Increase In Cyclic AMP Levels Research Articles

Toxicity and possible mechanisms of action of honokiol from Magnolia denudata seeds against four mosquito species

This study was performed to determine the toxicity and possible mechanism of the larvicidal action of honokiol, extracted from Magnolia denudata seeds, and its 10 related compounds against third-instar larvae of insecticide-susceptible Culex pipiens pallens, Aedes aegypti, and Aedes albopictus and Anopheles sinensis resistant to deltamethrin and temephos. Honokiol (LC50, 6.13–7.37 mg/L) was highly effective against larvae of all of the four mosquito species, although the toxicity of the compound was lower than that of the synthetic larvicide temephos. Structure–activity relationship analyses indicated that electron donor and/or bulky groups at the ortho or para positions of the phenol were required for toxicity. Honokiol moderately inhibited acetylcholinesterase and caused a considerable increase in cyclic AMP levels, indicating that it might act on both acetylcholinesterase and octopaminergic receptors. Microscopy analysis clearly indicated that honokiol was mainly targeted to the midgut epithelium and anal gills, resulting in variably dramatic degenerative responses of the midgut through sequential epithelial disorganization. Honokiol did not affect the AeCS1 mRNA expression level in Ae. aegypti larvae, but did enhance expression of the genes encoding vacuolar-type H+-ATPase and aquaporin 4, indicating that it may disturb the Na+, Cl− and K+ co-transport systems. These results demonstrate that honokiol merits further study as a potential larvicide, with a specific target site, and as a lead molecule for the control of mosquito populations.

Beta-2 adrenergic receptors increase TREG cell suppression in an OVA-induced allergic asthma mouse model when mice are moderate aerobically exercised

BackgroundThe potency of T regulatory (TREG) cells to inhibit T helper (Th)-driven immune cell responses has been linked to increased intracellular cyclic-AMP (cAMP) levels of TREG cells. In an ovalbumin (OVA)-driven allergic asthma mouse model, moderate aerobic exercise increases TREG cell function in a contact-dependent manner that leads to a significant reduction in chronic inflammation and restoration of lung function. However, the mechanism, whereby exercise increases TREG function, remains unknown and was the focus of these investigations. Exercise can communicate with TREG cells by their expression of β2-adrenergic receptors (β2-AR). Activation of these receptors results in an increase in intracellular levels of cyclic-AMP, potentially creating a potent inhibitor of Th cell responses.ResultsFor the allergic asthma model, female wildtype BALB/c mice were challenged with OVA, and exercised (13.5 m/min for 45 min) 3×/week for 4 weeks. TREG cells were isolated from all mouse asthma/exercise groups, including β2-AR−/− mice, to test suppressive function and intracellular cAMP levels. In these studies, cAMP levels were increased in TREG cells isolated from exercised mice. When β2-AR expression was absent on TREG cells, cAMP levels were significantly decreased. Correlatively, their suppressive function was compromised. Next, TREG cells from all mouse groups were tested for suppressive function after treatment with either a pharmaceutical β2-adrenergic agonist or an effector-specific cAMP analogue. These experiments showed TREG cell function was increased when treated with either a β2-adrenergic agonist or effector-specific cAMP analogue. Finally, female wildtype BALB/c mice were antibody-depleted of CD25+CD4+ TREG cells (anti-CD25). Twenty-four hours after TREG depletion, either β2-AR−/− or wildtype TREG cells were adoptively transferred. Recipient mice underwent the asthma/exercise protocols. β2-AR−/− TREG cells isolated from these mice showed no increase in TREG function in response to moderate aerobic exercise.ConclusionThese studies offer a novel role for β2-AR in regulating cAMP intracellular levels that can modify suppressive function in TREG cells.

Adenylyl cyclase 3/adenylyl cyclase-associated protein 1 (CAP1) complex mediates the anti-migratory effect of forskolin in pancreatic cancer cells.

