In vitro effects of lead ions on peripheral benzodiazepine receptors and adenylyl cyclase activity in the mantle of Mytilus galloprovincialis

As an extension of our previous work, where the density of peripheral benzodiazepine receptors (PBR) increased in mantle mitochondria of the marine mollusk Mytilus galloprovincialis Lmk. under chronic exposure to lead, the present study investigates the in vitro effects of an exogenous source of lead ions on PBR and on adenylyl cyclase (AC) complex in mantle membranes of mussels collected from a non-polluted coastal area. PBR binding experiments used the specific isoquinoline carboxamide derivative [3H]PK 11195, and AC activity was measured using a modified procedure adapted to M. galloprovincialis. Lead ions (Pb2+) dose-dependently decreased either the [3H]PK 11195 specific binding in mitochondria or basal AC velocity in plasma membranes of mussel mantle. The IC50 values for lead ions were 10 microM with [3H]PK 11195 binding and 25 microM with AC activity, with maximal inhibition values of 60% and 70%, respectively. Moreover, lead behaved as a non-competitive inhibitor on [3H]PK 11195 binding and as a 'mixed' inhibitor on AC activity. The present results suggest that some of the early effects induced by lead in mussel cell metabolism consist in significant changes of the PBR density and cyclic AMP production in the mantle of M. galloprovincialis.     

Circadian clocks regulate adenylyl cyclase activity rhythms in human RPE cells

Genes and components of the circadian clock may represent relevant drug targets for diseases involving circadian dysfunctions. By exploiting an established cell line derived from human retinal pigment epithelium (HRPE), the cell constituting the blood-retinal barrier that is essential to maintain the visual functions of the sensorineural retina, we showed serum-shock induction of rhythmic changes in forskolin-evoked adenylyl cyclase (AC) activity. In the presence of Ca2+ and protein kinase A, the forskolin-induced AC activity is significantly, but not completely inhibited, suggesting the involvement of both Ca2+-sensitive and Ca2+-insensitive AC isoforms in the regulation of circadian rhythmicity in these cells. Semi-quantitative RT-PCR showed circadian profile in the expression of three AC isoforms, the Ca2+-inhibitable AC5 and AC6 and the Ca2+-insensitive AC7, and the clock genes hPer1 and hPer2. Our results demonstrate for the first time circadian rhythmicity in a human cell line, identifying the isoforms involved in the circadian profile of AC activity and showing a rhythmicity of the clock gene mRNA expression in these cells. Therefore, the results reported here provide evidence for an intertwine between AC/[Ca2+]i signalling pathways and Per genes in the HRPE circadian clockwork.     

Involvement of the adenylyl cyclase signaling system in the action of insulin and mollusk insulin-like peptide

Involvement of the adenylyl cyclase signaling system in the mechanism of action of the mammalian insulin and epidermal growth factor as well as of insulin-like peptide isolated from the bivalve mollusk Anodonta cygnea has been studied. It was shown for the first time that insulin and insulin-like peptide exert in vitro the GTP-dependent stimulating action on the adenylyl cyclase activity. Epidermal growth factor has an analogous effect. Effectiveness of the peptides decreased in the order insulin-like peptide > epidermal growth factor > insulin in the foot smooth muscles of A. cygnea and insulin > epidermal growth factor > insulin-like peptide in the skeletal muscles of rat.     

Dexras1 blocks receptor-mediated heterologous sensitization of adenylyl cyclase 1

Dexras1/AGS1/RasD1 is a member of the Ras superfamily of monomeric G proteins and has been suggested to disrupt receptor-G protein signaling. We examined the ability of Dexras1 to modulate dopamine D_2L receptor regulation of adenylyl cyclase (AC) type 1 in HEK293 cells. Acute D_2L receptor-mediated inhibition of A23187-stimulated AC1 activity (IC₅₀, 4.0 ± 1.4 nM; 50 ± 3% inhibition) was not altered in the presence of Dexras1 (IC₅₀, 2.4 ± 1.3 nM, 50 ± 1% inhibition); however, Dexras1 blocked acute D_2L receptor-mediated activation of ERK 1/2 by approximately 50%. Heterologous sensitization of AC1 induced by persistent activation of D_2L receptors was completely blocked by Dexras1 under basal and A23187-stimulated conditions. The block of sensitization was concentration-dependent and was not observed with a nucleotide binding-deficient Dexras1G31V mutant. Sensitization of AC1 was Gβγ-dependent as demonstrated using the C-terminus of β-adrenergic receptor kinase (βARK-ct). These data suggest that Dexras1 selectively regulates receptor-mediated Gβγ signaling pathways.     

