Acute ethanol inhibits extracellular signal-regulated kinase, protein kinase B, and adenosine 3':5'-cyclic monophosphate response element binding protein activity in an age- and brain region-specific manner

BACKGROUND: As little as a single episode of exposure of the developing brain to ethanol can result in developmental neuropathology and mental retardation. Extracellular signal-regulated kinases (ERKs), protein kinase B (PKB), and adenosine 3':5'-cyclic monophosphate response element binding protein (CREB) are messenger molecules that play important roles in neuronal plasticity and survival. This study was undertaken to examine the effects of acute ethanol on ERK, PKB, and CREB activation in the brain. METHODS: Immunoblot analysis was used to determine the effects of a 1-hr exposure of ethanol on levels of phospho-ERC in primary cortical cultures and in the cerebral cortex, hippocampus, and cerebellum of postnatal day 5 (PN5), postnatal day 21 (PN21), and adult rats. RESULTS: In cortical cultures, ethanol (100 mM) significantly reduced activity-dependent activation of phospho-ERK, phospho-PKB, and phospho-CREB by approximately 50%. In PN5 rats, ethanol (3.5 g/kg) inhibited both phospho-ERK and phospho-PKB in the cerebral cortex and hippocampus but was without effect in the cerebellum. A similar brain region-specific inhibition of phospho-ERK was observed in PN21 rats, whereas in adult rats, ethanol inhibited phospho-ERK in all three brain regions. In contrast, ethanol had no effect on phospho-PKB in either PN21 or adult rats. Without exception, ethanol inhibited phospho-CREB in an identical brain region- and age-dependent manner as was observed for phospho-ERK. Finally, administration of the NMDA antagonist MK-801 (0.5 mg/kg) to PN5 rats had no effect on phospho-ERK or phospho-PKB levels in any brain region. CONCLUSION: The results demonstrate that acute ethanol inhibits ERK/PKB/CREB signaling in brain. This inhibition occurs in an age- and brain region-specific manner, with inhibition of PKB restricted to a time during the brain growth-spurt period. Furthermore, the lack of effect of MK-801 suggests that inhibition of NMDA receptors is unlikely to play a major role in binge ethanol inhibition of ERK/PKB/CREB signaling in vivo.     

Signaling through 3',5'-cyclic adenosine monophosphate and phosphoinositide-3 kinase induces sodium/iodide symporter expression in breast cancer

The sodium/iodide symporter (NIS) is a membrane transport glycoprotein normally expressed in the thyroid gland and lactating mammary gland. NIS is a target for radioiodide imaging and therapeutic ablation of thyroid carcinomas and has the potential for similar use in breast cancer treatment. To facilitate NIS-mediated radionuclide therapy, it is necessary to identify signaling pathways that lead to increased NIS expression and function in breast cancer. We examined NIS expression in mammary tumors of 14 genetically engineered mouse models to identify genetic manipulations associated with NIS induction. The cAMP and phosphoinositide-3 kinase (PI3K) signaling pathways are associated with NIS up-regulation. We showed that activation of PI3K alone is sufficient to increase NIS expression and radioiodide uptake in MCF-7 human breast cancer cells, whereas cAMP stimulation increases NIS promoter activity and NIS mRNA levels but is not sufficient to increase radioiodide uptake. This study is the first to demonstrate that NIS expression is induced by cAMP and/or PI3K in breast cancer both in vivo and in vitro.     

Progesterone increases basal 3',5'-cyclic adenosine monophosphate formation and down-regulates the agonist-induced inositol phosphates generation in human term placenta.

