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PubMed1997

Summer camps for children and adolescents with kidney disease.

Summer camps, sports camps, and residential camps are readily available to children and adolescents across the country. However, children and adolescents with end stage renal disease (ESRD) may not be able to participate in summer camp experiences because of specialized medical needs (e.g., dialysis or immunosuppressive medications) and concerns about abilities to keep up with camp activities. With enhancements in pediatric nephrology care in the past 10 years, patients can be expected to attend school full time and participate in peer activities. In addition, attendance at summer camps becomes a possibility for these children, particularly camps aimed at children with ESRD. Twenty pediatric nephrology centers in North America were surveyed about their participation in summer camp programs. This article reviews these and attempts to elucidate the values of summer camp programs for pediatric ESRD patients.

Adolescent↗
PubMed1999

Modulation of dihydropyridine-sensitive calcium channels in Drosophila by a cAMP-mediated pathway.

Drosophila has proved to be a valuable system for studying the structure and function of ion channels. However, relatively little is known about the regulation of ion channels, particularly that of Ca2+ channels, in Drosophila. Physiological and pharmacological differences between invertebrate and mammalian L-type Ca2+ channels raise questions on the extent of conservation of Ca2+ channel modulatory pathways. We have examined the role of cyclic adenosine monophosphate (cAMP) cascade in modulating the dihydropyridine (DHP)-sensitive Ca2+ channels in the larval muscles of Drosophila, using mutations and drugs that disrupt specific steps in this pathway. The L-type (DHP-sensitive) Ca2+ channel current was increased in the dunce mutants, which have high cAMP concentration owing to cAMP-specific phosphodiesterase (PDE) disruption. The current was decreased in the rutabaga mutants, where adenylyl cyclase (AC) activity is altered thereby decreasing the cAMP concentration. The dunce effect was mimicked by 8-Br-cAMP, a cAMP analog, and IBMX, a PDE inhibitor. The rutabaga effect was rescued by forskolin, an AC activator. H-89, an inhibitor of protein kinase-A (PKA), reduced the current and inhibited the effect of 8-Br-cAMP. The data suggest modulation of L-type Ca2+ channels of Drosophila via a cAMP-PKA mediated pathway. While there are differences in L-type channels, as well as in components of cAMP cascade, between Drosophila and vertebrates, main features of the modulatory pathway have been conserved. The data also raise questions on the likely role of DHP-sensitive Ca2+ channel modulation in synaptic plasticity, and learning and memory, processes disrupted by the dnc and the rut mutations.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Epitope-specific signaling through CD45 on T lymphocytes leads to cAMP synthesis in monocytes after ICAM-1-dependent cellular interaction.

We recently demonstrated that different CD45 monoclonal antibodies (mAb) are able to induce cellular aggregation in human peripheral blood mononuclear cells (PBMC) through LFA-1/ICAM-1 interactions. Such interactions could be down-modulated by protein kinase (PK) A/G inhibitors, but were unaffected by inhibitors of PKC, suggesting the involvement of PKA or PKG in CD45 mAb-induced adhesion. In this study we show that after incubation of PBMC with several (but not all) mAb to CD45, CD45RO and CD45RA, intracellular cAMP, but not cGMP concentrations readily increase, reaching a maximum 30 min after start of activation. As evidenced by several lines of investigation cAMP accumulation was independent of Fc receptor-associated signaling as well as tyrosine phosphatase activity of CD45. In highly pure T lymphocytes, CD45 mAb were unable to induce cAMP synthesis, but readily did so after addition of autologous monocytes. After paraformaldehyde fixation of both quiescent or IFN-gamma/TNF-alpha-preactivated monocytes, cAMP production was no longer detectable, suggesting monocytes as the cell of origin for the increased cAMP synthesis. Further, cAMP accumulation in monocytes occurred after reconstitution to T lymphocytes preincubated with CD45 mAb and extensively washed. Importantly, pretreatment of T lymphocyte/monocyte mixtures with LFA-1 mAb and/or ICAM-1 mAb down-regulated CD45 mAb-induced cAMP synthesis. Finally, we demonstrate that CD45 mAb are not only capable of inducing cAMP production, but also of directly stimulating PKA enzyme activity. Based on the data presented, we propose that CD45 signaling in T lymphocytes subsequently activates cAMP accumulation and PKA activation in monocytes via LFA-1/ICAM-1-dependent cellular interactions.

