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Factors that increase cAMP levels can induce lineage-specific differentiation of glioma cells into astrocyte-like cells. However, the differentiation pattern and underlying mechanisms remain unclear. Here, we find that cAMP/protein kinase A (PKA)/cAMP responsive element binding protein 1 (CREB1)-induced miR-221/222 suppression contributes to the neuron-like differentiation of gliomas. cAMP agonists selectively induced neuron- and astrocyte-like but not oligodendrocyte-like differentiation of C6 glioma cells. PKA inhibitors and CREB1 knockout blocked neuron-like differentiation of glioma cells. cAMP inhibited miR-221/222 in a PKA/CREB1-dependent manner. Importantly, both in vitro and in vivo assays demonstrated that transcriptional suppression of miR-221/222 is required for neuronal differentiation of glioma cells. Our findings suggest that increasing cAMP levels can induce bidirectional differentiation of glioma cells. Furthermore, the miR-221/222 cluster acts as an epigenetic brake during glioma differentiation.

Despite the importance of glutathione in Dictyostelium, the role of glutathione synthetase (gshB/GSS) has not been clearly investigated. In this study, we observed that increasing glutathione content by constitutive expression of gshB leads to mound-arrest and defects in 3',5'-cyclic adenosine monophosphate (cAMP)-mediated aggregation and developmental gene expression. The overexpression of gpaB encoding G protein alpha 2 (Gα2), an essential component of the cAMP signalling pathway, results in a phenotype similar to that caused by gshB overexpression, whereas gpaB knockdown in gshB-overexpressing cells partially rescues the above-mentioned phenotypic defects. Furthermore, Gα2 is highly enriched at the plasma membrane of gshB-overexpressing cells compared to wild-type cells. Therefore, our findings suggest that glutathione upregulates cAMP signalling via Gα2 modulation during Dictyostelium development.

During capacitation of mammalian sperm intracellular [Ca2+] and cyclic nucleotides increase, suggesting that CNG channels play a role in the physiology of sperm. Here we study the effect of capacitation, 8Br-cAMP (8-bromoadenosine 3′,5′-cyclic monophosphate) and 8Br-cGMP (8-bromoguanosine 3′,5′-cyclic monophosphate) on the macroscopic ionic currents of mouse sperm, finding the existence of different populations of sperm, in terms of the recorded current and its response to cyclic nucleotides. Our results show that capacitation and cyclic nucleotides increase the ionic current, having a differential sensitivity to cGMP (cyclic guanosine monophosphate) and cAMP (cyclic adenosine monophosphate). Using a specific inhibitor we determine the contribution of CNG channels to macroscopic current and capacitation.

A variety of immunoregulatory signals to effector T cells from monocytes, macrophages and regulatory T cells act through cyclic adenosine monophosphate. In the effector T cell, the protein kinase A (PKA) type I isoenzyme localizes to lipid rafts during T cell activation and modulates directly the proximal events that take place after engagement of the T cell receptor. The most proximal target for PKA phosphorylation is C-terminal Src kinase (Csk), which initiates a negative signal pathway that fine-tunes the T cell activation process. The A kinase anchoring protein Ezrin colocalizes PKA and Csk by forming a supramolecular signaling complex consisting of PKA, Ezrin, Ezrin/radixin/moesin (ERM) binding protein of 50kDa (EBP50), phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (GEMs) (PAG) and Csk.

Cholera toxin causes cyclic adenosine monophosphate (cAMP)-induced electrolyte and water secretion in the small intestine. The toxin-induced change in gene expression in rat small intestine was evaluated with microarray technique and the results were confirmed by semiquantitative polymerase chain reaction (PCR). The transporter CNT2 for nucleosides was upregulated between 6 and 18 h after challenge, whereas the level of GLUT1 transporter for glucose became elevated at 6 h. Both changes probably facilitate uptake of these nutrients in the gut. At 18 h, the major chloride channel in the villus, ClC2, was upregulated. Aquaporin 8 was downregulated at 6 h and two mucin-producing genes were upregulated 18 h after toxin challenge. The expression was back to normal after 72 h, which is the turnover time for intestinal epithelial cells.