Pancreatic cancer is one of the most lethal human malignancies. A better understanding of the intracellular mechanism of migration and invasion is urgently needed to develop treatment that will suppress metastases and improve overall survival. Cyclic adenosine monophosphate (cyclic AMP) is a second messenger that has shown to regulate migration and invasion of pancreatic cancer cells. The rise of cyclic AMP suppressed migration and invasion of pancreatic ductal adenocarcinoma cells. Cyclic AMP is formed from cytosolic ATP by the enzyme adenylyl cyclase (AC). There are ten isoforms of ACs; nine are anchored in the plasma membrane and one is soluble. What remains unknown is the extent to which the expression of transmembrane AC isoforms is both modified in pancreatic cancer and mediates the inhibitory effect of forskolin on cell motility. Using real-time PCR analysis, ADCY3 was found to be highly expressed in pancreatic tumor tissues, resulting in a constitutive increase in cyclic AMP levels. On the other hand, ADCY2 was down-regulated. Migration, invasion, and filopodia formation in two different pancreatic adenocarcinoma cell lines, HPAC and PANC-1 deficient in AC1 or AC3, were studied. We found that AC3, upon stimulation with forskolin, enhanced cyclic AMP levels and inhibited cell migration and invasion. Unlikely to be due to a cytotoxic effect, the inhibitory effects of forskolin involved the quick formation of AC3/adenylyl cyclase-associated protein 1 (CAP1)/G-actin complex, which inhibited filopodia formation and cell motility. Using Western blotting analysis, forskolin, through AC3 activation, caused phosphorylation of CREB, but not ERK. The effect of CREB phosphorylation is likely to be associated with long-term signaling changes. © 2016 Wiley Periodicals, Inc.

High throughput synthetic lethality screen reveals a tumorigenic role of adenylate cyclase in fumarate hydratase-deficient cancer cells

BackgroundSynthetic lethality is an appealing technique for selectively targeting cancer cells which have acquired molecular changes that distinguish them from normal cells. High-throughput RNAi-based screens have been successfully used to identify synthetic lethal pathways with well-characterized tumor suppressors and oncogenes. The recent identification of metabolic tumor suppressors suggests that the concept of synthetic lethality can be applied to selectively target cancer metabolism as well.ResultsHere, we perform a high-throughput RNAi screen to identify synthetic lethal genes with fumarate hydratase (FH), a metabolic tumor suppressor whose loss-of-function has been associated with hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Our unbiased screen identified synthetic lethality between FH and several genes in heme metabolism, in accordance with recent findings. Furthermore, we identified an enrichment of synthetic lethality with adenylate cyclases. The effects were validated in an embryonic kidney cell line (HEK293T) and in HLRCC-patient derived cells (UOK262) via both genetic and pharmacological inhibition. The reliance on adenylate cyclases in FH-deficient cells is consistent with increased cyclic-AMP levels, which may act to regulate cellular energy metabolism.ConclusionsThe identified synthetic lethality of FH with adenylate cyclases suggests a new potential target for treating HLRCC patients.

Characterization of the EP receptor subtype that mediates the inhibitory effects of prostaglandin E2 on IgE‐dependent secretion from human lung mast cells

Prostaglandin E2 (PGE2 ) has been shown to inhibit IgE-dependent histamine release from human lung mast cells. This effect of PGE2 is believed to be mediated by EP2 receptors. However, definitive evidence that this is the case has been lacking in the absence of EP2 -selective antagonists. Moreover, recent evidence has suggested that PGE2 activates EP4 receptors to inhibit respiratory cell function. The aim of this study was to determine the receptor by which PGE2 inhibits human lung mast cell responses by using recently developed potent and selective EP2 and EP4 receptor antagonists alongside other established EP receptor ligands. The effects of non-selective (PGE2 , misoprostol), EP2 -selective (ONO-AE1-259, AH13205, butaprost-free acid) and EP4 -selective (L-902,688, TCS251) agonists on IgE-dependent histamine release and cyclic-AMP generation in mast cells were determined. The effects of EP2 -selective (PF-04418948, PF-04852946) and EP4 -selective (CJ-042794, L-161,982) antagonists on PGE2 responses of mast cells were studied. The expression of EP receptor subtypes was determined by RT-PCR. Prostaglandin E2 , EP2 agonists and EP4 agonists inhibited IgE-dependent histamine release from mast cells. PGE2 and EP2 agonists, but not EP4 agonists, increased cyclic-AMP levels in mast cells. EP4 -selective antagonists did not affect the PGE2 inhibition of histamine release, whereas EP2 -selective antagonists caused rightward shifts in the PGE2 concentration-response curves. RT-PCR studies indicated that mast cells expressed EP2 and EP4 receptors. Although human lung mast cells may express both EP2 and EP4 receptors, the principal mechanism by which PGE2 inhibits mediator release in mast cells is by activating EP2 receptors.