Antibodies, Monoclonal↗
PubMed2004

Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high-cell-density cultures of Escherichia coli.

Guanosine-3',5'-tetraphosphate (ppGpp) and sigmaS, two regulators of the starvation response of Escherichia coli, have received increasing attention for monitoring cell physiological changes in production processes, although both are difficult to quantify. The kinetics of cAMP formation and degradation were not yet investigated in such processes, although the complex regulation of cAMP by synthesis, release, and degradation in connection with straightforward methods for analysis renders it a highly informative target. Therefore, we followed the cAMP concentration in various nonrecombinant and in four different recombinant glucose-limited fed-batch processes in different production scales. The intracellular cAMP concentration increases strongly at the end of the batch phase. Most cAMP is released to the cultivation medium. The rates of accumulation and degradation of extracellular cAMP are growth-rate-dependent and show a distinct maximum at a growth rate of about 0.35 h(-1). At very low growth rates, below 0.05 h(-1), extracellular cAMP is not produced but rather degraded, independent of whether this low growth rate is caused by glucose limitation or by the high metabolic load of recombinant protein production. In contrast to intracellular cAMP, which is highly unstable, analysis of extracellular cAMP is simpler and the kinetics of accumulation and degradation reflect well the physiological situation, including unlimited growth, limitation, and severe starvation of a production host.

Cell Culture Techniques↗
PubMed1988

The effect of caffeine, adenosine, and buffer ionic composition on the induction of cell-surface cAMP binding during starvation of Dictyostelium discoideum.

We have analyzed the effects of the cAMP relay inhibitor, caffeine, and the receptor antagonist, adenosine, on the regulation of the cell-surface cAMP receptor in suspension-starved Dictyostelium discoideum cells by measuring ammonium sulfate-stabilized binding of [3-H]cAMP to intact cells. When cells were starved in fast (230 r.p.m.) shaken suspension in 10 mM Na+/5 mM K+ phosphate buffer, pH 6.5, plus 1 mM CaCl2 and 2.5 mM MgCl2, and assayed for specific cAMP binding, receptor accumulation peaked at approximately 6 hours, reaching a maximum of 1.5 pmol cAMP bound/10(7) cells (saturation binding). Neither caffeine nor adenosine inhibited the accumulation of cAMP receptors. Similar results were obtained in caffeine-treated, slow shaken (90 r.p.m.) suspension cultures. These results suggest that starvation alone is sufficient stimulus to induce the cAMP receptor. We have also tested the effects of different buffer ionic compositions on the accumulation of cAMP receptors. Elevation of the monovalent ion concentration to 30-40 mM was found to significantly inhibit the induction of cAMP receptors.

Adenosine↗
PubMed1995

Involvement of cAMP in CD3 T cell receptor complex- and CD2-mediated apoptosis of human thymocytes.

During intrathymic T cell development, elimination of autoreactive T cell clones by programmed cell death (PCD or apoptosis) is an essential mechanism for self tolerance. The precise intracellular second messengers that lead to this process remain to be determined. In the present work, we show that treatment of freshly isolated thymocytes with an antagonist of the cAMP pathway, the Rp-cAMP, significantly decreases spontaneous death by apoptosis of human thymocytes in vitro. Addition of Rp-cAMP also rescues thymocytes from activation-induced apoptosis following the ligation of surface CD3/T cell receptor complex or CD2 antigens. A cAMP analog, the dibutyryl(Dibut)-cAMP increases PCD of human thymocytes in a dose-dependent manner. Growth and rescue from PCD of thymocytes in the presence of interleukin (IL)-2 or IL-4 are also enhanced by Rp-cAMP and inhibited by Dibut-cAMP. Finally, we detect substantial levels of intracellular cAMP in freshly isolated thymocytes. This study reveals the involvement of cAMP as a second messenger during the apoptosis of normal human thymocytes.

Apoptosis↗
PubMed1991

Negative regulation of transcription of the Saccharomyces cerevisiae catalase T (CTT1) gene by cAMP is mediated by a positive control element.