Whether the placenta is a target tissue for estrogens and progesterone, and their putative mechanism of action, is still a controversial question in the literature. The effect of progesterone and estradiol on 3',5'-cyclic adenosine monophosphate (cAMP) and inositol phosphates generation in human term placenta was investigated. Placental explants were incubated in vitro for up to 48 h in the absence and in the presence of estradiol, progesterone or both steroids (0.1 mumol/l final concentration in all cases), and were stimulated with terbutaline, a beta-adrenergic agonist, (0.1 mmol/l) or angiotensin II (1 mumol/l). The cAMP content was measured by a competitive protein binding assay, and the generation of labelled inositol phosphates formation in explants prelabelled with 3H-myo-inositol was measured by anion exchange chromatography. Progesterone increased significantly basal cAMP concentrations in comparison with control or estradiol-treated tissues (169 +/- 13, 72 +/- 8, and 69 +/- 2 pmol/g wet wt tissue, mean +/- SEM, respectively). However, following terbutaline stimulation cAMP levels (mean +/- SEM) increased to similar values under all conditions (182 +/- 33, 197 +/- 36, and 237 +/- 17 pmol/g wet wt tissue for control, estradiol-, and progesterone-treated tissues, respectively). Angiotensin II stimulated inositol phosphates generation in placental explants by an average of fivefold, but this increase was significantly reduced in the presence of progesterone (5.2 +/- 0.7, 3.7 +/- 0.4, and 2.2 +/- 0.3 fold increase vs non-angiotensin-stimulated tissues, for control, estradiol-, and progesterone-treated placenta, mean- +/- SEM, respectively). These data suggest that progesterone modulates the formation of second messengers in human placenta at term.     

Mitogen-activated protein kinase-dependent stimulation of proliferation of rat lactotrophs in culture by 3',5'-cyclic adenosine monophosphate.

Intracellular cAMP regulates cell proliferation as a second messenger of extracellular signals in a number of cell types. We investigated, by pharmacological means, whether an increase in intracellular cAMP levels changes proliferation rates of lactotrophs in primary culture, whether there are interactions between signal transduction pathways of cAMP and the growth factor insulin, and where the dopamine receptor agonist bromocriptine acts in the cAMP pathway to inhibit lactotroph proliferation. Rat anterior pituitary cells, cultured in serum-free medium, were treated with cAMP-increasing agents, followed by 5-bromo-2'-deoxyuridine (BrdU) to label proliferating pituitary cells. BrdU-labeling indices indicative of the proliferation rate of lactotrophs were determined by double immunofluorescence staining for PRL and BrdU. Treatment with forskolin (an adenylate cyclase activator) or (Bu)2cAMP (a membrane-permeable cAMP analog) increased BrdU-labeling indices of lactotrophs in a dose- and incubation time-dependent manner. The cAMP-increasing agents were also effective in increasing BrdU-labeling indices in populations enriched for lactotrophs by differential sedimentation. The stimulatory action of forskolin was observed, regardless of concentrations of insulin that were added in combination with forskolin. Inhibition of the action of endogenous cAMP by H89 or KT5720, a protein kinase A inhibitor, attenuated an increase in BrdU-labeling indices by insulin treatment. On the other hand, the specific mitogen-activated protein kinase inhibitor PD98059, which was effective in blocking the mitogenic action of insulin, markedly suppressed the forskolin-induced increase in BrdU-labeling indices. (Bu)2cAMP antagonized not only inhibition of BrdU labeling indices but also changes in cell shape induced by bromocriptine treatment, although forskolin did not have such an antagonizing effect. These results suggest that: 1) intracellular cAMP plays a stimulatory role in the regulation of lactotroph proliferation; 2) cAMP and insulin/mitogen-activated protein kinase signalings require each other for their mitogenic actions; and 3) the antimitogenic action of bromocriptine is, at least in part, caused by inhibition of cAMP production.     

Urinary excretion of calcium, hydroxyproline and 3',5'-cyclic adenosine monophosphate in primary hyperparathyroidism.

