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Objective: Elabela is a newly discovered peptide hormone. This study aimed to determine the functional effects and mechanisms of action of elabela in rat pulmonary artery and trachea., Materials and Methods: Vascular rings isolated from the pulmonary arteries of male Wistar Albino rats were placed in chambers in the isolated tissue bath system. The resting tension was set to 1 g. After the equilibration period, the pulmonary artery rings were contracted with 10 -6 M phenylephrine. Once a stable contraction was achieved, elabela was applied cumulatively (10 -10 -10 -6 M) to the vascular rings. To determine the vasoactive effect mechanisms of elabela, the specified experimental protocol was repeated after the incubation of signaling pathway inhibitors and potassium channel blockers. The effect and mechanisms of action of elabela on tracheal smooth muscle were also determined by a similar protocol., Results: Elabela exhibited a concentration-dependent relaxation in the precontracted rat pulmonary artery rings (p < .001). Maximal relaxation level was 83% (pEC 50 : 7.947 CI95(7.824-8.069)). Removal of the endothelium, indomethacin incubation, and dideoxyadenosine incubation significantly decreased the vasorelaxant effect levels of elabela (p < .001). Elabela-induced vasorelaxation levels were significantly reduced after iberiotoxin, glyburide, and 4-Aminopyridine administrations (p < .001). L-NAME, methylene blue, apamin, TRAM-34, anandamide, and BaCl 2 administrations did not cause a significant change in the vasorelaxant effect level of elabela (p = 1.000). Elabela showed a relaxing effect on precontracted tracheal rings (p < .001). Maximal relaxation level was 73% (pEC 50 : 6.978 CI95(6.791-7.153)). The relaxant effect of elabela on tracheal smooth muscle was decreased significantly after indomethacin, dideoxyadenosine, iberiotoxin, glyburide, and 4-Aminopyridine incubations (p < .001)., Conclusions: Elabela exerted a prominent relaxant effect in the rat pulmonary artery and trachea. Intact endothelium, prostaglandins, cAMP signaling pathway, and potassium channels (BK Ca , K V , and K ATP channels) are involved in the vasorelaxant effect of elabela. Prostaglandins, cAMP signaling pathway, BK Ca channels, K V channels, and K ATP channels also contribute to elabela-induced tracheal smooth muscle relaxant effect., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Nucleobindin (NUCB)-derived peptides, nesfatin-1 (NES-1) and nesfatin-1-like peptide (NLP) have several physiological roles in vertebrates. While NES-1 is implicated in stress, whether NUCB1/NLP and NUCB2/NES-1 have any effect on proopiomelanocortin (POMC) remains unknown. The main aim of this study was to determine if NES-1 and/or NLP affect POMC synthesis in mouse corticotrophs. Immunocytochemistry was employed to target NUCB colocalization with POMC in immortalized mouse tumoral corticotrophs (AtT-20 cells). The ability of NES-1 and NLP to modulate POMC mRNA and protein in AtT-20 cells was assessed by qPCR and Western blot, respectively. Moreover, cell-signaling molecules mediating the effect of NES-1 and NLP on POMC synthesis in mouse tumoral corticotrophs were studied using pharmacological blockers. Mouse tumoral corticotrophs showed immunoreactivity for both NUCB1/NLP and NUCB2/NES-1. Both NES-1 and NLP exerted a stimulatory effect on POMC transcript abundance and protein expression in a dose- and time-dependent manner. This effect was comparable to corticotropin-releasing factor (CRF, positive control) stimulation of POMC. Incubation of mouse tumoral corticotrophs with NES-1 or NLP upregulated the phosphorylation of protein kinase A (PKA) and cAMP-response element-binding protein (CREB). The stimulatory effect of these peptides on POMC transcript abundance and protein expression was blocked by the PKA inhibitor, H89, and an adenylate cyclase inhibitor, 2',3'-dideoxyadenosine (DDA). These pharmacological studies indicate that NES-1 and NLP act through the cAMP/PKA/CREB cellular pathway to stimulate POMC synthesis. Our results provide molecular evidence to support a stimulatory role for nucleobindin-derived peptides on POMC synthesis from corticotrophs. Collectively, this research indicates that corticotrophs produce NUCBs, and the encoded peptides NES-1 and NLP could elicit a direct action to stimulate the pituitary stress hormone. This stimulatory effect is mediated by an uncharacterized G protein-coupled receptor (GPCR) that utilizes the cAMP/PKA/CREB pathway., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Suppression of the production of granulocytic CSF under the effect of 5-fluorouracyl is related to disorders in the NF-κB-, cAMP-dependent signaling pathways and MAPK cascade. These secondary messengers are involved in the regulation of functional activity of nonadherent myelokaryocytes starting from day 10 of the experiment (initial period of the hemopoietic granulocytic stem regeneration after antimetabolite challenge). Granulocytic CSF does not play essential role in the formation of colony-stimulating activity of cells of the adherent and nonadherent fractions of the bone marrow. Only cAMP-dependent pathway is involved in the regulation of the realization of the granulocytic precursor growth potential in response to the challenge.