Morphine-related metabolites differentially activate adenylyl cyclase isozymes after acute and chronic administration

Morphine-3- and morphine-6-glucuronide are morphine’s major metabolites. As morphine-6-glucuronide produces stronger analgesia than morphine, we investigated the effects of acute and chronic morphine glucuronides on adenylyl cyclase (AC) activity. Using COS-7 cells cotransfected with representatives of the nine cloned AC isozymes, we show that AC-I and V are inhibited by acute morphine and morphine-6-glucuronide, and undergo superactivation upon chronic exposure, while AC-II is stimulated by acute and inhibited by chronic treatment. Morphine-3-glucuronide had no effect. The weak opiate agonists codeine and dihydrocodeine are also addictive. These opiates, in contrast to their 3-O-demethylated metabolites morphine and dihydromorphine (formed by cytochrome P450 2D6), demonstrated neither acute inhibition nor chronic-induced superactivation. These results suggest that metabolites of morphine (morphine-6-glucuronide) and codeine/dihydrocodeine (morphine/dihydromorphine) may contribute to the development of opiate addiction.     

Inhibition of melanogenesis by 5,7-dihydroxyflavone (chrysin) via blocking adenylyl cyclase activity

Due to its multiple biological activities, 5,7-dihydroxyflavone (chrysin) in propolis has gained attention as potentially useful therapeutics for various diseases. However, the efficacy of chrysin for the use of dermatological health has not been fully explored. To clarify the action mechanism of the skin protecting property of chrysin, we firstly investigated the molecular docking property of chrysin on the mammalian adenylyl cyclase, which is the key enzyme of cAMP-induced melanogenesis. We also examined the involvement of chrysin in alpha-MSH and forskolin-induced cAMP signaling within a cell based assay. In addition, we inquired into the inhibitory effect of chrysin on melanogenesis and found that the pretreatment with chrysin inhibited the forskolin-induced melanin contents significantly without annihilating the cell viability. These results strongly suggest that chrysin directly inhibits the activity of adenylyl cyclase, downregulates forskolin-induced cAMP-production pathway, consequently inhibiting melanogenesis. Thus, chrysin may also be used as an effective inhibitor of hyperpigmentation.     

EBV infection renders B cells resistant to growth inhibition via adenylyl cyclase

Cyclic AMP (cAMP) is an important physiological growth inhibitor of lymphoid cells, and the cAMP/protein kinase A (PKA) pathway is disrupted in several immunological disorders and cancers. Epstein Barr virus (EBV) infection of B lymphocytes is responsible for the development of lymphoproliferative disease as well as certain B-lymphoid malignancies. Here we hypothesized that EBV infection might render B lymphocytes resistant to cAMP/PKA-mediated growth inhibition. To test this, we assessed the growth-inhibitory response of cAMP-elevating compounds such as forskolin and isoproterenol, as well as the PKA activator 8-CPT-cAMP in normal B lymphocytes, EBV-infected B cells and in the EBV-negative B lymphoid cell line Reh. We could demonstrate that EBV infection indeed abolished cAMP-mediated growth inhibition of B cells. The defect was pinpointed to defective adenylyl cyclase (AC) activation by forskolin and isoproterenol, resulting in reduced formation of cAMP and lack of PKA activation and CREB phosphorylation. In contrast, 8-CPT-cAMP which directly activates PKA was able to inhibit EBV-infected B cell growth. The physiological implications of these results were underlined by the observation that the ability of forskolin to inhibit camptothecin-induced apoptosis was abolished in EBV-infected B cells. We conclude that EBV infection of B cells abrogates the activation of AC and thereby cAMP formation, and that this dysfunction renders the cells resistant to growth inhibition via the cAMP/PKA pathway.     