Urinary excretion of calcium (Ca), hydroxyproline (Hyp) and 3',5'-cyclic adenosine monophosphate (cAMP) was measured during fasting, and in the afternoon, over a 3 day period. Twelve hyperparathyroid patients, of whom 6 were re-studied after successful parathyroid surgery, and 10 control subjects participated, and were maintained on a collagen free diet for the duration of the study. Expressed as creatinine ratio values, Hyp was significantly higher in the morning than during the afternoon, whereas the Ca excretion pattern showed low morning and high afternoon values for all groups. cAMP excretion did not change during the two sampling periods. Large day to day variations for each parameter were observed in the individual patient. The value of cAMP measurements in the diagnosis of primary hyperparathyroidism was confirmed. The results may imply that a diurnal variation in Hyp excretion exists in primary hyperparathyroidism and that food intake produces a suppression of Hyp excretion, possibly secondary to suppression of parathyroid function or, in our view, to increased calcitonin excretion.     

C-Peptide induces vascular smooth muscle cell proliferation: involvement of SRC-kinase, phosphatidylinositol 3-kinase, and extracellular signal-regulated kinase 1/2

Increased levels of C-peptide, a cleavage product of proinsulin, circulate in patients with insulin resistance and early type 2 diabetes mellitus. Recent data suggest a potential causal role of C-peptide in atherogenesis by promoting monocyte and T-lymphocyte recruitment into the vessel wall. The present study examined the effect of C-peptide on vascular smooth muscle cells (VSMCs) proliferation and evaluated intracellular signaling pathways involved. In early arteriosclerotic lesions of diabetic subjects, C-peptide colocalized with VSMCs in the media. In vitro, stimulation of human or rat VSMCs with C-peptide induced cell proliferation in a concentration-dependent manner with a maximal 2.6+/-0.8-fold induction at 10 nmol/L human C-peptide (P<0.05 compared with unstimulated cells; n=9) and a 1.8+/-0.2-fold induction at 0.5 nmol/L rat C-peptide (P<0.05 compared with unstimulated cells; n=7), respectively, as shown by [H3]-thymidin incorporation. The proliferative effect of C-peptide on VSMCs was inhibited by Src short interference RNA transfection, PP2, an inhibitor of Src-kinase, LY294002, an inhibitor of PI-3 kinase, and the ERK1/2 inhibitor PD98059. Moreover, C-peptide induced phosphorylation of Src, as well as activation of PI-3 kinase and ERK1/2, suggesting that these signaling molecules are involved in C-peptide-induced VSMC proliferation. Finally, C-peptide induced cyclin D1 expression as well as phosphorylation of Rb in VSMCs. Our results demonstrate that C-peptide induces VSMC proliferation through activation of Src- and PI-3 kinase as well as ERK1/2. These data suggest a novel mechanism how C-peptide may contribute to plaque development and restenosis formation in patients with insulin resistance and early type 2 diabetes mellitus.     

Calcium-membrane interactions in the myocardium: effects of ouabain, epinephrine and 3',5'-cyclic adenosine monophosphate