A2A adenosine receptors are up-regulated in lymphocytes from amyotrophic lateral sclerosis patients

Adenosine, a purine nucleoside interacting with A1, A2A, A2B and A3 adenosine receptors (ARs), is a potent endogenous modulator of inflammatory and neuronal processes involved in the pathophysiology of several neurodegenerative diseases. In the present study, ARs were investigated in lymphocytes from patients with amyotrophic lateral sclerosis (ALS) and compared with age-matched healthy subjects. In ALS patients A2AARs were analysed by using RT-PCR, Western blotting and saturation binding experiments. The effect of A2AAR stimulation on cyclic AMP levels was evaluated in lymphocytes from ALS patients and healthy subjects.An up-regulation of A2AARs was observed in ALS patients with respect to healthy subjects while A1, A2B and A3AR affinity and density did not change. In ALS patients, the A2AAR density values correlated with the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R) scores. Furthermore, the stimulation of A2AARs mediated a significant increase in cyclic AMP levels in lymphocytes from ALS patients, with a higher potency than in lymphocytes from healthy subjects. In conclusion, the positive correlation between A2AAR density and ALSFRS-R scores could indicate a possible protective effect of this receptor subtype, representing an interesting starting point for the study of alternative therapeutic approaches for ALS based on A2AAR modulation.

5-hydroxy-7-methoxyflavone inhibits N-formyl-l-methionyl-l-leucyl-l-phenylalanine-induced superoxide anion production by specific modulate membrane localization of Tec with a PI3K independent mechanism in human neutrophils

Respiratory burst mediates crucial bactericidal mechanism in neutrophils. However, undesirable respiratory burst leads to pathological inflammation and tissue damage. This study investigates the effect and the underlying mechanism of 5-hydroxy-7-methoxyflavone (MCL-1), a lignan extracted from the leaves of Muntingia calabura L. (Tiliaceae), on N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP)-induced respiratory burst and cathepsin G release in human neutrophils. Signaling pathways regulated by MCL-1 to oppose fMLP-induced respiratory burst were evaluated by membrane localization of Tec induced by fMLP and by immunoblotting analysis of downstream phosphorylation targets of Tec. Briefly, MCL-1 specific inhibited fMLP-induced superoxide anion production in a concentration-dependent (IC50=0.16±0.01μM) and Tec kinase-dependent manner, however, MCL-1 did not affect fMLP-induced cathepsin G release. Further, MCL-1 suppressed fMLP-induced Tec translocation from the cytosol to the inner leaflet of the plasma membrane, and subsequently activation of phospholipase Cγ2 (PLCγ2). Moreover, MCL-1 attenuated PLCγ2 activity and intracellular calcium concentration notably through extracellular calcium influx. Consequently, fMLP-induced phosphorylation of protein kinase C (PKC) and membrane localization of p47phox were decreased by MCL-1 in a Tec-dependent manner, while the phosphorylation of extracellular signal-regulated kinase (ERK), p38, AKT and Src tyrosine kinase family remained unaffected. In addition, MCL-1 neither inhibited NADPH oxidase activity nor increased cyclicAMP levels. MCL-1 specific opposes fMLP-mediated respiratory burst by inhibition of membrane localization of Tec and subsequently interfered with the activation of PLCγ2, protein kinase C, and p47phox.

Effects of interferons on cortisol production in bovine adrenal fasciculata cells stimulated by adrenocorticotropin.

The effects of interferons (IFNs) IFN-alpha, IFN-beta and IFN-gamma on the production of cortisol in bovine adrenal fasciculata cells have been investigated. Pretreatment of the fasciculata cells with recombinant interferon-alpha-2b from man (over 300 units mL(-1)), but not with fibroblast IFN-beta or recombinant IFN-gamma from man, reduced the production of cortisol in cells stimulated with adrenocorticotropin (ACTH) (1 nM). IFN-alpha-2b inhibited ACTH-induced cortisol production in a concentration- (300-15000 units mL(-1)) and time- (2-24h) dependent manner. The inhibitory effect of IFN-alpha-2b on the production was abolished when the cells were simultaneously treated with anti-IFN-alpha antibody, and it was reversible. IFN-alpha-2b also inhibited dibutyryl cyclic AMP-induced production of cortisol but not pregnenolone-induced production. The effect of IFN-alpha-2b was not influenced by increases in external ACTH and Ca2+ concentrations and IFN-alpha-2b did not affect the ACTH-induced increase in cyclic AMP level in the cells. These results strongly suggest that IFN-alpha-2b reduces ACTH-induced production of cortisol in bovine adrenal fasciculata cells by affecting the early process of cortisol synthesis. The results also indicate that IFNs might not directly affect steroidogenesis in the adrenal cortex in-vivo, because of the requirement of high concentrations of IFN-alpha-2b for inhibition, and because of the ineffectiveness of IFN-beta and IFN-gamma.

