Cyclic AMP Content Research Articles

Prostaglandin E2 drives cyclooxygenase 2 expression via cyclic AMP response element activation in human pancreatic cancer cells

Cyclooxygenase-2 (COX-2) is constitutively expressed in most human primary carcinomas and with its synthesized product, prostaglandin E2 (PGE2), appears to play important roles in tumor invasion, angiogenesis, resistance to apoptosis, and suppression of host immunity. However, the molecular mechanisms that control COX-2 expression are unclear. The purpose of this study was to clarify the mechanism of basal and PGE2-mediated COX-2 expression in the highly metastatic L3.6pl human pancreatic cancer cell line. Using RNA interference to disrupt the expression of CREB and the NF-?B p65 subunit, we found that both are involved in maintaining basal COX-2 expression in L3.6pl cells. We also demonstrated that PGE2 increased the cyclic AMP concentration, thereby activating protein kinase A (PKA), which in turn phosphorylated the cyclic AMP response element binding protein (CREB), leading to interaction with the cyclic AMP response element in the promoter region of the COX-2 gene. Immunocytochemical analysis confirmed that PGE2 stimulated the translocation of PKA to the nucleus and increased the immunoreactivity of phosphorylated CREB. Pretreatment with the PKA selective inhibitor H-89 and the E-prostanoid receptor 2 inhibitor AH-6809 reduced COX-2 up-regulation by PGE2. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay results further suggested a role for CREB in COX-2 transcriptional control. Understanding the pathways that control COX-2 expression may lead to a better understanding of its dysregulation in pancreatic carcinomas and facilitate the development of novel therapeutic approaches.

Caffeine-mediated enhancement of glucocorticoid receptor activity in human osteoblastic cells

Caffeine-containing beverage consumption has been reported to be associated with an increased risk for osteoporosis. Since the glucocorticoid receptor (GR) is a major factor in the induction of osteoporosis we analyzed whether caffeine may act via altering GR function. Applying a reporter gene assay we provide evidence that caffeine drastically amplifies GR transcriptional activity in human osteoblastic cells. Substances that increase the intracellular cyclic AMP-concentration also strengthen the transactivity of the GR and coincubation with caffeine further reinforces this potentiation, indicating that caffeine-mediated enhancement of GR transcriptional function is due to the inhibitory activity of caffeine on the cyclic AMP phosphodiesterase. Our data suggest evidence for a hitherto unrecognized crosstalk between caffeine-modulated signalling and GR-initiated gene expression in human osteoblastic cells and could provide the molecular basis for the role of caffeine in osteoporosis.

Contribution of the Carboxyl Terminus of the VPAC1 Receptor to Agonist-induced Receptor Phosphorylation, Internalization, and Recycling

When exposed to vasoactive intestinal peptide (VIP), the human wild type VPAC1 receptor expressed in Chinese hamster ovary (CHO) cells is rapidly phosphorylated, desensitized, and internalized in the endosomal compartment and is not re-expressed at the cell membrane within 2 h after agonist removal. The aims of the present work were first to correlate receptor phosphorylation level to internalization and recycling, measured by flow cytometry and in some cases by confocal microscopy using a monoclonal antibody that did not interfere with ligand binding, and second to identify the phosphorylated Ser/Thr residues. Combining receptor mutations and truncations allowed identification of Ser250 (in the second intracellular loop), Thr429, Ser435, Ser448 or Ser449, and Ser455 (all in the distal part of the C terminus) as candidates for VIP-stimulated phosphorylation. The effects of single mutations were not additive, suggesting alternative phosphorylation sites in mutated receptors. Replacement of all of the Ser/Thr residues in the carboxyl-terminal tail and truncation of the domain containing these residues completely inhibited VIP-stimulated phosphorylation and receptor internalization. There was, however, no direct correlation between receptor phosphorylation and internalization; in some truncated and mutated receptors, a 70% reduction in phosphorylation had little effect on internalization. In contrast to results obtained on the wild type and all of the mutated or truncated receptors that still underwent phosphorylation, internalization of the severely truncated receptor was reversed within 2 h of incubation in the absence of the agonist. Receptor recovery was blocked by monensin, an endosome inhibitor.