Cardiac microsomes, which represent an enriched but not pure preparation of the heart's sarcoplasmic reticulum, can remove calcium from solution by 2 kinetically dissimilar mechanisms. In the presence of adenosine triphosphate (ATP), Ca⁺⁺ is taken up by cardiac microsomes by a process designated Ca-binding, which exhibits saturation kinetics. The rate and extent of Ca-binding, and the high affinity of the Ca-binding sites could allow this process to cause the intact cell to relax. When anions that permit Ca⁺⁺ to be precipitated within the mlcrosomal vesicles are included along with ATP, much larger amounts of Ca⁺⁺ are taken up by cardiac microsomes. This second process, designated Ca-uptake, does not follow saturation kinetics. Instead, the rate of Ca-uptake increases linearly with increasing Ca⁺⁺ concentration until Ca-uptake becomes inhibited at higher Ca⁺⁺ concentrations.The finding of 2 kinetically distinct Ca⁺⁺ transport processes in cardiac microsomes, both of which are highly active in the micromolar range of Ca⁺⁺ concentration, suggests that Ca⁺⁺ movements in the intact myocardial cell may be controlled by 2 mechanisms. It is suggested that one of these, possibly manifest in vitro as Ca-binding, represents an intracellular release site that initiates systole by delivering Ca⁺⁺ to the contractile proteins. The second process, possibly manifest in vitro as Ca-uptake, is suggested to represent the uptake of Ca⁺⁺ into an intracellular storage site whose Ca⁺⁺ content indirectly determines the amount of Ca⁺⁺ that is delivered to the contractile proteins. These 2 intracellular Ca⁺⁺ pools can be tentatively related to Ca⁺⁺ movements into and out of the myocardial cell, permitting the formulation of a model by which a number of inotropic interventions might modulate myocardial contractility.Cardiac glycosides had no detectible effect on either cardiac microsomal Ca-binding or Ca-uptake. Cyclic adenosine monophosphate (cAMP), which by itself was without effects on cardiac microsomes, more than doubled the rate of Ca-uptake in the presence of a cyclic AMP-dependent protein kinase. The resulting increase in rate of Ca-uptake could explain the actions of epinephrine to enhance contractility at the same time that systole is abbreviated.     

Enhanced effectiveness of pulsatile 3',5'-cyclic adenosine monophosphate in stimulating prolactin and alpha-subunit gene expression.

cAMP is involved in the regulation of secretory activity in lactotrope, thyrotrope, and gonadotrope cells. The present study examined whether pulsatile or intermittent changes in cAMP are more effective than a continuous stimulation in increasing pituitary hormone gene expression. Pituitaries from adult female rats were dissociated, plated for 48 h (7-8 x 10(6) cells per well) to allow attachment to Matrigel-coated plastic coverslips, then inserted into perifusion chambers (five to eight chambers per group). After 24 h of treatment, the cells were recovered, RNA extracted, and messenger RNAs (mRNAs) determined by dot blot hybridization. Perfused cells were exposed to either hourly pulses of monobutyryl cAMP (Bt cAMP, 0.01, 0.1, or 1 mM; 1 mM butyrate pulses to controls), or continuously to forskolin (10 microM). Bt cAMP pulses increased both PRL and alpha-subunit mRNAs, maximal after the 0.1 mM dose for PRL (51% increase vs. butyrate controls) and after the 1 mM dose for alpha (60% increase). However, forskolin was ineffective in increasing PRL or alpha mRNA concentrations. TSH, LH, and FSH beta-subunit mRNAs were not altered by Bt cAMP pulses or forskolin. To confirm the different effects of pulsatile vs. continuous cAMP on PRL and alpha-subunit mRNAs, the response to pulsatile 8-bromo cAMP (1 mM) or Bt cAMP (0.5 mM) was compared to continuous Bt cAMP (0.5 mM). PRL and alpha-subunit mRNAs were increased by both cAMP analogs given in a pulsatile manner but not by continuous Bt cAMP. PRL and LH secretory responses (determined in perifusate samples after 2 h and 22 h of treatment) revealed that both PRL and LH release was increased by cAMP stimulation, given either in a pulsatile or continuous manner. These results show that PRL and alpha-subunit gene expression were sensitive to changes in cAMP stimulation, whereas that of TSH, LH, and FSH beta were unaltered. Only intermittent cAMP stimuli were effective in increasing PRL and alpha mRNAs. These data suggest that pulsatile fluctuations in intracellular cAMP may be essential for maximal expression of the PRL and alpha genes. Thus, intermittent changes in intracellular second messengers may be a necessary part of the pathway involved in the transduction of signals from the plasma membrane to the nucleus.     

Angiotensin II potentiates agonist-induced 3',5'-cyclic adenosine monophosphate production by cultured bovine adrenal cells through protein kinase C and calmodulin pathways.

