones (e.g., beta-adrenergic agonists, PG) can exert their effect on cAMP production by stimulation of membrane-bound AC, whereas other agents such as lectins or arachidonic acid require additional intracellular components to elevate cAMP levels in macrophages. The mechanism of activation of AC by hormones in g.p. macrophage membranes appears to fit the model of a ternary complex, the components of which include the hormone receptor, AC, and guanine nucleotide regulatory protein, which transmits the signal from the receptor to AC.     

Are cyclic nucleotides involved in the initiation of mitogenic activation of human lymphocytes?

In order to obtain more insight into the possible role of cyclic AMP or cyclic GMP in modulating the initial cellular processes following activation of lymphocytes, we measured the effects of the T-cell mitogen concanavalin A and other substances including hormones on the cyclic nucleotide levels in human peripheral blood lymphocytes. The enzyme activities of the corresponding nucleotide cyclases, adenylate cyclase and guanylate cyclase were measured in both isolated plasma membranes or the cytosol of resting or concanavalin A stimulated rabbit thymocytes. Concanavalin A in a mitogenic concentration of about 5-10 micrograms/ml caused small, but consistent increases in cAMP but no changes in cGMP levels during the first hour of activation. Concomitantly, the specific activity of plasma membrane-bound adenylate cyclase was always increased at least 1.5-fold 30 min after stimulation of rabbit thymocytes with concanavalin A, but no effect could be detected on the specific activities of plasma membrane-bound or soluble guanylate cyclase. At high, supraoptimal concentrations of concanavalin A (more than 20 micrograms/ml) cAMP levels dramatically increased in human lymphocytes within minutes, but cGMP levels again were unaffected. Forskolin and beta-adrenergic hormones elevated cAMP in human lymphocytes, whereas cGMP levels were increased by the addition of sodium nitroprusside or alpha-adrenergic hormones. Sodium nitroprusside, in concentrations which elevated cGMP in human lymphocytes, had no influence on the incorporation of [3H]uridine into RNA of resting or concanavalin A stimulated human lymphocytes. Addition of forskolin resulted in an increase of cAMP levels and a dose-dependent decrease of [3H]uridine incorporation into RNA of concanavalin A-stimulated lymphocytes with no effect on resting lymphocytes. The data suggest that cGMP does not play a role in the initial phase of mitogenic activation of lymphocytes, whereas cAMP may be involved in the blast transformation process as an inhibitory signal.