Increased cAMP in monocytes augments Notch signaling mechanisms by elevating RBP-J and transducin-like enhancer of Split (TLE).

In cells of the innate immune system, pathological increases in intracellular cAMP attenuate immune responses and contribute to infections by bacteria such as Bacillus anthracis. In this work, cAMP from B. anthracis edema toxin (ET) is found to activate the Notch signaling pathway in both mouse macrophages and human monocytes. ET as well as a cell-permeable activator of PKA induce Notch target genes (HES1, HEY1, IL2RA, and IL7R) and are able to significantly enhance the induction of these Notch target genes by a Toll-like receptor ligand. Elevated cAMP also resulted in increased levels of Groucho/transducin-like enhancer of Split (TLE) and led to increased amounts of a transcriptional repressor complex consisting of TLE and the Notch target Hes1. To address the mechanism used by ET to activate Notch signaling, components of Notch signaling were examined, and results revealed that ET increased levels of recombinant recognition sequence binding protein at the Jκ site (RBP-J), a DNA binding protein and principal transcriptional regulator of Notch signaling. Overexpression studies indicated that RBP-J was sufficient to activate Notch signaling and potentiate LPS-induced Notch signaling. Further examination of the mechanism used by ET to activate Notch signaling revealed that C/EBP ß, a transcription factor activated by cAMP, helped activate Notch signaling and up-regulated RBP-J. These studies demonstrate that cAMP activates Notch signaling and increases the expression of TLE, which could be an important mechanism utilized by cAMP to suppress immune responses.

Role of Epac and protein kinase A in thyrotropin-induced gene expression in primary thyrocytes.

cAMP pathway activation by thyrotropin (TSH) induces differentiation and gene expression in thyrocytes. We investigated which partners of the cAMP cascade regulate gene expression modulations: protein kinase A and/or the exchange proteins directly activated by cAMP (Epac). Human primary cultured thyrocytes were analysed by microarrays after treatment with the adenylate cyclase activator forskolin, the protein kinase A (PKA) activator 6-MB-cAMP and the Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP (007) alone or combined with 6-MB-cAMP. Profiles were compared to those of TSH. Cultures treated with the adenylate cyclase- or the PKA activator alone or the latter combined with 007 had profiles similar to those induced by TSH. mRNA profiles of 007-treated cultures were highly distinct from TSH-treated cells, suggesting that TSH-modulated gene expressions are mainly modulated by cAMP and PKA and not through Epac in cultured human thyroid cells. To investigate whether the Epac-Rap-RapGAP pathway could play a potential role in thyroid tumorigenesis, the mRNA expressions of its constituent proteins were investigated in two malignant thyroid tumor types. Modulations of this pathway suggest an increased Rap pathway activity in these cancers independent from cAMP activation.

The cAMP analogue, dbcAMP affects release of steroid hormones by cultured rabbit ovarian cells and their response to FSH, IGF-I and ghrelin.

The aim of the present study was to examine possible involvement of cAMP-dependent intracellular mechanisms in control of ovarian cell steroidogenesis and its response to hormonal regulators. For this purpose, we examined the influence of administration of dbcAMP, a cAMP analogue (50 microg/animal) in vivo, on release of progesterone, testosterone and estradiol by isolated ovarian fragments, as well their response to hormonal regulators of ovarian steroidogenesis-FSH, IGF-I and ghrelin (all added at doses of 100 ng/ml). It was observed, that administration of dbcAMP resulted reduction in progesterone and testosterone, but not of estradiol release by isolated ovarian fragments. In ovarian tissue isolated from control animals, additions of hormones were able to reduce release of progesterone (FSH, IGF-I and ghrelin) and increase release of testosterone (ghrelin) but did not change estradiol output. Previous administration of dbcAMP modified action of exogenous hormones: it inverted inhibitory action of FSH, IGF-I and ghrelin on progesterone release to stimulatory action and induced stimulatory action of IGF-I on testosterone release and stimulatory effect of FSH on estradiol output. The present observations confirm involvement of peptide hormones FSH, IGF-I and ghrelin in the control of rabbit ovarian steroid hormones release and demonstrate the involvement of cAMP-dependent intracellular mechanisms in down-regulation of rabbit ovarian steroidogenesis and in modification, but not in mediating effect of FSH, IGF-I and ghrelin on ovarian steroid hormones release.

AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells.

Acidic luminal pH and low [HCO(3)(-)] maintain sperm quiescent during maturation in the epididymis. The vacuolar H(+)-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HCO(3)(-), induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and non-human primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer compared with that of controls perfused without drug. Moreover, preperfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by N(6)-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [(32)P]orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNA interference-mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knockdown. In summary, our findings suggest that AMPK inhibits PKA-mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK.

Activation and induction of cyclic AMP phosphodiesterase (PDE4) in rat pulmonary microvascular endothelial cells.

Regulation of the rolipram-sensitive cAMP-specific phosphodiesterase 4 (PDE4) gene family was studied in rat pulmonary microvascular endothelial cells (RPMVECs). Total PDE4 hydrolysis was increased within 10 min after addition of forskolin (10 microM), reached a maximum at 20-40 min, and then gradually declined in the cells. A similar activation of PDE4 activity was observed using a protein kinase A (PKA) activator, N(6)-monobutyryl cAMP. Both the forskolin and the N(6)-monobutyryl cAMP activated PDE4 activities were blocked by the PKA-specific inhibitor, H89. This forskolin-stimulated and PKA-mediated short-term activation of PDE4 activity was further confirmed by in vitro phosphorylation of 87kDa PDE4A6 and 83kDa PDE4B3 polypeptides using exogenous PKA Calpha. Increased immunoreactivity of phosphorylated PDE4A6 in situ was detected in Western blots by a PDE4A-phospho antibody specific to the putative PKA phosphorylation sites. Following long-term treatment of RPMVECs with rolipram and forskolin medium (RFM) for more than 60 days, PDE4 activity reached ten-fold higher values than control RPMVECS with twenty-fold increases detected in intracellular cAMP content. The RFM cells showed increased immunoreactivities of the constitutive 4A6 and 4B3 isoforms plus two novel splice variants at 101kDa (4B1) and 71kDa (4B2). Treatment with H89 did not inhibit the PDE4 elevation in RFM cells. In addition to the increased levels of PDE4 in RFM cells, immunofluorescence showed a translocation of PDE4A and 4B to a nuclear region, which was normally not observed in RPMVECs. The PDE4 activity in RFM cells decayed rapidly with an even faster decline of intracellular cAMP content when forskolin/rolipram were removed from the medium. These results suggest that both the activation (short-term) and induction (long-term) of PDE4A/4B isoforms in RPMVECs are closely modulated by the intracellular cAMP content via both post-translational and synthetic mechanisms.

Expression of aquaporin 8 and its up-regulation by cyclic adenosine monophosphate in human WISH cells.

OBJECTIVE: Water absorption across the fetal chorioamniotic membranes is a critical regulatory pathway for amniotic fluid volume homeostasis. Aquaporins are cell membrane proteins that significantly enhance membrane permeability to water by acting as water channels. We recently demonstrated that aquaporin 8 is expressed in human amnion, chorion, and placenta. Thus, aquaporin 8 expression represents a molecular mechanism of amniotic water absorption through intramembranous pathways. The current study sought to determine whether aquaporin 8 is expressed in human amnion-derived cell culture and to explore its regulation by second messenger cyclic adenosine monophosphate. STUDY DESIGN: Human amnion-derived WISH cells were cultured. Total RNA was isolated and reverse transcriptase-polymerase chain reaction was used to determine aquaporin 8 gene expression. To determine the effect of cyclic adenosine monophosphate on aquaporin 8 expression, WISH cells were cultured in the presence of either monobutyryl cyclic adenosine monophosphate or the cyclic adenosine monophosphate-elevating agent forskolin. Multiplex semiquantitative reverse transcriptase-polymerase chain reaction was carried out to quantify aquaporin 8 messenger RNA levels. RESULTS: Reverse transcriptase-polymerase chain reaction detected aquaporin 8 expression in WISH cells. After forskolin treatment for 2 hours, aquaporin 8 messenger RNA expression in WISH cells increased 4-fold (P <.001). Stimulation of aquaporin 8 gene expression by colforsin was observed throughout the study period of 20 hours. Incubation of WISH cells with monobutyryl cyclic adenosine monophosphate resulted in a 2-fold increase in aquaporin 8 messenger RNA level (P <.001). However, stimulation of aquaporin 8 gene expression by monobutyryl cyclic adenosine monophosphate attenuated to baseline level after 20 hours of monobutyryl cyclic adenosine monophosphate treatment. CONCLUSION: The current study demonstrates the expression of aquaporin 8 water channel in human amnion-derived WISH cells and aquaporin 8 expression up-regulation by second messenger cyclic adenosine monophosphate. Aquaporin 8 messenger RNA demonstrates a relatively short biologic half-life in vitro, which renders its rapid responsiveness to regulation