Functional reconstitution of G-protein-coupled receptor-mediated adenylyl cyclase activation by a baculoviral co-display system

Recently, evidence has accumulated in support of the heterologous expression of functional membrane proteins and their complexes on extracellular baculovirus particles (budded virus, BV). In this study, we attempted to apply this BV display system to detect G-protein-coupled receptor (GPCR) signaling. We infected Sf9 cells with a combination of four recombinant baculoviruses individually encoding the dopamine D1 receptor (DR-D1), G-protein -subunit (G_s), G-protein β₁γ₂ subunit dimer (Gβ₁γ₂), and adenylyl cyclase type VI (ACVI). The recovered BV fraction produced cAMP in response to the stimulation with dopamine. Co-expression of all three G-protein subunits in addition to receptor and ACVI led to a maximal response. BV co-expressing DR-D1, G_s, Gβ₁γ₂, and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other G_s-coupled receptors together with G_s, Gβ₁γ₂, and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, G_s and ACVI is reconstituted on BV. Since BV is essentially free of endogenous GPCRs, this BV co-display system should prove highly useful for the development of functional assay systems for GPCRs.     

Synthetic heteroprostanoids of A- and E-types as novel non-comprehensive inhibitors of adenylyl cyclase in rat hepatocytes

Treatment of rat hepatocyte plasma membranes with five novel synthetic heteroprostanoids of A- and E-types significantly decreased basal activity of adenylyl cyclase. Inhibition of forskolin-stimulated activity of the enzyme was seen as well. The maximal effective concentration for all substances tested was found at approximately 5x10(-6)-1x10(-5) M. The values of half maximal concentration (IC50) for all prostanoids were at the region of 0.7-1.1 microM. Prostanoids belonging to cyclopentenone group A (U-39, U-26) were less active than analogs of 11-deoxy-PGE1 (TA-227, TA-280, and TA-239). The strongest inhibitory effect of adenylyl cyclase activity (more than 3 times) was determined in the presence of prostanoid TA-227 possessing hydrophobic 15-phenyl ring and isoxazol group in omega-chain. The investigation of AC activity in the presence of different concentrations of prostanoids and varying concentrations of Mg x ATP has demonstrated that a non-comprehensive mechanism with particular effect takes place in case of AC inhibition by the heteroprostanoids.     

A bifunctional Galphai/Galphas modulatory peptide that attenuates adenylyl cyclase activity

Signaling via G-protein coupled receptors is initiated by receptor-catalyzed nucleotide exchange on Galpha subunits normally bound to GDP and Gbetagamma. Activated Galpha . GTP then regulates effectors such as adenylyl cyclase. Except for Gbetagamma, no known regulators bind the adenylyl cyclase-stimulatory subunit Galphas in its GDP-bound state. We recently described a peptide, KB-752, that binds and enhances the nucleotide exchange rate of the adenylyl cyclase-inhibitory subunit Galpha(i). Herein, we report that KB-752 binds Galpha(s) . GDP yet slows its rate of nucleotide exchange. KB-752 inhibits GTPgammaS-stimulated adenylyl cyclase activity in cell membranes, reflecting its opposing effects on nucleotide exchange by Galpha(i) and Galpha(s).     

A fluorimetric assay for real-time monitoring of adenylyl cyclase activity based on terbium norfloxacin

Adenylyl cyclases catalyze the production of the second messenger cyclic AMP from ATP. Until now, there has been no fluorescent adenylyl cyclase assay known that is applicable to high-throughput screening and kinetic determinations that can directly monitor the turnover of the unmodified substrate ATP. In this study, a fluorescence-based assay is described using the Ca(II)- and calmodulin-dependent adenylyl cyclase edema factor (EF) from Bacillus anthracis and Tb(III)-norfloxacin as probe for the enzyme activity. This assay can be used to study enzyme regulators, allows real-time monitoring of adenylyl cyclase activity, and does not substitute ATP by fluorescent derivatives. These derivatives must be judged critically due to their interference on the activity of enzymes. Furthermore, the new assay makes redundant the application of radioactively labeled substrates such as [α-³²P]ATP or fluorescently labeled antibodies such as anti-cyclic AMP. We determined the Michaelis-Menten constant (K_M), the v₀^max value of ATP turnover, and the IC₅₀ values for three inhibitors of EF by this newly developed fluorescent method.     