Heparin is the most commonly used in vitro capacitation inducer in the bovine. However, hyaluronic acid (HA) has been recently used for capacitation induction as well as for other reproductive biotechnologies, such as sperm selection and in vitro fertilization (IVF). Our aim was to induce sperm capacitation with heparin or HA in order to study mAC and TK intracellular signals and their relation with cleavage and blastocyst rates after IVF as well as with the oxidative status of the potential bovine embryos. 2,5-dideoxyadenosine and genistein were used as mAC and TK inhibitors, respectively. Sperm capacitation was analyzed using CTC technique, sperm plasma membrane and acrosome integrity were determined using trypan blue stain and differential interference contrast, and mitochondrial activity was evaluated using fluorochrome JC-1. Cleavage rate was analyzed 48h and blastocyst production 7-8 days after IVF, while cytosolic oxidative activity was determined using RedoxSensor Red CC-1 fluorochrome 7h after IVF. When mAC and TK inhibitors were added to sperm samples, only capacitation decreased significantly both in HA and heparin treated samples (P < 0.05), but plasma membrane and acrosome integrity percentages were not affected in any of these groups (P > 0.05). Sperm mitochondrial membrane potential only decreased in heparin treated samples in the presence of both inhibitors (P < 0.05). Oocytes activated with HA sperm treated samples with the addition of 2,5-dideoxyadenosine and genistein presented a lower cytosolic oxidative status than those activated with sperm treated with HA alone (P < 0.05). On the other hand, oocytes activated with heparin treated sperm samples presented a lower cytosolic oxidative status only in the presence of 2,5-dideoxyadenosine (P < 0.05). Therefore, mAC and TK present a differential participation in heparin and HA sperm induced capacitation and mitochondrial function as well as in IVF., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The effects of 4NO 2 PDPMe and 4APDPMe, which are thalidomide (Tha) analogs that act as selective phosphodiesterase (PDE-4) inhibitors, on estrous behavior (lordosis and proceptive behaviors) and on uterine contraction were studied in ovariectomized (OVX) estrogen-primed Sprague Dawley (SD) and in intact non-pregnant Wistar rats, respectively. We found that intracerebroventricular (ICV) infusion of either 4NO 2 PDPMe or 4APDPMe (20 to 80 μg) stimulated intense lordosis and proceptive behavior in response to mounts from a sexually active male, within the first 4 h after infusion, and persisting for up to 24 h. Inhibitors of the progesterone receptor (RU486, administered subcutaneously), the estrogen receptor (tamoxifen, ICV), the adenylate cyclase (AC)/ cyclic AMP (cAMP)/protein kinase A (PKA) pathway (administered ICV), and the mitogen activated protein kinase (MAPK) pathway (administered ICV) significantly decreased lordosis and proceptive behavior induced by Tha analogs. Uterine contractility studies showed that Tha analogs inhibited both the K + - and the Ca 2+ -induced tonic contractions in rat uterus. Tha analogs were equally effective, but 4APDPMe was more potent than 4NO 2 PDPMe. These results strongly suggest the central role of cAMP in both processes, sexual behavior, and uterine relaxation, and suggest that Tha analogs may also act as Ca 2+ -channel blockers., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The role of NF-κB, cAMP/PKA, JAKs/STAT3, ERK1/2, p38, JNK, and p53 signaling pathways in the realization of growth potential of mesenchymal, neural, erythroid, and granulomonocytic progenitor cells were examined in vitro. Using selective blockers of signaling molecules, we revealed some principal distinctions of their involvement in determination of proliferation-differentiation status of the progenitor cells of different functional classes. The most salient peculiarities were observed in the roles of cAMP/PKA, JNK, and JAKs/STAT3 signaling pathways in the control of functions of various types of the regeneration-competent elements. The specific features of intracellular signaling revealed in histogenetically and functionally different progenitor cells attest to visibility of differentiated pharmacological stimulation of regeneration in individual tissues and prospectiveness in the development of targeted remedies for regenerative medicine based on modifiers of activity of the intracellular signaling molecules.