Effect of Hydrogen Sulfide on Cyclic AMP Production in Isolated Bovine and Porcine Neural Retinae

Hydrogen sulfide (H(2)S) has been reported to exert pharmacological effects on neural and non-neural tissues from several mammalian species. In the present study, we examined the role of the intracellular messenger, cyclic AMP in retinal response to H(2)S donors, sodium hydrosulfide (NaHS) and sodium sulfide (Na(2)S) in cows and pigs. Isolated bovine and porcine neural retinae were incubated in oxygenated Krebs buffer solution prior to exposure to varying concentrations of NaHS, Na(2)S or the diterpene activator of adenylate cyclase, forskolin. After incubation at different time intervals, tissue homogenates were prepared for cyclic AMP assay using a well established methodology. In isolated bovine and porcine retinae, the combination of both phosphodiesterase inhibitor, IBMX (2 mM) and forskolin (10 microM) produced a synergistic increase (P < 0.001) in cyclic AMP concentrations over basal levels. NaHS (10 nM-100 microM) produced a time-dependent increase in cyclic AMP concentrations over basal levels which reached a maximum at 20 min in both bovine and porcine retinae. At this time point, both NaHS and Na(2)S (10 nM-100 microM) caused a significant (P < 0.05) dose-dependent increase in cyclic AMP levels in bovine and porcine retinae. For instance, NaHS (100 nM) elicited a four-fold and three-fold increase in cyclic AMP concentrations in bovine and porcine retinae respectively whilst higher concentrations of Na(2)S (100 microM) produced a much lesser effect in both species. In bovine and porcine retinae, the effects caused by forskolin (10 microM) on cyclic AMP production were not potentiated by addition of low or high concentrations of both NaHS and Na(2)S. We conclude that H(2)S donors can increase cyclic AMP production in isolated neural retinae from cows and pigs. Bovine retina appears to be more sensitive to the stimulatory effect of H(2)S donors on cyclic nucleotide production than its porcine counterpart indicating that species differences exist in the magnitude of this response. Furthermore, effects produced by forskolin on cyclic AMP formation were not additive with those elicited by H(2)S donors suggesting that these agents may share a common mechanism in their action on the adenylyl cyclase pathway.

Selective serotonin reuptake inhibitors pathway

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter that influences multiple processes, including autonomic function, motor activity, hormone secretion, cognition, and complex processes associated with affection, emotion, and reward [1]. In the terminal axon of the serotonergic neuron, free tryptophan (TRP) is converted to 5-HT [2]. 5-HT synthesis is a two-step process catalyzed by tryptophan hydroxylase (TPH) and aromatic decarboxylase (DDC). TPH is the rate-limiting enzyme and exists in two isoforms TPH1 and TPH2. The TPH2 isoform is the predominant form in neuronal tissue [3], [4]. The 5-HT uptake into presynaptic storage vesicles is mediated by the vesicular monoamine transporter (SLC18A2). The transporter accumulates serotonin into synaptic vesicles using a proton gradient across the vesicular membrane [5]. 5-HT that is not stored in vesicles is degraded by monoamine oxidase A (MAOA) to 5-hydroxyindoleacetic acid (5-HIAA). An action potential stimulates a calcium-dependent exocytotic release of serotonin from presynaptic vesicles into the synaptic cleft, where it interacts with both post- and presynaptic receptors. At the presynaptic side, 5-HT activates 5-hydroxytryptamine (serotonin) receptor 1A (HTR1A), B (HTR1B), and D (HTR1D), which results in an attenuation of the 5-HT exocytosis [2]. This feedback loop regulates the 5-HT concentration in the synaptic cleft and therefore, the extent of stimulation of various HTR receptor subclasses at the postsynaptic membrane [6]. Prolonged administration of selective serotonin reuptake inhibitors (SSRI) desensitizes these feedback loops. Thus, their regulatory effects on the serotonergic neurotransmission are weakened [7]. Postsynaptic HTR1 receptors (HTR1A, HTR1B, HTR1D, HTR1E, HTR1F) work together with HTR2 receptor subtypes (HTR2A, HTR2C) in mediating effector signals via activation of second messenger cascades [2]. Postsynaptic, the main signaling pathway for HTR1 receptor subtypes is via coupling of Gi/o protein alpha subunit (GNAI). This interaction decreases cyclic AMP formation by inhibiting adenylate cyclases (ADCY) [8]. After interaction with 5-HT, the main signaling linkage for the HTR2 receptor subpopulation is to activate phospholipase C (PLCB) through coupling of Gq/11 protein alpha (GNAQ) [8]. PLCB catalyzes the formation of myoinositol- 1, 4, 5-trisphosphate (IP3) and diacylglycerol (DAG) [9].The postsynaptic, ionotropic HTR3 receptor is a cation-specific ligand-gated ion channel, which does not activate a second messenger system [10]. The binding of 5-HT to this receptor depolarizes the postsynaptic membrane by sodium influx and potassium efflux, which is assumed to influence the activation of HTR2 receptors. HTR4, HTR6, and HTR7 primarily couple Gs protein alpha (GNAS), which results in an activation of adenylate cyclase, and consequently in an increase of cyclic AMP levels [9]. Further operational diversity is supported by the existence of a great number of splice and editing variants for several HTRs, their possible modulation by accessory proteins and chaperones, as well as their potential to form homo or heteromers both at the GPCR and at the ligand-gated channel level [11]. The amplification of all those second messenger signals in further downstream reactions leads to the mediation of neurotransmitter release from central serotonergic, noradrenergic, and dopaminergic neurons in the brain by regulating potassium channels, several protein kinases, and other calcium dependent signals. Chronic administration of antidepressant treatments have been reported to commonly increase the expression of brain-derived neurotrophic factor (BDNF), an activity-dependent secreted protein that is critical to organization of neuronal networks and synaptic plasticity [12], [13]. The solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 (SCL6A4) is responsible for terminating the action of 5-HT in the synaptic cleft. Released serotonin is transported back into the presynaptic terminals via this integral membrane protein. SCL6A4 is a member of the Na+/Cl−-dependent transporter family [14]. Four major classes of antidepressant drugs exist: monoamine oxidase inhibitors, selective serotonin reuptake inhibitors (SSRI), tricyclic compounds, and atypical antidepressant drugs [7]. To date, no comprehensive hypothesis concerning the antidepressant action for those therapies has been established [15]. Nonetheless, those drugs have one or more primary molecular targets in order to act. The molecular target for SSRI is SCL6A4, resulting in an inhibition of 5-HT reuptake in the presynapse from the synaptic cleft. The five SSRI fluoxetine, fluoxamine, paroxetine, sertraline, and citalopram vary in their pharmacological profile resulting in differential efficacy or side-effect profile for particular patients [16], [17], [18]. SSRI have a high affinity for 5-HT uptake transporters, low affinity for noradrenaline uptake transporters, and very low affinity for neurotransmitter receptors. The observation that current antidepressant therapies need a sustained treatment of 2–4 weeks to be effective suggests that adaptive changes in both serotonergic and noradrenergic neurotransmission and downstream neural adaptation (e.g. the BDNF receptor signaling pathway) rather than only the elevation in synaptic monoamine levels itself are responsible for the therapeutic effects [19], [20]. Several genetic polymorphisms have been associated with therapeutic SSRI response and also with adverse reaction, including genetic variants of the SCL6A4, HTR1A, HTR2A, HTR3B, TPH, BDNF, and G-protein beta3 subunit [10], [13], [21], [22].