The gene for mouse polyomavirus large tumor (LT) antigen, a potent oncoprotein, was expressed in Saccharomyces cerevisiae from the inducible GAL1 promoter. Substantial cell growth inhibition as well as colony and cell morphology changes dependent on cyclic adenosine monophosphate (cAMP) were observed. In contrast to cell and colony morphology alterations, the growth inhibition appeared to be transient, thus indicating the existence of an active adaptation of yeast cells to the LT antigen presence.

Extracellular superoxide dismutase (EC-SOD) is a secretory protein that is the major SOD isozyme in extracellular fluids. Plasma EC-SOD levels are distributed in two discrete groups with the rare group having an enzyme with glycine instead of arginine-213, which causes a 10-fold higher serum level. Within the common phenotype group, the urinary EC-SOD level was significantly correlated with the urinary excretion of N-acetyl-β-D-glucosaminidase (NAG), but not with serum EC-SOD. EC-SOD appears not to be leaked from the plasma by glomerular filtration, but rather to be secreted from the renal tubule or its surrounding tissues. The urinary EC-SOD level was also significantly correlated with the urinary cyclic adenosine monophosphate (cAMP) level. cAMP analogues and adenylate cyclase modulators significantly stimulated the expression of EC-SOD but not other SOD isozymes in cultured fibroblast cell lines. Moreover, injection of parathyroid hormone, in Ellsworth-Howard tests, increased urinary EC-SOD accompanied with the elevations of urinary cAMP and NAG. Together these observations suggest that factor(s) that stimulate the adenylate cyclase-cAMP system regulate the urinary EC-SOD level.

The signal transduction pathway of the cloned human glucagon-like peptide-1 (GLP-1) receptor was studied in voltage-clamped Xenopus oocytes. Binding of GLP-1(7–36)amide was associated with cAMP production, increased [Ca2+]i and activation of Ca2+-dependent Cl− current. The effect of GLP-1(7–36)amide reflects intracellular Ca2+ mobilization and was suppressed by injection of the Ca2+ chelator BAPTA and the inositol trisphosphate receptor antagonist heparin. The responses were not mimicked by the adenylate cyclase activator forskolin and unaffected by the protein kinase A (PKA) inhibitor Rp-cAMPS. We conclude that GLP-1 receptor expression in Xenopus oocytes evokes inositol trisphosphate-dependent intracellular Ca2+ mobilization independent of the cAMP/PKA signaling pathway.

The effects of the novel vasoactive regulatory peptide, adrenomedullin, on human oral keratinocytes was investigated. Adrenomedullin, acting via its specific receptor, stimulated a dose-dependent increase in DNA synthesis, and, in addition, stimulated further changes in the cell cycle resulting in the proliferation of keratinocytes. When cells were incubated in the presence of increasing concentrations of adrenomedullin, there was a rapid and dose-dependent rise in intracellular cyclic AMP levels. Stimulation of mitogenesis and cell proliferation in these cells were mimicked by the cell permeable cAMP analogue, di-butyryl cAMP. Adrenomedullin-stimulated mitogenesis was attenuated by the adenylyl cyclase inhibitor SQ22,536, but was unaffected by inhibitors of PKC, tyrosine kinase or the CGRP receptor antagonist, CGRP(8–37). These data identify adrenomedullin as a new mitogenic regulatory peptide of keratinocytes acting via the cAMP cascade.

The recently isolated, naturally occurring peptide hormones [Hyp3]-bradykinin and [Hyp3]-Lys-bradykinin were investigated for their agonist activity on solubilized binding sites from human fibroblasts. Both ligands competed with [3H]bradykinin binding in a dose-dependent fashion with potencies similar to bradykinin (BK) and Lys-BK. Biological activity was assessed by determination of inositol phosphate accumulation and cyclic 3',5'-adenosine monophosphate synthesis in intact cultured cells. Stimulation by the hydroxylated peptides resulted in a pronounced accumulation of both parameters with similar effectiveness as BK and Lys-BK. These results indicate that [Hyp3]-BK and [Hyp3]-Lys-BK are agonists at the bradykinin receptor system with properties comparable to their non-hydroxylated analogues. This suggests that hydroxylation of kinins does not alter receptor interaction or signal transduction in cultured human fibroblasts.