Suppressing actions of butyrate on growth hormone (GH) secretion induced by GH-releasing hormone in rat anterior pituitary cells

We assessed the inhibitory effects of butyrate on the growth hormone (GH) secretion in order to investigate the cellular mechanisms in rat somatotrophs. Isolated anterior pituitary cells were cultured in DMEM for several hours, either in the presence (1, 3, or 10 mM) or absence of butyrate, and then stimulated with 10 −7 M GHRH for 30 min, in the presence of butyrate at the concentrations used for the previous culture. The increase in GHRH-induced GH release was significantly reduced in a time-dependent and concentration-dependent manner in the cells previously cultured with butyrate. GH content (the sum of GH released into the medium induced by GHRH stimulation and the GH remaining in the cells after stimulation) was reduced by the culture of cells in the presence of butyrate, which was also inversely dependent on the concentrations used for the culture. Simultaneous addition of an L-type Ca 2+ channel blocker, nifedipine (10 pM), to the medium during 10 −9 M GHRH stimulation significantly reduced the stimulated GH release, which was further significantly decreased by a simultaneous addition of 10 mM butyrate. Butyrate blunted the GHRH (10 −9 M)-induced increase in cellular cyclic AMP and calcium ion concentrations, the activity of protein kinases (A and C), and GHmRNA expression. The expression of mRNA for GPR 41 and 43, known as receptors for short-chain fatty acids, was confirmed in the anterior pituitary cells. These findings suggest that butyrate inhibits GHRH-induced GH release as well as GH production, and the cellular inhibitory actions of butyrate occur in diverse cellular signaling pathways of rat somatotrophs.

Quantification of the glycogen cascade system: the ultrasensitive responses of liver glycogen synthase and muscle phosphorylase are due to distinctive regulatory designs

BackgroundSignaling pathways include intricate networks of reversible covalent modification cycles. Such multicyclic enzyme cascades amplify the input stimulus, cause integration of multiple signals and exhibit sensitive output responses. Regulation of glycogen synthase and phosphorylase by reversible covalent modification cycles exemplifies signal transduction by enzyme cascades. Although this system for regulating glycogen synthesis and breakdown appears similar in all tissues, subtle differences have been identified. For example, phosphatase-1, a dephosphorylating enzyme of the system, is regulated quite differently in muscle and liver. Do these small differences in regulatory architecture affect the overall performance of the glycogen cascade in a specific tissue? We address this question by analyzing the regulatory structure of the glycogen cascade system in liver and muscle cells at steady state.ResultsThe glycogen cascade system in liver and muscle cells was analyzed at steady state and the results were compared with literature data. We found that the cascade system exhibits highly sensitive switch-like responses to changes in cyclic AMP concentration and the outputs are surprisingly different in the two tissues. In muscle, glycogen phosphorylase is more sensitive than glycogen synthase to cyclic AMP, while the opposite is observed in liver. Furthermore, when the liver undergoes a transition from starved to fed-state, the futile cycle of simultaneous glycogen synthesis and degradation switches to reciprocal regulation. Under such a transition, different proportions of active glycogen synthase and phosphorylase can coexist due to the varying inhibition of glycogen-synthase phosphatase by active phosphorylase.ConclusionThe highly sensitive responses of glycogen synthase in liver and phosphorylase in muscle to primary stimuli can be attributed to distinctive regulatory designs in the glycogen cascade system. The different sensitivities of these two enzymes may exemplify the adaptive strategies employed by liver and muscle cells to meet specific cellular demands.

Platelet Aggregation and Adenosine Diphosphate/Adenosine Triphosphate Receptors: A Historical Perspective

The work of many investigators since adenosine diphosphate (ADP) was recognized as a platelet aggregating agent in 1961 has led to an appreciation of the important part that ADP plays in hemostasis and thrombosis. Recently, interest has focused on the platelet receptors for ADP and adenosine triphosphate (ATP). Platelets are unique because they have two P2Y receptors that must act in concert to achieve a normal aggregation response. The P2Y (1) receptor is responsible for mobilizing internal calcium, platelet shape change, and weak aggregation. The P2Y (12) receptor inhibits adenylyl cyclase, but the concentration of cyclic AMP is reduced only if it has been raised from its low basal levels by stimulation of adenylyl cyclase by an aggregation inhibitor such as adenosine or prostaglandin I (2). The abnormal bleeding of the rare patients whose platelets lack P2Y (12) and the beneficial clinical effects of ticlopidine and clopidogrel that block this receptor indicate that P2Y (12), in addition to inhibiting adenylyl cyclase, may have an as yet unidentified role that is needed for its cooperative aggregation effect with P2Y (1). ATP stimulates a rapid influx of calcium into platelets through the P2X (1) receptor, and it may synergize with ADP when these two nucleotides are released from platelets at a site of vessel injury.