Effects of rolipram and diazepam on the adaptive changes induced by morphine withdrawal in the hypothalamic paraventricular nucleus

A role for the cyclic AMP systems in the development of morphine dependence has been previously reported. In this study we investigated whether morphine dependence was inhibited by phosphodiesterase (PDE) 4 inhibitors rolipram and diazepam. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. On day 8, morphine withdrawal was precipitated by an injection of naloxone. In order to determine the effect of rolipram and diazepam rats were injected with these drugs once daily for seven days as well as 30 min before of naloxone injection. When opioid withdrawal was precipitated, an enhanced noradrenaline turnover and increased level of cyclic AMP and cyclic GMP in the hypothalamic paraventricular nucleus (PVN) were observed 30 min after naloxone administration. Moreover, c-Fos expression was induced in the PVN after naloxone-precipitated morphine withdrawal. Co-administration of rolipram or diazepam with morphine during the pre-treatment period, significantly reduced the signs of withdrawal, the enhancement of noradrenaline turnover and the increase in cyclic AMP. However, these inhibitors did not modify either levels of cyclic GMP or c-Fos expression in the PVN. These findings demonstrate that co-administration of rolipram or diazepam with morphine attenuate the withdrawal syndrome and suggest that these compounds may prevent the up-regulation of the cyclic AMP pathway and the associated increase in cyclic AMP level in morphine-withdrawn rats.

Tyramine as a possible neurotransmitter/neuromodulator at the spermatheca of the African migratory locust, Locusta migratoria