Hypertonicity increases cAMP in PMN and blocks oxidative burst by PKA-dependent and -independent mechanisms.

Hypertonic stress (HS) suppresses neutrophil (PMN) functions. We studied the underlying mechanism and found that HS rapidly (<1 min) increased intracellular cAMP levels by up to sevenfold. cAMP levels correlated with applied hypertonicity and the degree of neutrophil suppression. HS and cAMP-elevating drugs (forskolin and dibutyryl cAMP-acetoxymethyl ester) similarly suppressed extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase activation and superoxide formation in response to N-formylmethionyl-leucyl-phenylalanine (fMLP) stimulation. Inhibition of cAMP-dependent protein kinase A (PKA) with H-89 abrogated the suppressive effects of HS, restoring fMLP-induced ERK and p38 activation and superoxide formation. Inhibition of phosphodiesterase with 3-isobutyl-1-methylxanthine augmented cAMP accumulation and the suppressive effects of HS, while inhibition of adenylyl cyclase with MDL-12330A abolished these effects. These findings suggest that HS-activated cAMP/PKA signaling inhibits superoxide formation by intercepting fMLP-induced activation steps upstream of ERK and p38. In contrast to its effects in the presence of moderate hypertonicity levels (40 mM), H-89 was unable to rescue neutrophil functions from suppression by higher hypertonicity levels (100 mM), indicating that more severe HS suppresses neutrophils via secondary PKA-independent mechanisms.

Effects of cortisol and growth hormone on lipolysis in human adipose tissue.

The in vitro effects of cortisol and GH on basal and stimulated lipolysis in human adipose tissue were studied using a tissue incubation technique. After preincubation for 3 days in control medium containing insulin, adipose tissue pieces were exposed to cortisol for 3 days. GH was added to the cortisol-containing medium during the last 24 h (day 6). Adipocytes were then isolated, and lipolysis was studied in the absence and presence of isoprenaline, noradrenaline, forskolin, and N-6-monobutyryl-cAMP. Cortisol reduced the basal rate of lipolysis (P < 0.01) and the sensitivity to isoprenaline compared to the control values (P < 0.01). Addition of GH to the cortisol-containing medium increased the basal rate of lipolysis (P < 0.01) and the sensitivity to isoprenaline (P < 0.01) to the control level and increased the maximum isoprenaline-induced lipolytic activity (P < 0.01). Similar effects were obtained in the presence of noradrenaline. Maximum forskolin-induced lipolytic activity was reduced after exposure of the tissue to cortisol (P < 0.05), whereas addition of GH antagonized this effect (P < 0.01). Induction of the maximum lipolytic activity with N-6-monobutyryl-cAMP was not influenced by the preceding hormone exposure. Addition of GH alone during the last 24 h of incubation increased the basal rate of lipolysis (P < 0.05) and resulted in a borderline significant increase in the maximum isoprenaline-induced lipolytic activity (P = 0.055), suggesting that GH induces lipolysis also in the absence of glucocorticoids. Thus, cortisol and GH have opposite effects on the basal lipolytic activity in human adipose tissue in vitro as well as on the sensitivity to catecholamines, GH being the lipolytic and cortisol the antilipolytic agent. The present findings are in agreement with in vivo observations.