Up-regulation of gamma-glutamyl transpeptidase (GGT) activity in growth perturbed C6 astrocytes

Activity of gamma-glutamyl transpeptidase (GGT) was studied in astrocyte-like C6 glial cells modulated in growth and maturation by different concentration of serum and dibutyryl cyclic AMP (Db-cAMP) supplement in culture medium. After reduction of serum concentration from 10% to 0.1%, the number of GGT positive cells determined histochemically increased 3.1 times and the GGT activity/mg protein in whole cell lysates was 5.1 times higher. In cultures with 0.1% serum + Db-cAMP, the histochemically and biochemically assayed GGT activity exceeded 5.1 and 7.9 times the values measured in control 10% serum cultures, respectively. The up-regulation of GGT was accompanied by an inhibition of proliferation, enhanced differentiation and hypertrophy of cells. In addition, the process of metabolic perturbation and/or cellular stress was revealed in these cultures by the (i) growth-support release followed by shrinkage and death of a small number of cells and (ii) higher oxidation of 2′7′dichlorofluorescein diacetate to its fluorescent form in the adherent/viable cells. The observed up-regulation of GGT is considered to primarily reflect increased metabolism of glutathione and/or the maintenance of the redox potential in cells stressed by sub-optimal concentration of serum and Db-cAMP supplement. The concomitant cellular hypertrophy and differentiation and their relationship to increased activity of GGT await further investigation. The study suggests that up-regulation of GGT can contribute to adaptation of astrocytic cells to metabolic and/or oxidative perturbances occurring under various pathological conditions, including radiation- and drug-induced toxicity.

Retinal Ganglion Cells Are Autonomous Circadian Oscillators Synthesizing N-Acetylserotonin during the Day

Retinal ganglion cells send visual and circadian information to the brain regarding the environmental light-dark cycles. We investigated the capability of retinal ganglion cells of synthesizing melatonin, a highly reliable circadian marker that regulates retinal physiology, as well as the capacity of these cells to function as autonomous circadian oscillators. Chick retinal ganglion cells presented higher levels of melatonin assessed by radioimmunoassay during both the subjective day in constant darkness and the light phase of a light-dark cycle. Similar changes were observed in mRNA levels and activity of arylalkylamine N-acetyltransferase, a key enzyme in melatonin biosynthesis, with the highest levels of both parameters during the subjective day. These daily variations were preceded by the elevation of cyclic-AMP content, the second messenger involved in the regulation of melatonin biosynthesis. Moreover, cultures of immunopurified retinal ganglion cells at embryonic day 8 synchronized by medium exchange synthesized a [3H]melatonin-like indole from [3H]tryptophan. This [3H]indole was rapidly released to the culture medium and exhibited a daily variation, with levels peaking 8 h after synchronization, which declined a few hours later. Cultures of embryonic retinal ganglion cells also showed self-sustained daily rhythms in arylalkylamine N-acetyltransferase mRNA expression during at least three cycles with a period near 24 h. These rhythms were also observed after the application of glutamate. The results demonstrate that chick retinal ganglion cells may function as autonomous circadian oscillators synthesizing a melatonin-like indole during the day.