It has been proposed that the pICln protein forms a nucleotide-sensitive plasma membrane anion channel with a GXGXG motif being an essential component of the extracellular nucleotide-binding site. To evaluate this hypothesis, we have performed voltage-clamp experiments on Xenopus laevis oocytes injected with RNA encoding a rat mutant pICln in which the three glycines of the putative nucleotide-binding site have been changed into alanines (G54A; G56A; G58A). The injected oocytes displayed outwardly rectifying anion currents, which were voltage-dependently blocked by extracellular cAMP, but which were not affected by removal of extracellular Ca2+. Furthermore, the mutation did not affect the voltage-dependent inactivation. We therefore conclude that there is no evidence in favour of an extracellular nucleotide-binding site in pICln.

Phosphodiesterases (PDEs) are enzymes that degrade intracellular cAMP. In the present study, 3-isobutyl-1-methylxanthine (IBMX) and pentoxifylline, PDE inhibitors, induced osteoclast formation in cocultures of mouse bone marrow cells and calvarial osteoblasts. These inhibitors induced the expression of the osteoclast differentiation factor, TNF-related activation induced cytokine (TRANCE, identical to RANKL, ODF, and OPGL), in calvarial osteoblasts. IBMX induced phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in osteoblasts. Induction of TRANCE expression by IBMX was partially suppressed by the inhibitors of protein kinase A (PKA), ERK, and p38 MAPK, suggesting that activation of ERK and p38 MAPK, as well as PKA, is involved in TRANCE expression by IBMX. Osteoblasts expressed PDE4, a PDE subtype, and rolipram, a selective inhibitor of PDE4, induced TRANCE expression. These results suggest that PDE4 is a key regulator of TRANCE expression in osteoblasts, which in turn controls osteoclast formation.

The Ras/cAMP pathway in the yeast S. cerevisiae couples the cell cycle of this unicellular organism to the availability of nutrients. Glucose derepressed S. cerevisiae cells respond to glucose addition by an intracellular rise in cAMP. In the prevailing model, yeast Ras plays a similar role to that of heterotrimeric G-proteins coupled to cell surface receptors. A crucial element of this model is that the exchanger, Cdc25 is activated by glucose. Such activation would result in a glucose-dependent rise in GTP-bound Ras concentration. We here show, in contrast to this view, that Cdc25 cannot be the receiver of the glucose signal. We suggest that the Ras-GTP/cyclase complex is the molecular element directly receiving the signal while Cdc25-dependent exchange constitutes a prerequisite for complex formation.

We have cloned and sequenced a novel cDNA (RPR7) encoding a receptor for pituitary adenylate cyclase activating polypeptide (PACAP). RPR7 was identified by PCR of rat pituitary cDNA, and full-length clones were isolated from a rat olfactory bulb cDNA library. When expressed in COS cells, RPR7 was functionally coupled to increases in intracellular cyclic adenosine monophosphate (cAMP) in response to stimulation by PACAP-38, PACAP-27, vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI). The order of potency of these ligands was PACAP-38 ~ PACAP-27 > VIP > PHI, suggesting that the receptor corresponds to the pharmacologically characterised PACAP Type I receptor.

Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions of the human gastric inhibitory polypeptide (GIP) promoter into hamster insulinoma (HIT T15) cells indicated that the region between −180 and +14 is sufficient for basal promoter activity. Two CRE-BP1 binding sites were identified in this promoter region by DNase I footprinting with the bacterially expressed cAMP response element (CRE) binding protein, CRE-BP1. Mutation analyses showed that these two CREs are required for the basal promoter activity, and furthermore that one site, at nucleotide-158, contributed mainly to the cAMP inducibility of the GIP promoter in HIT T15 cells. Interestingly, the GIP promoter activity was repressed by the c-jun proto-oncogene product, possibly through the CREs.

Phorbol 12-myristate 13-acetate (100 nM), a potent protein kinase C and macrophage activator, has a biphasic affect on 25(OH)D3-1 alpha-hydroxylase activity in synovial fluid macrophages from arthritis patients. After 5 h, 1 alpha, 25(OH)D3 synthesis fell from 5.2 +/- 0.1 to 1.6 +/- 0.2 pmol/h per 10(6) cells, however, after 24 h and 48 h, synthesis increased to 17.4 +/- 0.3 and 22.3 +/- 1.4 pmol/h per 10(6) cells, respectively. Although an independent short-term mechanism is suggested, protein kinase C may promote macrophage activation, thus increasing long-term 25(OH)D3-1 alpha-hydroxylase expression. Intracellular calcium and cAMP are unlikely to activate the enzyme, since 0.1 microM of the calcium ionophore, A23187, and 1 mM dibutyryl-cAMP inhibited synthesis by 87% and 79%, respectively, after 24 h.