Transcription of the CTT1 (catalase T) gene of Saccharomyces cerevisiae is controlled by oxygen via heme, by nutrients via cAMP and by heat shock. Nitrogen limitation triggers a rapid, cycloheximide-insensitive derepression of the gene. Residual derepression in a cAMP-nonresponsive mutant with attenuated protein kinase activity (bcy1 tpk1w tpk2 tpk3) demonstrates the existence of an alternative, cAMP-independent nutrient signaling mechanism. Deletion analysis using CTT1-lacZ fusion genes revealed the contribution of multiple control elements to derepression, not all of which respond to the cAMP signal. A positive promoter element responding to negative control by cAMP was inactivated by deletion of a DNA region between base pairs -340 and -364. Upstream fragments including this element confer negative cAMP control to a LEU2-lacZ fusion gene. Northern analysis of CTT1 expression in the presence or absence of heme, in RAS2+ (high cAMP) and ras2 mutant (low cAMP) strains and in cells grown at low temperature (23 degrees C) and in heat-shocked cells (37 degrees C) shows that CTT1 is only induced to an appreciable extent when at least two of the three factors contributing to its expression (oxidative stress signaled by heme, nutrient starvation (low cAMP) and heat stress) activate the CTT1 promoter.

Base Sequence↗
PubMed1985

Reversible effects of dibutyryl cAMP on laminin and type IV collagen secretion from retinoic acid treated F9 cells.

The effects of dibutyryl cAMP on the differentiation of embryonal carcinoma F9 cells were studied mainly using the secretion of laminin and type IV collagen as the marker. For this purpose, F9 cells were labeled with 35S-methionine and radioactive proteins in the medium were analyzed by SDS-polyacrylamide gel electrophoresis. Treatment of F9 cells with retinoic acid alone induced differentiation into cells secreting type IV collagen. The combination of retinoic acid and dibutyryl cAMP stimulated laminin secretion in addition to type IV collagen secretion. This effect of dibutyryl cAMP was observed only 16 h after adding dibutyryl cAMP. Immunofluorescence staining demonstrated that the majority of the cells in culture were converted into cells secreting laminin under these conditions. In contrast to the irreversible effect of retinoic acid, the effect of dibutyryl cAMP on laminin and type IV collagen secretion was reversible at least during the first 5 days of maintaining cells in the medium containing retinoic acid plus dibutyryl cAMP. Removal of dibutyryl cAMP from the culture medium decreased the protein secretion to the basal levels within 2 days. This reversibility was not due to a change in cell number. An in vitro translation assay also suggested the reversible effect of dibutyryl cAMP on the levels of laminin mRNA. Coinciding with variations of the protein secretion, a reversible and homogeneous change in the morphology of retinoic acid generated F9 cells was observed by dibutyryl cAMP.

Animals↗
PubMed1991

Regulation of glucose transport as well as glucose transporter and immediate early gene expression in 3T3-L1 preadipocytes by 8-bromo-cAMP.

In the present study we have examined the ability of 8-bromoadenosine cyclic 3',5'-phosphate (8-bromo-cAMP; the membrane permeant analog of cAMP which can activate protein kinase A) to mimic hormone action and stimulate glucose transport and glucose transporter (GLUT-1) gene expression as well as the expression of several growth-related protooncogenes in quiescent 3T3-L1 fibroblasts. 8-Bromo-cAMP induced a rapid and prolonged increase in the rate of hexose transport. Early activation of hexose transport (within 30 min) was associated with increased plasma membrane immunoreactive glucose transporters, which corresponded to a doubling in the number of D-glucose-displaceable, plasma membrane cytochalasin B binding sites. The time course for 8-bromo-cAMP-induced hexose transport preceded the accumulation of GLUT-1 mRNA, which peaked between 4 and 8 h after exposure to the agent, and subsequently declined to approach basal (control) levels. Expression of the immediate-early genes c-fos and jun-B was induced by 8-bromo-cAMP on a rapid, but sustained time course, whereas induction of c-jun expression was delayed. Alterations in specific mRNAs following exposure to 8-bromo-cAMP were due to increased gene transcription (as judged by nuclear transcription run-on assays), although with respect to GLUT-1, an increase in mRNA stability was also observed. Treatment of the cells with forskolin resulted in the induction of GLUT-1 expression as well as expression of the immediate early genes. Exposure of quiescent 3T3-L1 fibroblasts to 8-bromo-cAMP resulted in a substantial increase in rates of total protein and RNA synthesis, but had little effect on DNA synthesis. The results demonstrate that 8-bromo-cAMP initiated a G0/G1 transition, but did not permit progression into S-phase. The results further suggest that increased cytosolic cAMP results in the stimulation of glucose transport by three distinct mechanisms to include translocation of pre-existing transporters, increased transcription of the GLUT-1 gene and increased stability of GLUT-1 mRNA.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2004