T cell receptor induced intracellular redistribution of type I protein kinase A

The productive activation of CD4(+) T lymphocytes, leading to proliferation and cytokine secretion, requires precise temporal regulation of intracellular cyclic AMP concentrations. The major effector molecule activated by cyclic AMP in mammalian cells is the cyclic AMP-dependent protein kinase A (PKA). The type I PKA isozyme mediates the inhibitory effects of cyclic AMP on T-cell activation. Using laser scanning confocal microscopy, we demonstrated that the regulation of PKA type I activity involves spatial redistribution of PKA type I molecules following T-cell receptor (TCR) stimulation. In resting T cells, PKA type I was located in membrane proximal regions and distributed equally across the cell. Shortly after antigen engagement, T cells and antigen-presenting cells formed an area of intense contact, known as the immunological synapse. TCR concentrated at the synapse, whereas PKA type I molecules redistributed to the opposite cell pole within 10 min after T-cell stimulation. Type I PKA redistribution was solely dependent on TCR signalling, because we observed the same temporal and spatial distribution after antibody-mediated cross-linking of the TCR-associated CD3 complex. Segregation of TCR and PKA type I molecules was maintained for at least 20 min. Thirty minutes after stimulation, PKA type I partially colocalized with the TCR. After 60 min, PKA type I distribution again approached the resting state. Considering that initial TCR signals lead to increases in intracellular cyclic AMP, PKA type I molecules may be targeted towards localized cyclic AMP accumulations or transported away from these areas, depending on the requirements of the cellular response.

A pore-forming toxin produced by Aeromonas sobria activates cAMP-dependent Cl − secretory pathways to cause diarrhea

Aeromonas sobria hemolysin (ASH) is one of the major virulence factors produced by A. sobria, a human pathogen that causes diarrhea. We investigated the effects of ASH on Cl − transport in human colonic epithelial cells. ASH increased short-circuit currents (Isc) and 125I efflux from Caco-2 cells, indicating ASH activate Cl − secretion. Additions of inhibitors of cyclic AMP dependent Cl − channels, glybenclamide and NPPB suppressed the Isc and 125I efflux increases induced by ASH. And ASH increased the intracellular cyclic AMP concentration. Moreover, ASH stimulated fluid accumulation in the iliac loop test, and glybenclamide and NPPB suppressed this fluid accumulation. Thus, cAMP-dependent Cl − secretory pathway could be related with diarrhea induced by A. sobria.

Suppression of respiratory burst in human neutrophils by new synthetic pyrrolo-benzylisoquinolines

Reactive oxygen species produced by neutrophils contribute to the pathogenesis of inflammatory diseases. In this study, the inhibition of superoxide anion (O 2 −) generation in human neutrophils by new synthetic pyrrolo-benzylisoquinoline derivatives was determined. We found that KW-2, KW-5, and KW-7 (8,9-dimethoxyl-1-( R-phenyl)-5,6-dihydro-pyrrolo[2,1- a]isoquinoline-2,3-dione; where R is 3-chloro, 3-bromo, and 4-methoxy, respectively) were the most effective inhibitors of formyl- l-methionyl- l-leucyl- l-phenylalanine (FMLP)-induced O 2 − release in human neutrophils. KW-2, KW-5, and KW-7 displayed no antioxidant or O 2 −-scavenging ability. The inhibition of O 2 − generation was reversed by the protein kinase (PK)A inhibitor, N-(2-(( p-bromocinnamyl)amino)ethyl)-5-isoquinolinesulfonamide (H89), but not by the PKG inhibitor (8 R,9 S,11 S)-(−)-2-methyl-9-methoxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cyclocta(cde)trinen-1-one (KT5823), or the soluble guanylate cyclase (sGC) inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ). KW derivatives increased cellular cyclic AMP concentrations through the inhibition of phosphodiesterase (PDE) activity but not the elevation of adenylate cyclase (AC) activity. These results indicate that inhibition of FMLP-induced respiratory burst in human neutrophils by KW derivatives are cyclic AMP/PKA-dependent and are due to inhibition of PDE. The new chemical skeleton of PDE inhibitors may protect against the progression of inflammation.