Role of an adenylyl cyclase isoform in ethanol's effect on cAMP regulated gene expression in NIH 3T3 cells

Previous research has indicated that the cyclic AMP (cAMP) signal transduction system plays an important role in the predisposition to and development of ethanol abuse in humans. Our laboratory has demonstrated that ethanol is capable of enhancing adenylyl cyclase (AC) activity. This effect is AC isoform-specific; type 7 AC (AC7) is most enhanced by ethanol. Therefore, we hypothesized that the expression of a specific AC isoform will play a role on the effect of ethanol on cAMP regulated gene expression. We employed NIH 3T3 cells transfected with AC7 or AC3 as a model system. To evaluate ethanol's effects on cAMP regulated gene expression, a luciferase reporter gene driven by a cAMP inducing artificial promoter was utilized. Stimulation of AC activity leads to an increase in the reporter gene activity. This increase was enhanced in the presence of ethanol in cells expressing AC7, while cells expressing AC3 did not respond to ethanol. cAMP reporter gene expression was increased in the presence of 8-bromo-cAMP; this expression was not enhanced by ethanol. These observations are consistent with our hypothesis. The basal level of CREB phosphorylation was high and did not change by cAMP stimulation or in the presence of ethanol. However, there were significant changes in the TORC3 amount in nuclei depending on stimulation conditions. The results suggest that nuclear translocation of TORC3 plays a more important role than CREB phosphorylation in the observed changes in the cAMP driven reporter gene activity.     

Thrombospondin-1 C-terminal-derived peptide protects thyroid cells from ceramide-induced apoptosis through the adenylyl cyclase pathway

Thrombospondin-1, a multi-modular matrix protein is able to interact with a variety of matrix proteins and cell-surface receptors. Thus it is multifunctional. In this work, we examined the role of thrombospondin-1 in ceramide-induced thyroid apoptosis. We focused on the VVM containing sequence localized in the C-terminal domain of the molecule. Primary cultured thyroid cells synthesize thrombospondin-1 depending on their morphological organization. As it leads thyrocytes to organize into monolayers before inducing apoptosis ceramide can modulate this organization. Here, we established that C₂-ceramide treatment decreased thrombospondin-1 expression by interfering with the adenylyl cyclase pathway, thus leading to apoptosis. Furthermore, we demonstrated that the thrombospondin-1-derived peptide 4N1 (RFYVVMWK) abolished ceramide-induced thyroid cell death by preventing intracellular cAMP levels from dropping. Finally, we reported that 4N1-mediated inhibition of ceramide-induced apoptosis was consistently associated with a down-regulation of the caspase-3 processing. Integrin-associated protein receptor (IAP or CD47) was identified as a molecular relay mediating the observed 4N1 effects. Taken together, our results shed light for the first time on anti-apoptotic activities of the thrombospondin-1-derived peptide 4N1 and provide new information on how thrombospondin-1 may control apoptosis of non-tumoral cells.     

Brain-specific natriuretic peptide receptor-B deletion attenuates high-fat diet-induced visceral and hepatic lipid deposition in mice

C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor-B (NPR-B), are abundantly distributed in the hypothalamus. To explore the role of central CNP/NPR-B signaling in energy regulation, we generated mice with brain-specific NPR-B deletion (BND mice) by crossing Nestin-Cre transgenic mice and mice with a loxP-flanked NPR-B locus. Brain-specific NPR-B deletion prevented body weight gain induced by a high-fat diet (HFD), and the mesenteric fat and liver weights were significantly decreased in BND mice fed an HFD. The decreased liver weight in BND mice was attributed to decreased lipid accumulation in the liver, which was confirmed by histologic findings and lipid content. Gene expression analysis revealed a significant decrease in the mRNA expression levels of CD36, Fsp27, and Mogat1 in the liver of BND mice, and uncoupling protein 2 mRNA expression was significantly lower in the mesenteric fat of BND mice fed an HFD than in that of control mice. This difference was not observed in the epididymal or subcutaneous fat. Although previous studies reported that CNP/NPR-B signaling inhibits SNS activity in rodents, SNS is unlikely to be the underlying mechanism of the metabolic phenotype observed in BND mice.Taken together, CNP/NPR-B signaling in the brain could be a central factor that regulates visceral lipid accumulation and hepatic steatosis under HFD conditions. Further analyses of the precise mechanisms will enhance our understanding of the contribution of the CNP/NPR-B system to energy regulation.     