8-Cl-cAMP and its metabolite, 8-Cl-adenosine induce growth inhibition in mouse fibroblast DT cells through the same pathways: protein kinase C activation and cyclin B down-regulation.

8-Chloro-cyclic AMP (8-Cl-cAMP) is known to be most effective in inducing growth inhibition and differentiation of a number of cancer cells. Also, its cellular metabolite, 8-Cl-adenosine was shown to induce growth inhibition in a variety of cell lines. However, the signaling mechanism that governs the effects of 8-Cl-cAMP and/or 8-Cl-adenosine is still uncertain and it is not even sure which of the two is the key molecule that induces growth inhibition. In this study using mouse fibroblast DT cells, it was found that adenosine kinase inhibitor and adenosine deaminase could reverse cellular growth inhibition induced by 8-Cl-cAMP and 8-Cl-adenosine. And 8-Cl-cAMP could not induce growth inhibition in the presence of phosphodiesterase (PDE) inhibitor, but 8-Cl-adenosine could. We also found that protein kinase C (PKC) inhibitor could restore this growth inhibition, and both the 8-Cl-cAMP and 8-Cl-adenosine could activate the enzymatic activity of PKC. Besides, after 8-Cl-cAMP and 8-Cl-adenosine treatment, cyclin B was down-regulated and a CDK inhibitor, p27 was up-regulated in a time-dependent manner. These results suggest that it is not 8-Cl-cAMP but 8-Cl-adenosine which induces growth inhibition, and 8-Cl-cAMP must be metabolized to exert this effect. Furthermore, there might exist signaling cascade such as PKC activation and cyclin B down-regulation after 8-Cl-cAMP and 8-Cl-adenosine treatment.

2-Chloroadenosine↗
PubMed2006

Rap1 and p38 MAPK mediate 8-chloro-cAMP-induced growth inhibition in mouse fibroblast DT cells.

8-Cl-cAMP, which is known to induce differentiation, growth inhibition, and apoptosis in various cancer cells, has been investigated as a putative anti-cancer drug. Previously, we reported that 8-Cl-cAMP and its metabolite 8-Cl-adenosine induce growth inhibition and apoptosis through p38 mitogen-activated protein kinase (MAPK) activation. To further investigate the signal mechanisms that regulate the cellular effects of 8-Cl-cAMP, we focused on a small GTPase Rap1 that is known to be involved in growth inhibition and reverse-transformation. 8-Cl-cAMP and 8-Cl-adenosine could increase Rap1 activity, which was blocked by ABT702-an adenosine kinase inhibitor. This suggests that 8-Cl-cAMP-induced Rap1 activation is also dependent on the metabolic degradation of 8-Cl-cAMP. Overexpression of a constitutively active mutant form of Rap1 (Rap1V12) attenuated cellular growth and soft-agar colony formation, which was basically the same effect as that observed with the 8-Cl-cAMP treatment. Furthermore, the Rap1V12 transfectant showed a high level of p38 MAPK activation. However, 8-Cl-cAMP-induced Rap1 activation was not diminished by SB203580, a p38 MAPK inhibitor, suggesting that Rap1 activation might act upstream of p38 MAPK activation during 8-Cl-cAMP-induced growth inhibition.

3T3 Cells↗
PubMed1990

Stimulation of adrenergic development in neural crest cultures by a reconstituted basement membrane-like matrix is inhibited by agents that elevate cAMP.