Interactions between signal transducing systems may be important in the integrated control of cellular processes in basal and hormonally regulated cells. The cultured bovine adrenal fasciculata cell provides a model to study the interactions between the cAMP and calcium-sensitive phospholipid dependent protein kinase C. In this study, angiotensin II (A-II) and phorbol ester (PMA) potentiated the stimulatory actions of ACTH in a dose-dependent manner on cAMP production. At maximal concentrations, A-II and PMA also potentiated the effects of cholera toxin and forskolin on cAMP production. Both staurosporine, a protein kinase C inhibitor, and desensitization of protein kinase C by a 24-h pretreatment with PMA blunted the effect of PMA, but only partially inhibited (34%) the effect of A-II. Neither nifedipine, a specific calcium channel antagonist, nor pretreatment of cells with pertussis toxin modified the amplifying effects of A-II or PMA. In contrast, trifluoperazine, a calmodulin inhibitor, reduced the potentiating effect of A-II by about 35%, but association with staurosporine blunted its effects. Moreover, the steroidogenic effects of ACTH plus A-II were more than additive, but this synergism was blunted in the presence of both inhibitors. In conclusion, PMA and A-II potentiated agonist-induced cAMP production by bovine adrenal fasciculata cells. The data suggest that the effects of PMA were mediated exclusively by protein kinase C, whereas those of A-II were mediated by both protein kinase C and calmodulin.     

Regulation of tetrahydrobiopterin biosynthesis in cultured adrenal cortical tumor cells by adrenocorticotropin and adenosine 3',5'-cyclic monophosphate

Y-1 adrenal cortical tumor cells in culture, which contain substantial amounts of tetrahydrobiopterin [6R-(L-erythro-1',2'-dihydroxypropyl)5,6,7,8-tetrahydropterin] (BH4) and GTP cyclohydrolase (GTP-CH), were used to study the regulation of BH4 biosynthesis by ACTH and cAMP. ACTH produced a dose-dependent increase in steroidogenesis, BH4 levels and GTP-CH activity. Maximal stimulation of BH4 biosynthesis occurred at the same concentration of ACTH that caused maximal stimulation of steroidogenesis. ACTH-(1-24) was more potent than ACTH-(1-39). The stimulation of BH4 biosynthesis by ACTH was dependent on cell density, being greater at lower cell densities, but was independent of time in culture. The lack of stimulation by ACTH at higher cell densities was due to an increase in the specific activity of GTP-CH in the control cells as density increased. This increase may be due in part to the increased release of steroids, since exogenous steroids added to low density cultures also resulted in an increase in the specific activity of the enzyme. Addition of steroids had no effect on ACTH-dependent stimulation of BH4 biosynthesis at low cell densities. (Bu)2cAMP, 8-bromo-cAMP, and forskolin all produced time- and dose-dependent increases in BH4 levels, GTP-CH activity, and steroidogenesis. Maximum increases in GTP-CH and BH4 occurred at concentrations similar to those required for maximal stimulation of steroidogenesis. In the Kin-8 mutant of Y-1 cells, which has a type 1 cAMP-dependent protein kinase with an altered regulatory subunit, ACTH was unable to increase BH4 levels or GTP-CH activity at a concentration that produced maximal stimulation of BH4 and steroid biosynthesis in the parent Y-1 line. These studies indicate that Y-1 cells in culture are useful for studying the regulation of BH4 biosynthesis in the adrenal cortex.     

Differential effects of the 3',5'-cyclic adenosine monophosphate and protein kinase C pathways on the response of isolated rat osteoclasts to calcitonin.