Tyramine-like immunoreactivity was identified in neurons of the VIIIth abdominal ganglion and in axons projecting to the spermatheca of adult females of Locusta migratoria. Tyramine-like immunoreactive processes were also found throughout all regions of the spermatheca and tyramine-like immunoreactive bipolar or multipolar neurons were present on the spermathecal sac. HPLC coupled with electrochemical detection revealed more tyramine than octopamine present in spermathecal tissue. Electrical stimulation of the ventral ovipositor nerve resulted in a significant increase in calcium-dependent release of tyramine from the spermatheca. Both tyramine and octopamine increase the frequency and basal tonus of spermathecal contractions in a dose-dependent manner, with octopamine having a lower threshold. When tyramine is applied along with a half maximal octopamine dose, there is an additive effect on contractions of the spermatheca with slight synergistic effects at lower doses of tyramine. High concentrations of tyramine (10 −4 M) stimulated increases in cyclic AMP levels of the spermatheca; an effect blocked by phentolamine. Phentolamine has a higher affinity (and thus a lower IC 50 value ≅5.6 × 10 −8 M) than yohimbine (IC 50 ≅1.1 × 10 −4 M) in reducing tyramine-induced spermathecal contractions. Taken together, these results suggest that tyramine may be a co-transmitter with octopamine at the spermatheca, with both neuroactive chemicals acting on an octopamine receptor.

Inhibition of Basal and Stimulated Release of Endothelin-1 from Guinea Pig Tracheal Epithelial Cells in Culture by Beta 2-adrenoceptor Agonists and Cyclic AMP Enhancers

The effects of cyclic AMP-related compounds and beta adrenoceptor agonists on the basal and lipopolysaccharide (LPS)-stimulated release of endothelin-1 (ET-1) from guinea-pig tracheal epithelial cells (GPTEpCs) in culture were studied. Forskolin (a potent activator of adenylyl cyclase), 8-bromo-cyclic AMP (a cyclic AMP analogue), salbutamol and salmeterol (two beta 2-adrenoceptor agonists), were used to increase cyclic AMP levels. Cultured GPTEpCs released ET-1 continuously over a 24 h incubation period. The values reached 1,938 +/- 122 pg/mg of total cell proteins after 24 h. LPS (10 microg/ml) significantly stimulated the release of ET-1 by 1.6- to 1.8-fold, up to 1,262 +/- 56 pg/mg total cell proteins after an 8 h incubation period. Compound 8-bromo-cyclic AMP (10(-5), 10(-4) and 10(-3) M) reduced the basal release of ET-1 from GPTEpCs by up to 31% (P < 0.01) and the LPS stimulated release by up to 42% (P < 0.05), after an 8 h incubation period. Forskolin (10(-6), 10(-5) and 10(-4) M) also inhibited the basal release of ET-1 by up to 28% (P < 0.05) and LPS-stimulated release of ET-1 by up to 50% (P < 0.05), after an 8 h incubation period. At the concentration of 10(-5) M, forskolin increased cyclic AMP levels in GPTEpCs by 17-fold (P < 0.001) in the medium, 15 min after the beginning of the incubation. Salbutamol (10(-8) to 10(-6) M) had no effect on the basal production and release of ET-1 after 8 h. Conversely, this short acting beta 2-adrenoceptor agonist significantly reduced LPS-mediated increase of ET-1 production by up to 55% (P < 0.05) after an 8 h incubation period. Salmeterol (10(-9) M to 10(-5) M) inhibited basal and LPS-stimulated production and release of ET-1 after an 8 h incubation period (between 44 and 51%, P < 0.01). Both salbutamol and salmeterol (10(-6) M) increase cyclic AMP levels by five- and twofold, respectively (P < 0.05). In summary, these observations indicate that beta 2-adrenoceptor agonists or cyclic AMP enhancers can modulate both basal and more markedly, the enhanced production of ET-1 from LPS-activated guinea pig airway EpCs. In addition, these compounds increase cyclic AMP levels in the cells. It is suggested that there is a correlation between cyclic AMP increase and inhibition of ET-1 release by guinea pig airway EpCs. Since ET-1 production was shown to be elevated in asthmatic subjects and in patients suffering from other inflammatory lung disorders, the inhibition of its production by beta adrenoceptor agonists, such as salbutamol and salmeterol, could be added to their therapeutical benefits.

Phosphodiesterase 4 inhibitors, rolipram and diazepam block the adaptive changes observed during morphine withdrawal in the heart

In this study, we investigated whether morphine dependence was inhibited by phosphodiesterase (PDE) 4 inhibitors rolipram and diazepam, since a role for the cyclic AMP systems in the development of morphine dependence was reported. Dependence of morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone. In order to determine the effect of rolipram or diazepam the animals were injected with these drugs for seven days and 30 min before the administration of naloxone. When opioid withdrawal was precipitated, enhancement of noradrenaline (NA) turnover in the heart was observed 30 min after naloxone administration. Moreover, morphine withdrawal induces Fos expression, increase in cyclic AMP and cyclic GMP levels. Co-administration of rolipram or diazepam with morphine during the pre-treatment period significantly reduces the signs of withdrawal symptoms, the enhancement of NA turnover, the increase in cyclic AMP and the Fos expression. However, these inhibitors did not modify the levels of cyclic GMP. These findings demonstrated that co-administration of rolipram or diazepam with morphine abolish the development of morphine dependence and suggest that these compounds prevent the up-regulation of the cyclic AMP pathway and the associated increase in cyclic AMP level after naloxone administration.