Previous work (Maxwell and Forbes: Development 101:767-776, 1987) has shown that an overlay of reconstituted basement membrane-like (RBM) gel dramatically increased the number of catecholamine-positive (CA+) cells which differentiated in neural crest cultures. We report here that this increase was inhibited when cultures were grown for 7 days in the presence of agents that elevate cAMP, such as 8-bromo-cAMP and 3-isobutyl-1-methylxanthine. The action of 8-bromo-cAMP was dose dependent with a half-maximal effect at about 50 microM. The development of CA+ cells was dramatically reduced when 8-bromo-cAMP was present from days 0-4 in vitro, but was relatively unaffected if 8-bromo-cAMP was present from days 4-7 in vitro. The development of tyrosine hydroxylase immunoreactive cells was also inhibited by 8-bromo-cAMP. The addition of 8-bromo-cAMP increased the number of melanocytes and resulted in either no change or only modest reductions in the number of E/C8 and neurofilament immunoreactive cells, indicating that the effect on CA+ cell ontogeny was selective. In contrast to the effect of 8-bromo-cAMP, addition of 8-bromo-cGMP did not inhibit CA+ cell development in the presence of the RBM gel nor did it stimulate CA+ cell development in the absence of the RBM gel overlay. Our results suggest that cAMP may be an important regulator of phenotypic expression in at least some neural crest cell lineages.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1990

Induction and inhibition of meiotic maturation of amphibian (Rana dybowskii) follicular oocytes by forskolin and cAMP in vitro.

Previous studies have demonstrated that direct or indirect elevation of cAMP levels in cultured amphibian ovarian follicles simultaneously stimulated production of oocyte maturation-inducing steroid (progesterone) by the follicles and inhibited oocyte maturation induced by endogenous or exogenous hormone. The duration of cAMP stimulation influenced arrest and reinitiation of oocyte meiotic maturation in ovarian follicles of Rana dybowskii. Addition of forskolin (adenylate cyclase stimulator) to cultured follicles inhibited both progesterone- and frog pituitary homogenate (FPH)-induced oocyte maturation. Similar inhibitory results were obtained when hormone-treated follicles were cultured in the continual presence of cAMP. Oocyte maturation increasingly occurred in follicular oocytes when cAMP or forskolin addition was delayed following treatment with FPH or progesterone. Transient exposure (6-8 hr) of ovarian follicles to forskolin or cAMP markedly stimulated oocyte maturation as well as accumulation of progesterone as measured by radioimmunoassay within the ovarian follicles. Forskolin was more effective than cAMP, at the dose tested, in stimulating progesterone production and accumulation by the follicles. The data demonstrate that transient manipulation (elevation) of cAMP levels in cultured follicles, without added FPH or steroid, was sufficient to initiate oocyte maturation. Results suggest that, with transient exposure to forskolin or exogenous cAMP, there is a sequential increase and decrease in endogenous cAMP levels in the somatic cells and germ cell components of the ovarian follicle. These changes appear to mediate production of maturation-inducing steroid and secondarily allow its effects on the oocyte to be expressed.

Adenylyl Cyclase Inhibitors↗
PubMed2006

Differing mechanisms of cAMP- versus seawater-induced oocyte maturation in marine nemertean worms I. The roles of serine/threonine kinases and phosphatases.

Unlike in most animals, oocytes of marine nemertean worms initiate maturation (=germinal vesicle breakdown, GVBD) following an increase, rather than a decrease, in intraoocytic cAMP. To analyze how serine/threonine (Ser/Thr) kinase cascades involving mitogen-activated protein kinase (MAPK), maturation-promoting factor (MPF), cAMP-dependent protein kinase (PKA), and phosphatidylinositol 3-kinase (PI3K) regulate nemertean GVBD, oocytes of Cerebratulus sp. were treated with pharmacological modulators and stimulated with cAMP-elevating drugs or seawater (SW) alone. Both cAMP elevators and SW triggered GVBD while activating MAPK, its target p90Rsk, and MPF. Similarly, neither cAMP- nor SW-induced GVBD was affected by several Ser/Thr phosphatase inhibitors, and both stimuli apparently accelerated GVBD via a MAPK-independent, PI3K-dependent mechanism. However, inhibitors of Raf-1, a kinase that activates MAPK kinase, blocked GVBD and MAPK activation during SW-, but not cAMP-induced maturation. In addition, MPF blockers more effectively reduced GVBD and MAPK activity in SW versus in cAMP-elevating treatments. Moreover, the two maturation-inducing stimuli yielded disparate patterns of PKA-related MAPK activations and phosphorylations of putative PKA substrates. Collectively, such findings suggest that in maturing oocytes of Cerebratulus sp., Ser/Thr kinase cascades differ during cAMP- versus SW-induced GVBD in several ways, including MAPK activation modes, MPF-feedback loops, and PKA-related signaling pathways. Additional differences in cAMP- versus SW-induced oocyte maturation are also described in the accompanying study that deals with the roles of tyrosine kinase signaling during GVBD.