1,9-Dideoxyforskolin inhibits proteoglycan synthesis and xyloside-initiated glycosaminoglycan (GAG) synthesis in chick embryo chondrocytes. Dideoxyforskolin does not affect the length of xyloside-initiated GAG chains secreted into the medium but chains from the dense proteoglycan secreted into the medium appear slightly longer. Incorporation of labeled serine into the dense proteoglycan and subsequent digestion with Pronase revealed a dramatic decrease in percent of total radioactivity associated with GAG chains in the proteoglyean from cultures treated with forskolin or dideoxyforskolin. These observations suggest that these diterpenes have a specific inhibitory effect on chain initiation reactions and thus may be useful tools in the study of proteoglycan synthesis and processing.

Some properties were examined of the isolated membranes of zymogen granules from the rat pancreas. It was possible to detect an endogenous protein kinase activity capable of phosphorylating 8 to 10 membrane protein constituents as well as added histones. Cyclic adenosine monophosphate (AMP), and cyclic guanosine monophosphate (GMP), to a lesser extent, exerted moderate stimulatory effects. These effects of cyclic nucleotides on membrane phosphorylation became more apparent upon addition of a partially purified soluble protein kinase also extracted from the rat pancreas., SCOPUS: ar.j, info:eu-repo/semantics/published

Vasodilator-stimulated phosphoprotein (VASP), an actin binding protein localized to areas of focal contacts, is a substrate for the cyclic adenosine monophosphate/cyclic guanosine monophosphate (cAMP/cGMP)-dependent protein kinases (PKA, PKG). In this study, we show that serum stimulation of vascular smooth muscle cells (SMCs) induces VASP phosphorylation on Ser157, in a mechanism not dependent on PKA or PKG. We tested the possibility that protein kinase C (PKC), a regulator of cytoskeletal function, is involved. PKC inhibition or down-regulation prevented serum-induced phosphorylation of VASP at Ser157 in rat vascular SMCs. Additionally, recombinant PKCα directly phosphorylated Ser157 on VASP. In summary, our data support the hypothesis that PKC phosphorylates VASP and mediates serum-induced VASP regulation.

Extracellular superoxide dismutase (EC-SOD) is synthesized in mesenchymally derived cells and prevents the oxygen radical-induced injury. We studied whether kidney mesangial cells (MCs) produce EC-SOD and how its production is associated with chemokine secretion. Under unstimulated condition, MCs produced EC-SOD, and its production was correlated positively with cyclic adenosine monophosphate (cAMP), but negatively with interleukin (IL)-6 or IL-8 production. By prednisolone or phorbol myristate acetate treatment, EC-SOD levels were correlated negatively with levels of IL-6 and IL-8. The presence of adenylate cyclase inhibitor 2′,3′-dideoxyadenosine lost the prednisolone effect. The stimulation of EC-SOD production might be one of the important effects of prednisolone via cAMP pathway in MCs.

In contrast to the ribosomal RNA gene expression increasing with growth rate, transcription of the lac operon is downregulated by cell growth rate. In continuous culture, growth rate regulation of lac promoter was independent of carbon substrate used and its location on the chromosome. Since the lac operon is activated by cyclic adenosine monophosphate (cAMP), which decreases with increasing cell growth rate, expression of plac-lacZ reporter fusion was analyzed in cya mutant under various growth conditions. The results demonstrated that expression of plac-lacZ in cya mutant was both lower and growth rate independent. In addition, ppGpp (guanosine tetraphosphate) was not involved in the mechanism of growth rate regulation of the lac promoter. Thus, the results of this study indicate that cAMP mediates the growth rate-dependent regulation of lac operon expression in Escherichia coli.