Cyclic adenosine 3',5'-monophosphate regulates GLUT4 and GLUT1 glucose transporter expression and stimulates transcriptional activity of the GLUT1 promoter in muscle cells.

We have previously reported that innervation-dependent basal contractile activity regulates in an inverse manner the expression of GLUT1 and GLUT4 glucose transporters in skeletal muscle. Based on the facts that muscle innervation decreases and muscle denervation increases cAMP levels, we investigated whether cAMP might mediate the effects of innervation/denervation on glucose transporter expression. Treatment of L6E9 myotubes with 8-bromo-cAMP, forskolin, or monobutyryl-8-bromo-cAMP led to a marked decrease in GLUT4 protein levels; 8-bromo-cAMP also diminished GLUT4 messenger RNA (mRNA), suggesting pretranslational repression. In contrast, L6E9 myoblasts and myotubes responded to 8-bromo-cAMP or forskolin by increasing the cell content of GLUT1 protein. Induction of GLUT1 protein was a consequence of the activation of different mechanisms in myoblast and myotube cells; whereas 8-bromo-cAMP treatment caused a substantial increase in GLUT1 mRNA in myoblasts, no change in GLUT1 mRNA was detected in myotubes. The increase in GLUT1 mRNA in L6E9 myoblasts induced by 8-bromo-cAMP was the result of transcriptional activation, as concluded from transfection analysis of 2.1 kilobases of the rat GLUT1 gene promoter fused to the bacterial chloramphenicol acetyltransferase gene. Furthermore, the stimulatory effect of 8-bromo-cAMP on the transcriptional activity of the GLUT1 promoter required a 33-bp sequence lying 5' upstream of the transcription start site. In all, cAMP inversely regulates GLUT4 and GLUT1 glucose transporter expression in muscle cells. Furthermore, our results suggest that down-regulation of GLUT4 expression and up-regulation of GLUT1 expression in muscle associated with denervation are partly attributable to cAMP.

Somatostatin inhibits interleukin 6 release from rat cortical type I astrocytes via the inhibition of adenylyl cyclase.

Interleukin 6 is a pleiotropic cytokine produced in the central nervous system (CNS) that has been involved in both direct neurotrophic activities and in the regulation of the production of acute phase proteins both at peripheral and central levels. In rat cortical type I astrocytes, interleukin 6 release is under the control of cAMP-protein kinase A and calcium-phospholipids-protein kinase C systems. Somatostatin is a neuropeptide, acting as a neurotransmitter, highly concentrated within the CNS, where it has been involved in the modulation of learning and memory processes. The aim of this study was to characterize the effects of somatostatin on the release of interleukin 6 from rat cortical type I astrocytes and the intracellular mechanisms involved in this activity. Our results show that somatostatin, in a concentration-dependent manner, inhibited basal and forskolin-stimulated interleukin 6 release from rat cortical type I astrocytes in culture. The EC50 of the inhibitory action was calculated to be approximately 10 nM. Furthermore, this effect of somatostatin was completely abolished by pretreating cortical astrocytes with pertussis toxin that, uncoupling, by ADP-rybosylating, the inhibitory GTP-binding protein from the receptors, prevents the activation of the intracellular effectors such as the adenylyl cyclase enzyme. To identify the intracellular mechanism mediating the effects of somatostatin on the interleukin 6 release, we evaluated the peptide modulation of basal and stimulated intracellular accumulation of cAMP. In our experimental conditions somatostatin significantly inhibited both basal and forskolin-stimulated cAMP accumulation. Conversely, somatostatin did not affect the increase of interleukin 6 release induced by dibutyryl-cAMP, a nonhydrolizable cAMP analog that, bypassing the effects of somatostatin on adenylyl cyclase activity, directly activated protein kinase A. These observations support the hypothesis that somatostatin inhibitory activity on interleukin 6 release is mediated by its effects on cAMP production. Somatostatin analog SMS 201-995 did not affect interleukin 6 production either in basal or stimulated conditions. Since, SMS 201-995 was reported to bind with high affinity only to somatostatin receptors type 2, 3 and 5, the lack of effect of this compound on interleukin 6 release suggests that the inhibitory action of somatostatin could be mediated by the activation of either type 1 or type 4 somatostatin receptors. In conclusion, our data demonstrate that the release of interleukin 6 from rat cortical type I astrocytes is inhibited by somatostatin through the activation of a somatostatin receptor coupled to the inhibition of adenylyl cyclase via a G-protein sensitive to pertussis toxin.