Androstadienes↗
PubMed2000

Wortmannin, a PI3-kinase inhibitor: promoting effect on insulin secretion from pancreatic beta cells through a cAMP-dependent pathway.

To determine the role of phosphatidylinositol 3-kinase (PI3-kinase) in the regulation of insulin secretion, we examined the effect of wortmannin, a PI3-kinase inhibitor, on insulin secretion using the isolated perfused rat pancreas and freshly isolated islets. In the perfused pancreas, 10(-8) M wortmannin significantly enhanced the insulin secretion induced by the combination of 8.3 mM glucose and 10(-5) M forskolin. In isolated islets, cyclic AMP (cAMP) content was significantly increased by wortmannin in the presence of 3.3 mM, 8.3 mM, and 16.7 mM glucose with or without forskolin. In the presence of 16.7 mM glucose with or without forskolin, wortmannin promoted insulin secretion significantly. On the other hand, in the presence of 8.3 mM glucose with forskolin, wortmannin augmented insulin secretion significantly; although wortmannin tended to promote insulin secretion in the presence of glucose alone, it was not significant. To determine if wortmannin increases cAMP content by promoting cAMP production or by inhibiting cAMP reduction, we examined the effects of wortmannin on 10(-4) M 3-isobutyl-1-methylxantine (IBMX)-induced insulin secretion and cAMP content. In contrast to the effect on forskolin-induced secretion, wortmannin had no effect on IBMX-induced insulin secretion or cAMP content. Moreover, wortmannin had no effect on nonhydrolyzable cAMP analog-induced insulin secretion in the perfusion study. These data indicate that wortmannin induces insulin secretion by inhibiting phosphodiesterase to increase cAMP content, and suggest that PI3-kinase inhibits insulin secretion by activating phosphodiesterase to reduce cAMP content.

1-Methyl-3-isobutylxanthine↗
PubMed2001

Gprk2 controls cAMP levels in Drosophila development.

G protein-coupled receptors mediate their biological responses through the generation of second messengers, such as cAMP. The down-regulation of their activity (desensitization) is carried out, in part, by the family of G protein-coupled receptor kinases, which phosphorylate activated receptors. The Gprk2 gene in Drosophila melanogaster is a putative member of this family. The GPRK2 protein is expressed most abundantly in the ovaries and in the mushroom bodies, the brain region that is implicated in learning and memory in insects. Many of the genes that are involved in learning in Drosophila are members of a cAMP-signaling pathway and are also expressed in the mushroom bodies. These observations suggest that the Gprk2 gene may be involved in a cAMP-mediated pathway. To investigate this possibility, we tested for a genetic interaction between Gprk2 and dunce (which encodes cAMP-specific phosphodiesterase). A mutant allele of Gprk2, called gprk2(6936), has decreased fertility as a result of reduced levels of egg laying and hatching, and developing egg chambers display defects in the formation of anterior structures. Similarly, many alleles of dunce are sterile, with an ovary phenotype that resembles gprk2(6936). Introduction of a single copy of a hypomorphic or null allele of dunce into the gprk2(6936) background suppressed all of these defects to a significant degree. Suppression was also observed when a single copy of gprk2(6936) was introduced into a dunce background. Like mutants of rutabaga (which encodes a calcium/calmodulin-dependent adenylate cyclase), gprk2(6936) has reduced levels of cAMP. Ovaries from gprk2(6936) females contain about one third of the normal amount of cAMP. In addition, in every mutant combination where fertility is increased, cAMP levels are closer to wild type levels. These results suggest that Gprk2 is functioning in a cAMP-signaling pathway and that the underlying basis of the interaction between Gprk2 and dunce is a normalization of cAMP levels.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2002

Follicle-stimulating hormone induces a gap junction-dependent dynamic change in [cAMP] and protein kinase a in mammalian oocytes.