Identification and characterization of a novel family of Drosophilaβ‐adrenergic‐like octopamine G‐protein coupled receptors

Insect octopamine receptors carry out many functional roles traditionally associated with vertebrate adrenergic receptors. These include control of carbohydrate metabolism, modulation of muscular tension, modulation of sensory inputs and modulation of memory and learning. The activation of octopamine receptors mediating many of these actions leads to increases in the levels of cyclic AMP. However, to date none of the insect octopamine receptors that have been cloned have been convincingly shown to be capable of directly mediating selective and significant increases in cyclic AMP levels. Here we report on the identification and characterization of a novel, neuronally expressed family of three Drosophila G-protein coupled receptors that are selectively coupled to increases in intracellular cyclic AMP levels by octopamine. This group of receptors, DmOct beta1R (CG6919), DmOct beta2R (CG6989) and DmOct beta3R (CG7078) shows homology to vertebrate beta-adrenergic receptors. When expressed in Chinese hamster ovary cells all three receptors show a strong preference for octopamine over tyramine for the accumulation of cyclic AMP but show unique pharmacological profiles when tested with a range of synthetic agonists and antagonists. Thus, the pharmacological profile of individual insect tissue responses to octopamine might vary with the combination and the degree of expression of the individual octopamine receptors present.

Binding of (-)-[ 3 H]-CGP12177 at two sites in recombinant human � 1 -adrenoceptors and interaction with �-blockers

To verify the hypothesis that the non-conventional partial agonist (-)-CGP12177 binds at two beta(1)-adrenoceptor sites, human beta(1)-adrenoceptors, expressed in CHO cells, were labelled with (-)-[(3)H]-CGP12177. We compared the binding affinity and antagonist potency of 12 clinically used beta-blockers against the cyclic AMP-enhancing effects of (-)-isoprenaline and (-)-CGP12177.(-)-[(3)H]-CGP12177 bound to a high affinity site (H; K(H)=0.47 nM) and low affinity site (L); K(L)=235 nM). (-)-[(3)H]-CGP12177 dissociated from the beta(1)-adrenoceptors with a fast component (k(off)=0.45 min(-1)), consistent with the L-site, and a slow component (k(off)=0.017-0.033 min(-1)), consistent with the H-site. (-)-Isoprenaline and (-)-CGP12177 caused 96-fold and 12-fold maximal increases in cyclic AMP levels with -logEC(50)M of 8.2 and 7.6. (-)-CGP12177 antagonised the effects of (-)-isoprenaline with a pK(B) of 9.9. The beta-blockers antagonised the effects of (-)-isoprenaline more than the effects of (-)-CGP12177 with potency ratios: (-)-atenolol 1,000, (+/-)-metropolol 676, (-)-pindolol 631, (-)-timolol 589, (+/-)-carvedilol 204, (+/-)-oxprenolol 138, (+/-)-sotalol 132, (-)-propranolol 120, (+/-)-bisoprolol 95, (+/-)-alprenolol 81, (+/-)-nadolol 68 and (-)-bupranolol 56. In intact cells the binding constants of beta-blockers, estimated from competition with 3-5 nM (-)-[(3)H]-CGP12177 (binding to the H-site), correlated with the corresponding affinities estimated from antagonism of the (-)-isoprenaline effects. We conclude that (-)-[(3)H]-CGP12177 binds at two sites in the recombinant beta(1)-adrenoceptor. (-)-CGP12177 is an antagonist of catecholamine effects through the H-site and a non-conventional partial agonist through the L-site. beta-blockers are more potent antagonists through the H-site than the L-site.

Antiplatelet effects of KW-7, a new inhibitor of cyclic nucleotide phosphodiesterases

The antiplatelet effect of a new synthetic compound, 8,9-dimethoxyl-1-(4-methoxy-phenyl)-5,6-dihydro-pyrrolo[2,1- a]isoquinoline-2,3-dione (KW-7), was determined in rabbit platelets. KW-7 concentration-dependently prevented platelet aggregation caused by arachidonic acid, collagen, platelet-activating factor, and thrombin. KW-7 induced a substantial increase in cyclic AMP levels and a smaller increase in cyclic GMP levels in platelets. In platelet homogenates, KW-7 inhibited both cyclic AMP- and cyclic GMP-phosphodiesterase activities. The antiplatelet effect of KW-7 was reversed by SQ22536 (an inhibitor of adenylate cyclase) and H89 (an inhibitor of protein kinase A) but not by ODQ (an inhibitor of soluble guanylate cyclase). These data suggest that the antiplatelet effect of KW-7 is cyclic AMP-dependent, and is through inhibition of platelet phosphodiesterases. In addition, KW-7 inhibited arachidonic acid-stimulated thromboxane production; this effect was associated with an increase in prostaglandin D 2 levels indicating KW-7 is also an inhibitor of thromboxane synthase. The dual inhibition of KW-7 on phosphodiesterase and thromboxane synthase might provide an attractive target in developing antiplatelet drugs.