Calcitonin (CT) activates both the cAMP and the protein kinase C (PKC) pathways in the kidney cell line LLC-PK1. Although CT also activates cAMP in osteoclasts, its effects on PKC in this cell type are unknown. In order to determine whether the response of osteoclasts to CT also involves the PKC pathway, the effects of activators and inhibitors of PKC on bone resorption and cell surface area were analyzed in isolated rat osteoclasts. As expected, CT inhibited in a dose-dependent manner bone resorption by rat osteoclasts cultured for 24 h on devitalized bovine bone slices and this effect could be mimicked by cAMP. The inhibitory effect of CT could however also be mimicked by phorbol-12,13-dibutyrate (PDBu) and blocked by the PKC inhibitor sphingosine, as well as by the less specific inhibitors H7 and H8, none of which had detectable effects in the absence of CT. No changes in the number of attached osteoclasts were observed under any of these conditions. These results indicate that CT activates PKC in osteoclasts and that this activation, like the activation of cAMP-dependent protein kinase, leads to an inhibition of bone resorption. Quantitative time-lapse videomicroscopy showed that the CT-induced retraction of osteoclasts also involved activation of the PKC pathway and could therefore be induced by phorbol esters. In contrast, (Bu)2 cAMP (1-200 microM) failed to induce rapid cell retraction. It is concluded that, in osteoclasts, CT receptors are coupled to both the cAMP-dependent protein kinase and the PKC pathways. Although these two second messengers can have additive inhibitory effects on bone resorption, only activation of the PKC pathway induces rapid cell retraction. These two effects of calcitonin on osteoclasts are therefore independent and may be functionally unrelated.     

Hyperglycemia enhances adipogenic induction of lipid accumulation: involvement of extracellular signal-regulated protein kinase 1/2, phosphoinositide 3-kinase/Akt, and peroxisome proliferator-activated receptor gamma signaling

The molecular events of hyperglycemia-triggered increase in adipogenic induction of lipid accumulation remain unclear. We examined the effects of hyperglycemia on adipogenic induction of lipid accumulation and its involved signaling molecules, such as phosphoinositide 3-kinase (PI3K), ERKs, and peroxisome proliferator-activated receptor gamma (PPAR gamma). Bone marrow-derived mesenchymal stem cells (MSCs) isolated from FVB/N mice were capable of differentiating into adipocytes in adipogenic medium. The effects of high glucose (HG) (25.5 mm) were assessed in vitro by RT-PCR, ELISA, flow cytometry, immunostaining, and immunoblotting. The in vivo effect of hyperglycemia was further studied in streptozotocin (STZ)-induced diabetic FVB/N mice. Exposure of MSCs to HG enhanced adipogenic induction of lipid accumulation as compared with 5.5 mm glucose. HG increased PPAR gamma expression and PI3K activity and its downstream effector Akt phosphorylation during adipogenesis. Inhibition of PI3K/Akt activity with PI3K inhibitor LY294002 or by expressing the dominant negative p85 or Akt prevented the HG-enhanced PPAR gamma-dependent adipogenic induction of lipid accumulation. Moreover, HG increased the phosphorylation of ERK1/2 during adipogenesis. MAPK/ERK inhibitor PD98059 inhibited the PI3K activity, Akt phosphorylation, and lipid accumulation triggered by HG. PI3K inhibitor LY294002 did not affect the HG-increased ERK1/2 phosphorylation during adipogenesis. We next observed that adipogenic induction of lipid accumulation of MSCs isolated from STZ-induced diabetic mice is enhanced. Moreover, triglyceride, PPAR gamma expression, phosphorylated Akt and ERK1/2, and marrow fat in bones of STZ-diabetic mice were also increased. These results suggest that hyperglycemia enhances the adipogenic induction of lipid accumulation through an ERK1/2-activated PI3K/Akt-regulated PPAR gamma pathway.     