The second messenger cyclic adenosine 5'monophosphate (cAMP) has been implicated in controlling meiotic maturation. To date, there have been no direct measurements of cAMP in living mammalian oocytes. Here, we have used the fluorescently labelled cAMP-dependent protein kinase A (PKA), FlCRhR, to monitor cAMP in mouse oocytes. In cumulus-enclosed oocytes, follicle-stimulating hormone (FSH) stimulated an increase in the oocyte [cAMP] that was prevented by using the gap junction inhibitor, carbenoxolone. The FSH-induced increase in oocyte [cAMP] was suppressed in a time-dependent manner by prior exposure to ATP, while epidermal growth factor had no effect on basal or stimulated levels of cAMP. Finally, using confocal microscopy, we show that the regulatory and catalytic subunits of the microinjected PKA are distributed in a punctate manner with a stronger accumulation in the perinuclear region. On an increase in [cAMP], in response to phosphodiesterase inhibition or FSH, the catalytic subunit diffused throughout the cytoplasm and germinal vesicle, while the regulatory subunit remained anchored. These experiments show that increases in cAMP in ovarian somatic cells are communicated via gap junctions to the oocyte, where it can lead to a redistribution of the catalytic subunit of PKA.

Animals↗
PubMed1998

Differential modulation of CYP2E1 activity by cAMP-dependent protein kinase upon Ser129 replacement.

Many toxic compounds are activated by cytochrome P450 (CYP) 2E1 to reactive metabolites, which represents a potential hazard for cellular homeostasis. Therefore knowledge about CYP2E1 regulation could be of great biological importance. It has been shown that CYP2E1 is controlled transcriptionally and post-translationally by phosphorylation. In the present study we investigated the role of serine-129 (Ser129) in the protein kinase A (PKA) recognition sequence motif Arg-Arg-Phe-Ser129. To gain further insights into the possible relevance of Ser129 for CYP2E1 function, Ser129 was replaced by alanine (Ala) or glycine (Gly) by site-directed mutations of the cDNA coding for CYP2E1. The mutant cDNAs were transfected into Chinese hamster lung fibroblast V79 cells. Despite the mutation in the PKA phosphorylation motif, all strains produced catalytically active CYP2E1. However, there was a marked change in the substrate preference: The Gly129-containing strains hydroxylated p-nitrophenol (PNP) to a markedly higher extent than the wild-type cDNA-containing cells, while they demethylated N-nitrosodimethylamine (NDMA) to a markedly lower extent than the wild-type cells. All the strains activated NDMA to mutagenic products. Treatment with the membrane-permeating cAMP derivative db-cAMP reduced markedly both the PNP hydroxylase and the NDMA demethylase activities as well as the mutation frequency induced by NDMA in the Ser129-containing strain. This decrease in activity was not accompanied by a decrease in CYP2E1 content. In addition, the catalytic activities of CYP2E1 were decreased in microsomes from rat hepatocytes treated with db-cAMP. Also in this case, the decrease in activities was not accompanied by a decrease in enzyme protein. These findings argue that involvement of Ser129 and its phosphorylation is not in determining CYP2E1 protein level, but rather in controlling its catalytic activity. In contrast, in the strains containing Ala129 or Gly129, treatment with db-cAMP caused a marked increase in both PNP hydroxylase and NDMA demethylase. In these strains a similar db-cAMP-mediated increase was also observed in the mutation frequency, resulting from the treatment with the promutagen NDMA, which is activated by CYP2E1. Our results show that CYP2E1 in V79 cells responds in two separate ways to db-cAMP exposure depending on the amino acid residue present in the PKA recognition sequence. The enzyme is committed to a negative regulation by db-cAMP if Ser129 is the target amino acid for PKA, leading to a decrease in the metabolic activation to mutagenic and carcinogenic species. On the other hand, Ala129 or Gly129 substitution directed CYP2E1 toward a positive regulation by increasing its catalytic activities and metabolic activation to mutagenic intermediates in the presence of db-cAMP. We also obtained evidence that cAMP-mediated downregulation of wild-type (Ser129) CYP2E1 was not accompanied by its destruction but instead by its stabilization, which shows that Ser129 is not involved in CYP2E1 degradation but dictates requirements for its specific activities.

Amino Acid Substitution↗