Dopamine D1 receptors mediate CREB phosphorylation via phosphorylation of the NMDA receptor at Ser897-NR1.

Addictive drugs such as amphetamine and cocaine stimulate the dopaminergic system, activate dopamine receptors and induce gene expression throughout the striatum. The signal transduction pathway leading from dopamine receptor stimulation at the synapse to gene expression in the nucleus has not been fully elucidated. Here, we present evidence that D1 receptor stimulation leads to phosphorylation of the transcription factor Ca2+ and cyclic AMP response element binding protein (CREB) in the nucleus by means of NMDA receptor-mediated Ca2+ signaling. Stimulation of D1 receptors induces the phosphorylation of Ser897 on the NR1 subunit by protein kinase A (PKA). This phosphorylation event is crucial for D1 receptor-mediated CREB phosphorylation. Dopamine cannot induce CRE-mediated gene expression in neurons transfected with a phosphorylation-deficient NR1 construct. Moreover, stimulation of D1 receptors or increase in cyclic AMP levels leads to an increase in cytosolic Ca2+ in the presence of glutamate, but not in the absence of glutamate, indicating the ability of dopamine and cyclic AMP to facilitate NMDA channel activity. The recruitment of the NMDA receptor signal transduction pathway by D1 receptors may provide a general mechanism for gene regulation that is fundamental for mechanisms of drug addiction and long-term memory.

Dopamine D2-like receptor-mediated opening of K+ channels in opossum kidney cells.

(1) This study examined the effects of dopamine D(1)- and D(2)-like receptor activation upon basolateral K(+) (I(K)) currents and changes in membrane potential in opossum kidney (OK) cells. (2) The addition of amphotericin B (3 micro g ml(-1)) to the apical side resulted in a rapid increase in I(K), this effect being markedly inhibited by the addition of the K(+) channel blockers barium chloride (1 mM) or glibenclamide (10 micro M), but not apamin (1 micro M). The K(+) channel opener pinacidil increased the amphotericin B-induced I(K). The selective D(2)-like receptor agonist quinerolane increased, in a concentration dependent manner (EC(50)=136 nM), I(K) across the basolateral membrane, this effect being abolished by pre-treatment with pertussis toxin (PTX), S-sulpiride (selective D(2)-like receptor antagonist) and glibenclamide. The selective D(1)-like receptor agonist SKF 38393 did not change I(K). Both H-89 (PKA inhibitor) and chelerythrine (PKC inhibitor) failed to prevent the stimulatory effect of quinerolane upon I(K). (3) Quinerolane did not change basal levels of cyclic AMP and also failed to affect the forskolin-induced increase in cyclic AMP levels. (4) The stimulation of D(2)-like receptor was associated with a rapid hyperpolarizing effect, whereas D(1)-like receptor activation was accompanied by increases in cell membrane potential. The hyperpolarizing effect of quinerolane (EC(50)=129 nM) was prevented by pre-treatment with PTX, S-sulpiride and glibenclamide. (5) It is concluded that stimulation of dopamine D(2)-like, but not D(1)-like, receptors coupled to PTX-sensitive G proteins of the G(i/o) class produce membrane hyperpolarization through opening of K(ATP) channels.

Adenosine regulates the survival of avian retinal neurons and photoreceptors in culture.

Adenosine modulates the survival of chick embryo retinal neurons in culture. When cultures were incubated for 3 days and refed with fresh medium, a large proportion of neurons died in the subsequent 3 days of culture. This cell death was prevented by preincubation of cultures for at least 24 h with adenosine plus the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), an adenosine uptake blocker nitrobenzylthioinosine (NBI), the adenosine A2A receptor agonist 2-[4-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680), or the permeant cyclic AMP analog 8-bromo cyclic AMP, but not the A1 receptor agonist cyclohexyladenosine (CHA). Adenosine deaminase induced cell death when added to culture medium, and this effect was prevented by EHNA. Cell death was not observed when the medium was replaced by a conditioned medium from sister cultures. The data strongly suggest that adenosine regulates the survival of developing retinal neurons by a long-term activation of A2A receptors and the increase of cyclic AMP levels.