Genistein activates the 3',5'-cyclic adenosine monophosphate signaling pathway in vascular endothelial cells and protects endothelial barrier function

The soy phytoestrogen, genistein, has an array of biological actions, including weak estrogenic effects, inhibition of tyrosine kinase, and cellular antioxidant activity. Recent studies showed that genistein may improve vascular function, but the mechanism is unclear. We show that genistein stimulates intracellular cAMP accumulation in intact bovine aortic endothelial cells and human umbilical vein endothelial cells over an incubation period of 30 min. Increases in intracellular cAMP are evoked by as low as 10 nm genistein but not by estrogen. These increases in cAMP may result primarily from enhanced adenylate cyclase activity by a mechanism that does not involve genomic actions or estrogen receptors. The cAMP induced by genistein activates cAMP-dependent protein kinase (PKA) in bovine aortic endothelial cells. The activation of PKA phosphorylates and activates cAMP response element-binding protein, leading to up-regulation of cAMP response element-containing gene expression. In addition, activation of PKA protects thrombin-induced endothelial monolayer permeability, a novel cardioprotective effect of genistein mediated by the cAMP/PKA cascade. These findings demonstrate that a nongenomic action of genistein leads to activation of the cAMP/PKA signaling system to protect the vascular barrier function and alter the expression of cAMP-regulated genes, thereby providing a novel mechanism underlying some of the cardiovascular protective effects proposed for soy phytoestrogens.     

Effects of ouabain on insulin release, adenosine 3',5'-monophosphate and guanine 3',5'-monophosphate in pancreatic islets.

Isolated pancreatic islets of noninbred ob/ob mice were used to test the hypothesis that adenylate cyclase responds to changes of the transmembrane milieu or electric field in intact beta-cells. In the presence of a phosphodiesterase inhibitor, ouabainstimulated both the release of insulin and the islet content of cAMP. Ouabain had no noticeable effect on the islet content of cGMP. These results support the hypothesis at test. However, because ouabain also had some stimulatory effect on cAMP in islet homogenates, a direct action of ouabain on adenylate cyclase cannot be ruled out.     

Control of FSH receptor mRNA expression in rat granulosa cells by 3',5'-cyclic adenosine monophosphate, activin, and follistatin.

FSH is required to maintain FSH and LH/hCG receptors at elevated steady-state levels after receptor induction. Although this function of FSH is mediated by cAMP, how cAMP level is related to the maintenance of gonadotropin receptors is unknown. To investigate cAMP's effect on changes in the levels of FSH receptor mRNAs in rat granulosa cells, total RNA from cells was prepared and analyzed by Northern blots. Incubation with 8-Br-cAMP for 24 h produced a dose-related increase in FSH receptor mRNA in granulosa cells of DES-primed immature rats. On the other hand, 8-Br-cAMP, washed at 24 h, exerted inverse dose-related effects on FSH receptor mRNA levels at 96 h. The addition of 1 mM cAMP resulted in higher levels of FSH receptor mRNA than that induced by 0.2 mM cAMP at 24 h, while 0.2 mM cAMP is as effective as 1-2 mM cAMP for the induction of FSH receptor mRNA at 96 h. To further analyze cAMP's role in the production of activin in granulosa cells, we measured activin levels in the culture medium after the addition of 8-Br-cAMP. The levels of activin A were suppressed by the addition of 8-Br-cAMP in a dose-dependent manner. In addition, the procedure by which 8-Br-cAMP was removed after 24 h incubation showed that the level of activin in the medium increased after medium change. With regard to the actions of activin A on gonadotropin receptors, our laboratory has demonstrated that activin A increases the levels of FSH receptor mRNAs. Therefore, cAMP has a negative effect on FSH receptor expression by suppressing the activin level. Since follistatin production is up-regulated by cAMP in this system, we examined the effect of follistatin on FSH receptor mRNA level, which is induced by activin and FSH. Cotreatment with follistatin (0-100 ng/ml) and activin (50 ng/ml) in the presence of FSH (30 ng/ml) caused a significant reduction in FSH receptor mRNA levels induced by activin. Based on these observations, it is possible that cAMP has both stimulatory and inhibitory effects on the expression of gonadotropin receptors, and the overall influence of cAMP on their expression might be determined by the integration of such opposing effects.