Production, uptake, and metabolic effects of cyclic AMP in the bivascularly perfused rat liver.

Production, uptake, and metabolic effects of cyclic AMP (cAMP) were measured in the bivascularly perfused rat liver in anterograde and retrograde perfusion. Glucagon, cAMP, N6,2'-O-dibutyryl cAMP and N6-monobutyryl cAMP were infused into the portal vein (anterograde perfusion), the hepatic vein (retrograde perfusion), or the hepatic artery (anterograde and retrograde perfusion) in order to reach different cell populations. The following results were obtained: (1) cAMP release caused by glucagon was directly proportional to the cell spaces that were accessible via the hepatic artery in anterograde and retrograde perfusion; since the metabolic effects of glucagon were not proportional to the accessible cell spaces, this observation also implies a disproportion between cAMP release and metabolic effects of the hormone; (2) when cAMP and N6,2'-O-dibutyryl cAMP were given to all liver cells (e.g. when infused into the portal vein), their metabolic effects were qualitatively and quantitatively the same and qualitatively equal to the effects of glucagon; (3) the changes caused by cAMP were a function of the cell spaces that can be reached via the hepatic artery in anterograde and retrograde perfusion; this behaviour contrasts markedly with that of glucagon, whose metabolic effects were practically independent of the accessible cell spaces; and (4) the effects of N6,2'-O-dibutyryl cAMP and N6-monobutyryl cAMP were independent of the cell spaces that were accessible via the hepatic artery in anterograde and retrograde perfusion; in this respect their behaviour was equal to that of glucagon. It is apparent that exogenously added cAMP mimicked the metabolic effects of glucagon in the liver only when it was supplied to all liver cells. Since glucagon, N6,2'-O-dibutyryl cAMP, and N6-monobutyryl cAMP were able to produce a full response even when given to only 30% of the liver parenchyma, it was concluded that cAMP production under the stimulus of glucagon or in consequence of the metabolic transformation of N6,2'-O-dibutyryl cAMP and N6-monobutyryl cAMP occurs in a compartment to which exogenous cAMP has no access. cAMP generated within this compartment is possibly able to diffuse from cell to cell.

Cryptic receptors for insulin-like growth factor II in the plasma membrane of rat adipocytes--a possible link to cellular insulin resistance.

To further elucidate the mechanisms for short-term regulation of the receptor for insulin-like growth factor II (IGF-II), we investigated effects of insulin, cAMP and phosphatase inhibitors on cell surface 125I-IGF-II binding in rat adipocytes. Preincubation with the serine/threonine phosphatase inhibitor okadaic acid (OA, 1 microM) or the non-hydrolysable cAMP analogue N6-mbcAMP (4 mM) markedly impaired insulin-stimulated 125I-IGF-II binding. Furthermore, addition of OA enhanced the inhibitory effect exerted by N6-mbcAMP. N6-mbcAMP also induced an insensitivity to insulin which was normalized by concomitant addition of the tyrosine phosphatase inhibitor vanadate (0.5 mM). In contrast, vanadate did not affect the impairment in maximal insulin-stimulated 125I-IGF-II binding produced by either OA or N6-mbcAMP. Phospholipase C (PLC), which cleaves phospholipids at the cell surface, markedly enhanced cell surface 125I-IGF-II binding in a concentration-dependent manner. Scatchard analysis demonstrated that the effect of PLC was due to an increased number of binding sites suggesting that "cryptic' IGF-II receptors are associated with the plasma membrane (PM). PLC (5 U/ml) also reversed the N6-mbcAMP-induced decrease of 125I-IGF-II binding at a low insulin concentration (10 microU/ml). Taken together, these data indicate that cAMP, similar to its effects on the glucose transporter GLUT 4 and the insulin receptor, may increase the proportion of functionally cryptic IGF-II receptors in the PM through mechanisms involving serine phosphorylation, possibly of a docking or coupling protein. Tyrosine phosphorylation appears to exert an opposite effect promoting the full cell surface expression of receptors.