?2-Adrenoceptor-mediated intrinsic sympathomimetic activity of carteolol: an in vivo study

The intrinsic sympathomimetic activity (ISA) of a beta-adrenoceptor blocker can be mediated by beta(1)- or beta(2)-adrenoceptors. The aim of this study was to characterize the ISA of the beta-adrenoceptor blocker carteolol in healthy volunteers. Two approaches were employed. First, we assessed the effects of carteolol (20, 40 or 80 mg p.o.) on blood pressure, heart rate and heart-rate corrected duration of electromechanical systole (QS(2)c, a measure of cardiac contractility) in the volunteers. Carteolol dose-dependently increased systolic blood pressure, heart rate and contractility and decreased diastolic blood pressure. The beta(1)-adrenoceptor blocker bisoprolol did not attenuate these carteolol effects, but rather enhanced the effects on heart rate and systolic blood pressure. Second, we treated volunteers for 7 days with 1 x 20 mg/day carteolol and assessed lymphocyte beta(2)-adrenoceptor density (by (-)-[(125)I]-iodocyanopindolol binding) and functional responsiveness (by 10 muM isoprenaline-induced increase in lymphocyte cyclic AMP content). Carteolol significantly reduced lymphocyte beta(2)-adrenoceptor density and function. After withdrawal of carteolol lymphocyte beta(2)-adrenoceptor density and function recovered only very slowly and had not returned to control levels 11 days after carteolol withdrawal. In conclusion, the fact that, on the one hand, the cardiovascular effects of carteolol were not attenuated by the beta(1)-adrenoceptor blocker bisoprolol and, on the other, carteolol significantly decreased lymphocyte beta(2)-adrenoceptor density and function is in favour of the idea that the ISA of carteolol is mediated by beta(2)-adrenoceptors. Involvement of an additional receptor site (e.g. the propranolol-resistant state of the beta(1)-adrenoceptor), however, cannot be excluded.

Functional characterization of human neuropeptide Y receptor subtype five specific antagonists using a luciferase reporter gene assay

Neuropeptide Y (NPY) has several receptors; one of them, the neuropeptide Y5 receptor (NPY5) seems involved in feeding behavior in mammals. Although this particular receptor has been extensively studied in the literature, the difficulties encountered to obtain a stable cell line expressing this recombinant receptor have impaired the development of tools necessary to establish its molecular pharmacology. We thus established a method for the functional study of new ligands. It is based upon the cotransfection in human melatonin receptor 1 (MT1)-overexpressing HEK293 cells of three plasmids encoding melanocortin receptor (MC5), neuropeptide Y5 receptor (NPY5) and a cyclic AMP response element-controlled luciferase. Once challenged with αMSH, the MC5 receptor activates the cyclic AMP response, through the coupling protein subunit G s. In contrast, NPY5 agonists, through the NPY5 receptor which is negatively coupled to the same pathway, counteract the αMSH-mediated effect on cyclic AMP level. Using appropriate controls, this method can pinpoint compounds with antagonistic activity. Simple and straightforward, this system permits reproducible measurements of agonist or antagonist effects in the presence of neuropeptide Y, the natural agonist. This method has the advantage over already existing methods and beyond its apparent complexity, to enhance the cyclic AMP concentration at a ‘physiological’ level, by opposition to a forskolin-induced adenylate cyclase activation. Finally, to further validate this assay, we showed results from (1) a series of natural peptidic agonists that permitted the standardization and (2) a series of potent nonpeptidic antagonists (affinity >10 −9 M) that form a new class of active NPY5 receptor antagonists.

A Natural Variant Type II G Protein-coupled Receptor for Vasoactive Intestinal Peptide with Altered Function

The vasoactive intestinal peptide (VIP) and its G protein-coupled receptors VPAC1 and VPAC2 prominently mediate diverse physiological functions in the neural, endocrine, and immune systems. A deletion variant of mouse VPAC2 has been identified in immune cells that lacks amino acids 367-380 at the carboxyl-terminal end of the seventh transmembrane domain. When expressed at equivalent levels in a human Jurkat T cell line, which has very low endogenous expression of human VPAC1 and VPAC2, wild-type and deletion-variant VPAC2 bound the same amount of 125I-VIP with similar affinity. Unlike wild-type VPAC2, however, deletion-variant VPAC2 did not transduce VIP-elicited increases in intracellular concentration of cyclic AMP, chemotaxis, or suppression of generation of interleukin-2. Natural deletion of part of the last transmembrane domain of VPAC2 thus abrogates signaling functions without apparent alterations of expression or ligand binding.