Reversal of defective G-proteins and adenylyl cyclase/cAMP signal transduction in diabetic rats by vanadyl sulphate therapy

Vanadium salts exhibit a wide variety of insulinomimetic effects. In the present studies, we have examined the modulation of G-protein levels and adenylyl cyclase activity in the liver of streptozotocin-induced chronic diabetic rats (STZD) by vanadyl sulfate treatment and compared it with that of insulin. The basal enzyme activity, as well as the stimulatory effects of guanine nucleotides, glucagon, N-Ethylcarboxamideadenosine (NECA), isoproterenol, forskolin and sodium fluoride (NaF) on adenylyl cyclase were significantly increased in STZ-D rat liver as compared to control. In addition, the levels of stimulatory (Gs) as well as inhibitory (Gi_-2 and Gi_-3) as determined by immunoblotting techniques were also significantly higher in the STZ-D rat liver, however, the inhibitory effects of oxotremorine and low concentration of GTPS on adenylyl cyclase were not different in the two groups. Vanadyl sulfate and insulin treatments restored the augmented basal enzyme activity, the stimulations exerted by stimulatory inputs on adenylyl cyclase and the G-protein levels to various degrees, however, vanadyl sulfate was more effective than insulin. In addition, unlike vanadyl sulfate, insulin was unable to improve the stimulation exerted by glucagon and isoproterenol on adenylyl cyclase activity in STZD rats. These results suggest that vanadyl sulfate mimics the effects of insulin to restore the defective levels of G-proteins and adenylyl cyclase activity. From these results it may be suggested that one of the mechanisms by which vanadyl sulfate improves the glucose homeostasis in STZ-D rats may be through its ability to modulate the levels of G-proteins and adenylyl cyclase signal transduction system.Abbreviations NECA N-ethylcarboxamideadenosine - Iso Isoproterenol - Glu Glucagon - FSK forskolin - GTPS guanosine 5-[-thio]triphosphate - Gs stimulatory guanine nucleotide regulatory protein - Gi inhibitory guanine nucleotide regulatory protein - STZ streptozotocin This work was supported by grants from Medical Research Council and Canadian Diabetes Association.     

A new family of adenylyl cyclase genes in the male germline of Drosophila melanogaster

We describe the cloning and characterization of a new gene family of adenylyl cyclase related genes in Drosophila. The five adenylyl cyclase-like genes that define this family are clearly distinct from previously known adenylyl cyclases. One member forms a unique locus on chromosome 3 whereas the other four members form a tightly clustered, tandemly repeated array on chromosome 2. The genes on chromosome 2 are transcribed in the male germline in a doublesex dependent manner and are expressed in postmitotic, meiotic, and early differentiating sperm. These genes therefore provide the first evidence for a role for the cAMP signaling pathway in Drosophila spermatogenesis. Expression from this locus is under the control of the always early, cannonball, meiosis arrest, and spermatocyte arrest genes that are required for the G₂/M transition of meiosis I during spermatogenesis, implying a mechanism for the coordination of differentiation and proliferation. Evidence is also provided for positive selection at the locus on chromosome 2 which suggests this gene family is actively evolving and may play a novel role in spermatogenesis. Received: 26 September 1999 / Accepted: 27 October 1999     

Stimulation of the adenylyl cyclase activity in human endometrial membranes by VIP and related peptides