The presence and regulation of a hormone-sensitive cyclic GMP-inhibited cyclic nucleotide phosphodiesterase (cGI PDE) in rat brown adipose cells was investigated. cDNA clones for two cGI PDE isoforms, cGIP1 and cGIP2, have been isolated. Using a rat cGIP1 (RcGIP1) cDNA probe, RcGIP1 mRNA (approximately 5.3 kb) was detected in Northern blots of both brown and white adipose RNA. cGI PDE was detected in both microsomal and plasma membrane fractions of brown and white adipose cells by Western blotting using anti-RcGIP1 peptide antibody. When cells were incubated with insulin before membrane preparation, cGI PDE activity in the microsomal fraction was increased by 2- to 2.5-fold within 10 min. Isoproterenol also stimulated the activity of cGI PDE in the microsomal fraction by 1.5-fold. In cells incubated with both insulin and isoproterenol, microsomal cGI PDE activity was similar to that in microsomal fractions isolated from cells incubated with insulin alone. These results suggest that the hormonal regulation of cGI PDE, presumably a cGIP1 isoform, in rat brown adipose cells is similar to that in white adipose cells.

Vasoactive intestinal polypeptide (VIP) has been shown to exert a hyperglycemic effect in vivo and to induce glucogenolysis in rabbit liver slices [l] but the biochemical evidence supporting an effect of VIP on glucose metabolism in liver cells is scarce [2-31 except for a recently described effect [4]. This effect is thought to be cyclic adenosine monophosphate (CAMP)-dependent since numerous studies have shown that VIP can stimulate rat liver adenylate cyclase through the interaction with a specific membrane receptor [5-81. VIP seems to share this membrane binding site with secretin but not with glucagon [6] or gastric inhibitory polypeptide (GIP) 131. Although the VIP effect on membrane adenylate cyclase was clearly documented, the action of VIP on CAMP levels in isolated liver cells is still unclear [4,8]. The aim of this study was to systematically investigate the actions and mechanism of action of VIP, secretin and GIP, three glucagon-related polypeptides [9], on glucose and lactate metabolism in isolated rat liver cells and to compare them to the known, CAMP-mediated, effects of glucagon.

Erythropoietin (Epo) mRNA expression is suppressed by interleukin 1 (IL-1). Cyclic adenosine monophosphate (cAMP) can increase Epo mRNA and Epo protein levels in IL-1 treated HepG2 cells to some extent. To identify molecular mechanisms of this reaction we investigated three transcription factors (NF-kappaB, GATA-2 and HIF-1) that control the Epo gene. Western blot analyses and electrophoretic mobility shift assays (EMSAs) revealed that IL-1 strongly activated NF-kappaB, which is a likely suppressor of the Epo promoter. Treatment of the cells with dibutyryl-cAMP (Bt2-cAMP) inhibited the activation of NF-kappaB by IL-1. Bt2-cAMP increased GATA-2 DNA binding. Since GATA-2 is a suppressor of the Epo promoter, GATA-2 activation was unlikely to cause the increase of Epo mRNA expression in IL-1 treated cells. Furthermore, Western blots, EMSAs and reporter gene studies showed that Bt2-cAMP was without effect on the hypoxia-inducible transcription factor HIF-1. Thus, NF-kappaB is probably the primary transcription factor by which cAMP counteracts the inhibition of Epo gene expression by IL-1.

Human tracheal glands are considered as the principle secretory structures in the bronchotracheal tree. In earlier studies, we successfully performed primary cultures of human tracheal gland (HTG) serous cells and noted that these cells were responsive to many secretagogues including purinergic agonists but not to the inflammatory mediator adenosine. In this study, we demonstrate that adenosine was capable of inducing stimulation of protein secretion by HTG serous cells which had previously been cultured in pro-inflammatory conditions (induced by lipopolysaccharide (LPS)). This stimulation was inhibited by 8-phenyltheophylline but not by dipyridamole, which is indicative of a P1 purinoceptor. This inducible receptor is of the adenosine A2 subtype [rank potency order: 5′-(N-ethyl)-carboxamidoadenosine (NECA) > adenosine > N6-(phenylisopropyl)-adenosine (PIA); and stimulation of adenylyl cyclase]. The adenosine-induced protein secretion was concentration-dependent, however, increased intracellular cyclic adenosine monophosphate (cAMP) was not dependent on the concentration of adenosine. The adenosine-induced secretion and the ATP-induced secretion were shown to be additive. This study concludes that there is evidence of a LPS-inducible adenosine A2 receptor in human tracheal gland serous cells.