Myosin heavy chain isoforms expression and cyclic AMP concentrations in hypoxia-induced hypertrophied right ventricle in rats

We have previously demonstrated that the relative expression of myosin heavy chain-beta (MHC-β) in both ventricles of rats exposed to long-term hypobaric hypoxia correlated significantly with the relative ventricular mass. In the present study, we investigated whether an increased expression of MHC-β was accompanied by a reduction in cyclic AMP (cAMP) activity in hypoxia-induced hypertrophied right ventricle (RV). We used male Wistar–Kyoto rats born and raised at simulated altitudes (2200 m: H2 group or 4000 m: H4 group) compared to age-matched sea level controls (SC group). There were no significant differences between the groups in basal and forskolin-stimulated adenylyl cyclase (AC) activities. The basal and IBMX-inhibited phosphodiesterase (PDE) activities were slightly higher in both hypoxic groups ( p>0.05), except that the H2 group had a higher basal PDE activity than the SC group ( p<0.05). The AC/PDE activity ratios were significantly decreased in both hypoxic groups ( p<0.05), suggesting that low concentrations of cellular cAMP were maintained in the RV under hypoxic conditions. However, there were no correlations between MHC-β expression and either AC activity, PDE activity, or AC/PDE activity ratio. These results provided evidence against the causal role for cAMP concentration in the expression of MHC-β associated with hypoxia-induced ventricular hypertrophy.

Caffeine suppresses TNF-α production via activation of the cyclic AMP/protein kinase A pathway

This study investigated the effect of in vitro exposure to caffeine, and its major metabolite paraxanthine, at concentrations relevant to typical caffeine consumption in humans, on lipopolysaccharide (LPS)-stimulated cytokine production in human whole blood. In addition, a role for the cyclic AMP/protein kinase A (PKA) pathway in the immunomodulatory effect of caffeine was investigated. Diluted whole blood (taken following ≥15 h abstinence from caffeine-containing food and beverages) was preincubated with caffeine or paraxanthine (10–100 μM) and stimulated with LPS (1 ∝g/ml) for 24 h. The proinflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-1β and IL-12, and the antiinflammatory cytokine IL-10 were measured in cell-free supernatants. Whilst caffeine and paraxanthine had little or no effect on IL-10, IL-1β, or IL-12 production, TNF-α production was suppressed in all individuals studied. The effect was statistically significant at 100 μM and consistent across seven experiments performed. Although not statistically significant, a similar effect was observed with paraxanthine. Caffeine (100 μM) also increased intracellular cyclic AMP concentrations in LPS-stimulated monocytes isolated from whole blood. Moreover, the effect of caffeine on TNF-α production was abolished by pretreatment with the protein kinase A inhibitor Rp-8-Br-cAMPS (10−4 and 10−5M). To conclude, this study demonstrates that concentrations of caffeine that are relevant to human consumption consistently suppress production of the proinflammatory cytokine TNF-α in human blood and that this effect is mediated by the cyclic AMP/protein kinase A pathway.

Suppressing effects of bisphenol A on the secretory function of ovine anterior pituitary cells.

We investigated the action of bisphenol A (BPA) on cellular GH release and content, cell number, GHmRNA expression, and concentrations of cellular cyclic AMP ([cAMP]c) and calcium ion ([Ca2+]c) in primary cultured ovine anterior pituitary cells. The following results were found: (1) BPA as well as nonylphenol (NP) at 10(-6) to 10(-3) M significantly and concentration-dependently suppressed basal and GHRH-stimulated GH release, and the cellular GH content, (2) BPA suppressed the cell number in a time- and concentration-dependent manner, (3) 10(-4)M BPA suppressed GHmRNA expression to 68% of control (BPA-free), and abolished GHRH (10(-8) M)-induced increases in [cAMP]c and [Ca2+]c. From these findings we conclude that BPA possesses a suppressing action on GH synthesis and release, and this suppressing action is probably related to impairment of cellular signal transduction systems in ovine anterior pituitary cells.

Sensitization of Dictyostelium chemotaxis by phosphoinositide-3-kinase-mediated self-organizing signalling patches.