Vasoactive intestinal peptide (VIP) has been shown to stimulate adenylyl cyclase activity in human endometrial membranes. The effect was dependent on the time and temperature of incubation as well as on the concentration of endometrial membrane proteins in the medium. In the presence of 1 M GTP, half-maximal stimulation of adenylyl cyclase activity was observed at 25.0±7.0 nM VIP, whereas the maximal activity (at 1 M VIP)corresponded to an increase of about 140% with respect to basal values (7.5±0.6 pmol cyclic AMP/min/mg of protein). However, the maximal stimulation of adenylyl cyclase activity was obtained with helodermin (1 M) that increased the activity by 170% over the basal. The relative potency of VIP-related peptides upon the adenylyl cyclase activity was: helodermin (ED₅₀=1.8±1.4 nM)>VIP(ED₅₀=25.0±7.0 nM)>PHI (ED₅₀=725.0±127.2 nM). Secretin had a faint effect upon the adenylyl cyclase activity and glucagon was completely inefficient at this level. The presence of _s and _i subunits of G proteins in human endometrium was detected by immunoblot. Preliminary results showed the presence of two classes of¹²⁵I-VIP receptors in human endometrial membranes with the following stoichoimetric parameters: high affinity receptor (Kd=2.0 nM, binding capacity 0.1 pmol VIP/mg protein) and low affinity receptor (Kd=0.43 M, binding capacity 13.1 pmol VIP/mg protein). The present results together with the known presence of VIP in human uterus and the actions of this neuropeptide in the adjacent myometrial tissue support the idea that VIP and related peptides may have a role in human endometrium.     

Impaired water reabsorption in mice deficient in the type VI adenylyl cyclase (AC6)

Adenylyl cyclase (AC) type VI (AC6) is a calcium-inhibitable enzyme which produces cAMP upon stimulation. Herein, we characterized the specific role of AC6 in the kidneys using two AC6-knockout mouse lines. Immunohistochemical staining revealed that AC6 exists in the tubular parts of the nephron and collecting duct. Activities of AC evoked by forskolin or a selective agonist of the V2 vasopressin receptor were lower in the kidneys of AC6-null mice compared to those of wildtype mice. Results of a metabolic cage assay and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) showed for the first time that AC6 plays a critical role in regulating water homeostasis.     

Nitric oxide regulates adenylyl cyclase activity in rat striatal membranes

The regulation of adenylyl cyclase activity by nitric oxide (NO) was studied in rat (Sprague-Dawley) striatal membranes. Three chemically distinct NO donors attenuated forskolin-stimulated activity but did not alter basal activity. Maximum inhibition resulted in a 50% decrease in forskolin-stimulated activity, consistent with the presence of multiple isoforms of adenylyl cyclase and our previous findings that only the forskolin-stimulated activity of the type-5 and -6 isoform family of enzymes is inhibited by NO. To monitor primarily the type-5 isoform, we examined the ability of NO donors to attenuate D(1)-agonist-stimulated adenylyl cyclase activity. Under those conditions, complete inhibition was observed. The data indicate that NO attenuates neuromodulator-stimulated cAMP signaling in the striatum.     

Novel natriuretic peptides from the venom of the inland taipan (Oxyuranus microlepidotus): isolation, chemical and biological characterisation

Three natriuretic-like peptides (TNP-a, TNP-b, and TNP-c) were isolated from the venom of Oxyuranus microlepidotus (inland taipan) and were also present in the venoms of Oxyuranus scutellatus canni (New Guinea taipan) and Oxyuranus scutellatus scutellatus (coastal taipan). They were isolated by HPLC, characterised by mass spectrometry and Edman analysis, and consist of 35-39 amino acid residues. These molecules differ from ANP/BNP through replacement of invariant residues within the 17-membered ring structure and by inclusion of proline residues in the C-terminal tail. TNP-c was equipotent to ANP in specific GC-A assays or aortic ring assays whereas TNP-a and TNP-b were either inactive (GC-A over-expressing cells and endothelium-denuded aortic rings) or weakly active (endothelium-intact aortic rings). TNP-a and TNP-b were also unable to competitively inhibit the binding of TNP-c in endothelium-denuded aortae (GC-A) or endothelium-intact aortae (NPR-C). Thus, these naturally occurring isoforms provide a new platform for further investigation of structure-function relationships of natriuretic peptides.