Cyclic nucleotide-induced termination of vitellogenin uptake by Hyalophora cecropia follicles.

Endocytosis of vitellogenin by isolated follicles of Hyalophora cecropia terminated after membrane-permeable analogs of cAMP or cGMP were added to the culture medium. Depending on the concentration of the analog, a lag period of 30 min to 3 h preceded termination. Forskolin and IBMX both stimulated a rise in endogenous cAMP, and this also induced termination, as did pharmacological activation of the cyclic nucleotide-dependent protein kinases PKA and PKG. Inhibitors of PKA or PKG protected follicles from the corresponding cyclic nucleotide effect. When cAMP or cGMP was added to homogenates of vitellogenic follicles, a 32 kDa polypeptide was phosphorylated; inhibition of PKA, prevented phosphorylation of this protein. The rate of vitellogenin uptake did not accelerate significantly when PKA or PKG was inhibited in culture, which suggests that these kinases are normally inactive or operating below threshold during the several days of vitellogenesis. They seem thus not to be involved in the steady-state modulation of protein uptake. A more likely function of this control pathway in follicle development would be to trigger the termination of vitellogenesis, which normally occurs spontaneously in follicles of this species as they reach a length of 2 mm.

Cyclic AMP-induced differentiation increases the synthesis of extracellular superoxide dismutase in rat C6 glioma.

The effect of membrane permeable cAMP analogues on the expression of extracellular superoxide dismutase (EC-SOD) was studied in rat C6 glioma. EC-SOD is constitutively expressed but stimulation with cAMP analogues still increased the EC-SOD transcription and the secreted SOD activity. The potency to enhance EC-SOD expression is correlated with the ability of the cAMP analogue to induce cAMP-dependent differentiation in C6. The increase in EC-SOD mRNA and in secreted activity depended on the concentration of the cAMP analogues and on the cultivation time. Twenty-four hours after addition of 0.5 mM N6, O'2-dibutyryl cAMP (dbcAMP) or N6-monobutyryl cAMP (N6-mbcAMP) EC-SOD mRNA expression increased approximately twofold, while stimulation for 68 h with 0.5 mM N6-mbcAMP or 1 mM 8-Chloro cAMP (ClcAMP) and 1 mM dbcAMP enhanced the mean secreted activity/cell three- and fivefold, respectively. O'2-monobutyryl cAMP (O'2-mbcAMP) did not affect EC-SOD synthesis. The enhancement in EC-SOD activity did not require activation of protein kinase A. ATP, TGF-beta, IFN-gamma, and LPS did not affect EC-SOD synthesis. The presented data point to a cAMP-dependent pathway for the enhanced expression of EC-SOD by glial cells in brain.

Mechanism of action of epidermal growth factor-induced porcine oocyte maturation.