The leading edge of Dictyostelium cells in chemoattractant gradients can be visualized using green fluorescent protein (GFP) tagged to the pleckstrin-homology (PH) domain of cytosolic regulator of adenylyl cyclase (CRAC), which presumable binds phosphatidylinositol-(3,4,5)triphosphate [PtdIns(3,4,5)P(3)]. Uniform cyclic AMP (cAMP) concentrations induce persistent translocation of PH(Crac)-GFP from the cytosol to multiple patches, which are similar to the single patch of PH(Crac)-GFP at the leading edge in a cAMP gradient. We show that cAMP determines the probability of patch formation (half-maximal effect at 0.5 nM cAMP) but not the size, lifetime or intensity of patches, indicating that patches are self-organizing structures. A pseudopod is extended from the area of the cell with a PH(Crac)-GFP patch at about 10 seconds after patch formation. Cells treated with the F-actin inhibitor latrunculin A are round without pseudopodia; uniform cAMP still induces localized patches of PH(Crac)-GFP. Inhibition of phosphoinositide-3-kinase (PI3K) activity with LY294002 inhibits PH(Crac)-GFP patches and inhibits chemotaxis towards nanomolar cAMP but has no effect at higher cAMP concentrations. Thus, very low cAMP concentrations induce self-organizing PH(Crac)-GFP patches that serve as a spatial cue for pseudopod formation, which enhances the sensitivity and amplitude of chemotactic movement.

Musclin, a Novel Skeletal Muscle-derived Secretory Factor

Skeletal muscle is involved in the homeostasis of glucose and lipid metabolism. We hypothesized that the skeletal muscle produces and secretes bioactive factor(s), similar to adipocytokines secreted by fat tissue. Here, we report the identification of a novel secretory factor, musclin, by signal sequence trap of mouse skeletal muscle cDNAs. Musclin cDNA encoded 130 amino acids, including NH(2)-terminal 30-amino acid signal sequence. Musclin protein contained a region homologous to natriuretic peptide family, and KKKR, a putative serine protease cleavage site, similar to the natriuretic peptide family. Full-length musclin protein and KKKR-dependent cleaved form were secreted in media of musclin cDNA-transfected mammalian cell cultures. Musclin mRNA was expressed almost exclusively in the skeletal muscle of mice. Musclin mRNA levels in skeletal muscle were markedly low in fasted, increased upon re-feeding, and were low in streptozotocin-treated insulin-deficient mice. Musclin mRNA expression was induced at late stage in the differentiation of C2C12 myocytes. In myocytes, insulin increased, while epinephrine, isoproterenol, and forskolin reduced musclin mRNA, all of which are known to increase the cellular content of cyclic AMP, a counter-regulator to insulin. Pathologically, overexpression of musclin mRNA was noted in the muscles of obese insulin-resistant KKAy mice. Functionally, recombinant musclin significantly attenuated insulin-stimulated glucose uptake and glycogen synthesis in myocytes. In conclusion, we identified musclin, a novel skeletal muscle-derived secretory factor. Musclin expression level is tightly regulated by nutritional changes and its physiological role could be linked to glucose metabolism.

Calmodulin-Dependent Cyclic Nucleotide Phosphodiesterase (PDE1) Is a Pharmacological Target of Differentiation-Inducing Factor-1, an Antitumor Agent Isolated from Dictyostelium

The differentiation-inducing factor-1 (DIF-1) isolated from Dictyostelium discoideum is a potent antiproliferative agent that induces growth arrest and differentiation in mammalian cells in vitro. However, the specific target molecule(s) of DIF-1 has not been identified. In this study, we have tried to identify the target molecule(s) of DIF-1 in mammalian cells, examining the effects of DIF-1 and its analogs on the activity of some candidate enzymes. DIF-1 at 10-40 micro M dose-dependently suppressed cell growth and increased the intracellular cyclic AMP concentration in K562 leukemia cells. It was then found that DIF-1 at 0.5-20 micro M inhibited the calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase (PDE1) in vitro in a dose-dependent manner. Kinetic analysis revealed that DIF-1 acted as a competitive inhibitor of PDE1 versus the substrate cyclic AMP. Because DIF-1 did not significantly affect the activity of other PDEs or CaM-dependent enzymes and, in addition, an isomer of DIF-1 was a less potent inhibitor, we have concluded that PDE1 is a pharmacological and specific target of DIF-1.