EGF has been reported to promote oocyte maturation in several species, although the mechanism of action is not yet known. The present study is designed to determine the pathway used by EGF to enhance porcine oocyte maturation. Oocytes were aspirated from 2-5 mm follicles and cultured with various treatments in Medium-199 at 37 degrees C, 100% relative humidity, and 5% CO2 for 48 hr for the maturation study and 3 hr for intracellular cAMP measurement. Although treatment with 100 IU/ml hCG stimulated both intracellular cAMP formation and oocyte maturation, 10 ng/ml EGF stimulated oocyte maturation only. Dibutyryl cAMP (dbcAMP) inhibited oocyte maturation at 10(-5), 10(-4), and 10(-3) M concentration s in the control medium. However, in the presence of 10 ng/ml EGF, dbcAMP inhibited oocyte maturation only at a concentration of 10(-3) M. Increasing concentrations of EGF (i.e., 25 and 50 ng/ml) were ineffective in overcoming the inhibitory effect of dbcAMP at 10(-3) M. In contrast, EGF reversed the decreased maturation rate caused by transforming growth factor-beta. Phorbol myristate acetate (PMA), a tumor-promoting phorbol ester, enhanced the spontaneous maturation rate; 4 alpha-phorbol dideconoate, an inactive phorbol ester, did not show this effect. PMA- and EGF-stimulated porcine oocyte maturation is reversed by calphostin-C, a PKC inhibitor. In conclusion, EGF's promotional activity on porcine oocyte maturation is independent of the cAMP pathway and probably mediated by the PKC pathway.

Protein kinase activity-dependent inhibition of urokinase-type plasminogen activator gene transcription by cyclic AMP in human pre-B lymphoma cell line RC-K8.

We investigated the effects of cAMP on the urokinase-type plasminogen activator (uPA) production in human pre-B lymphoma cell line RC-K8 that is consistently secreting uPA in the conditioned medium. Both Bt2cAMP and PGE1 inhibited the uPA accumulation in a dose-dependent manner. Northern blot analysis and nuclear run-on assay revealed that uPA gene transcription was repressed by Bt2cAMP and the repression was negated by inhibition of de novo protein synthesis by cycloheximide. Pretreatment with H89 (N-[2-(p-bromocinnamyl-amino) ethyl]-5-isoquinoline sulfonamide), a specific cAMP-dependent protein kinase (PKA) inhibitor, strongly inhibited both the PKA activation and the supression of uPA mRNA accumulation induced by cAMP. H85 (N-[2-(N-formyl-p-chlorocinnamyl-amino) ethyl]-5-isoquinoline sulfonamide), which closely resembles H89 in its chemical structure but is not a selective inhibitor of PKA, showed little effect on the regulation of uPA gene regulation by Bt2cAMP. These results suggest that cAMP represses uPA gene transcription in human pre-B lymphoma cells through PKA pathway and in which de novo protein synthesis is required.

Regulation of calcium-activated nonselective cation channel activity by cyclic nucleotides in the rat insulinoma cell line, CRI-G1.

The regulation of a calcium-activated nonselective cation (Ca-NS+) channel by analogues of cyclic AMP has been investigated in the rat insulinoma cell line, CRI-G1. The activity of the channel is modulated by cyclic AMP in a complex way. In the majority of patches (83%) tested concentrations of cyclic AMP of 10 microM and above cause an inhibition of channel activity which is immediately reversible on washing. In contrast, lower concentrations of cyclic AMP, between 0.1 and 1.0 microM, produce a transient activation of channel activity in most patches (63%) tested. One group of analogues, including N6-monobutyryl cyclic AMP and N6, 2'-O-dibutyryl cyclic AMP reduced the activity of the Ca-NS+ channel at all concentrations tested and 2'-O-Monobutyryl cyclic AMP produced inhibition in all patches tested except one, at all concentrations. A second group produced dual concentration-dependent effects on Ca-NS+, low concentrations stimulating and high concentrations inhibiting channel activity. 6-Chloropurine cyclic AMP and 8-bromo cyclic AMP produced effects similar to those of cyclic AMP itself. In contrast, 8-[4-chlorophenylthio] cyclic AMP also showed a dual action, but with a high level of activation at all concentrations tested up to 1 mM. Ca-NS+ channel activity was also predominantly activated by low concentrations of Sp-cAMPS. The activating effects of both Sp-cAMPS and cyclic AMP are antagonized by Rp-cAMPS, which by itself only produced a weak inhibition of Ca-NS+ channel activity even at concentrations of 10 microM and above. The results are discussed in terms of a model in which cyclic AMP, and other cyclic nucleotides, modulate the activity of the Ca-NS+ channel by binding to two separate sites.

Acquisition of a transcriptionally permissive state during the 1-cell stage of mouse embryogenesis.

Zygotic gene transcription initiates during the 2-cell stage of mouse embryogenesis. To learn more of the nature and timing of events leading up to transcriptional activation, we evaluated the ability of enucleated 1-cell-stage embryos to support transcription of the 2-cell-stage-specific gene(s) encoding the 70,000-Da transcription-requiring complex (TRC). Nuclei were transplanted from transcriptionally inhibited alpha-amanitin or N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8)-treated 2-cell-stage embryos to either late or early enucleated 1-cell-stage recipients. Expression of the TRC gene(s) was much greater following transfer to late 1-cell than early 1-cell-stage recipients. In addition, treatment of early 1-cell-stage recipients with N6-monobutyryl cyclic AMP following transplantation of a nucleus from an H8-treated donor increased the rate of TRC synthesis to a value similar to that observed for late 1-cell-stage recipients. These results indicate that during the first cell cycle and prior to initiation of zygotic gene expression, the embryonic cytoplasm undergoes a transition from a transcriptionally nonpermissive to permissive state.

Confocal microscopy of genome exposure in normal, cancer, and reverse-transformed cells.

Genome exposure studies were carried out on malignant CHO-K1 and C6 rat glioma cells and their respective, phenotypically normal counterparts (reverse-transformed CHO-K1, and both reverse-transformed C6 glioma and normal rat fibroblasts). Cells were subjected to the nick-translation technique previously developed to make visible the exposed (i.e., DNase I-sensitive) nuclear DNA, and examined by both epifluorescence and confocal microscopy. The confocal microscopy, by permitting examination of sections throughout the nucleus, made possible clearer identification of the regions of exposed and sequestered DNA in the cells studied. A peripheral shell of exposed DNA with some discontinuities was displayed in the great majority of the cells with normal phenotype, but in none of the cancer cells. Both types of cells displayed regions of exposed DNA in the nuclear interior, particularly surrounding the nucleoli. In accordance with previous theoretical proposals we postulate: the peripheral nuclear shell of exposed DNA contains differentiation-specific genes that include the specific growth-control genes and that are functional in normal cells but not in cancer; the exposed genes surrounding the nucleoli may represent housekeeping genes active in both normal and cancer cells; and the DNase I-resistant DNA in the interior of the nucleus we postulate to consist for the most part of genes specific to alternative differentiation states and to be sequestered and inactive. Previous differences in evaluation of roles of peripheral and internal DNA sensitivity to DNAse I hydrolysis appear to be reconciled by this formulation. Identification of exposed DNA may be useful in cancer diagnosis.

Effects of cyclic adenosine 3',5'-monophosphate on amino acid transport and incorporation into protein in the rat aorta.

To assess the effects of cyclic AMP on amino acid transport and incorporation into aortic tissue protein, rat aortic rings were incubated with the cyclic AMP analog, N6-monobutyryl cyclic AMP (MBcAMP), the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (MIX), and radiolabeled amino acids. Subsequently, the aortic rings were homogenized in 5% trichloroacetic acid (TCA) and processed for liquid scintillation counting. Radioactivity present in the TCA supernatant following centrifugation was used to estimate amino acid transport. TCA-precipitable radioactivity was used as a measure of amino acid incorporation into protein. MBcAMP induced an increase in the uptake of [3H]alpha-aminoisobutyric acid into aortic rings and an increase in the incorporation of radiolabeled proline and leucine into TCA-precipitable protein. Similar effects were observed with low concentrations of MIX (0.025-0.25 mM); however, at higher concentrations of MIX, there was an attenuation of the effect or frank inhibition. Maximum stimulation of transport was observed within 90-120 min of the addition of MIX or MBcAMP to the incubation medium, whereas the effect on amino acid incorporation was not detectable until after 12 h of exposure to MIX or MBcAMP. The effects of cyclic AMP on transport were observed in both the tunica media and the tunica adventitia, whereas the effects on amino acid incorporation into protein were observed only in the tunica media. These data are consistent with a possible role for cyclic AMP in promoting changes in the tunica media that could lead to the development of vascular hypertrophy.