Neuropeptide S (NPS) is an important anxiolytic substance of the brain. However, the signaling pathways downstream of NPS receptor (NPSR) activation, underlying the behavioral effect of NPS, remain largely unknown. Here, we show that bilateral microinfusion of NPS (0.2 nmol/0.5 μl) into the medial amygdala (MeA) of male adult Wistar rats reduced anxiety-related behavior on both the elevated plus-maze and the open field. Moreover, as shown in amygdala tissue micropunches intracerebroventricular infusion of NPS (1 nmol/5 μl) (1) evoked phosphorylation and synthesis of CaMKIIα in relation to reference protein β-tubulin representing Ca 2+ influx, and (2) induced phosphorylation of mitogen-activated protein kinase ERK1/2. The NPS-induced anxiolysis was prevented by local inhibition of phospholipase C signaling using U73122 (0.5 nmol/0.5 μl) in the MeA, indicating the behavioral relevance of this pathway. Conversely, local pharmacological blockade of adenylyl cyclase signaling using 2',5'-dideoxyadenosine (12.5 nmol/0.5 μl) failed to inhibit the anxiolytic effect of NPS infused into the MeA. Hence, NPS promotes acute anxiolysis within the MeA dependent on NPSR-mediated phospholipase C signaling. Taken together, our study extends the knowledge about the intracellular signaling mechanisms underlying the potent anxiolytic profile of NPS.

Diabetes mellitus is a chronic disease in which the pancreas no longer produces enough insulin. Pancreatic alpha cell mass increases in response to insufficient insulin secretion. However, the reason for this increase is not clear. It is possible that the increased alpha-cells may stimulate compensatory insulin release in response to the insufficient insulin such as insulin resistance. In this study, we investigated whether glucagon and glucagon-like peptide-1 (GLP-1), hormones produced by alpha cells, contribute to insulin secretion in INS-1 cells, a beta cell line. We confirmed that alpha cell area in the pancreatic islets and glucagon secretion were increased in HFD-induced obese mice. Co-treatment with glucagon and exendin-4 (Ex-4), a GLP-1 receptor agonist, additively increased glucose-stimulated insulin secretion in INS-1 cells. In parallel, cAMP production was also additively increased by co-treatment with these hormones. The increase of insulin secretion by Ex-4 in the presence of high glucose was inhibited by 2'5'-dideoxyadenosine, a transmembrane adenylyl cyclase inhibitor, but not by KH-7, a soluble adenylyl cyclase inhibitor. The increase of insulin secretion by glucagon in INS-1 cells was inhibited by both 2'5'-dideoxyadenosine and KH-7. We suggest that glucagon and GLP-1 produced from alpha cells additively increase cAMP and insulin secretion in the presence of high glucose via distinct adenylyl cyclases in INS-1 cells, and this may contribute to the compensatory increase of insulin secretion by an increase of pancreatic alpha cell mass under conditions of insulin resistance., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Glucagon-like peptide-1 receptors (GLP-1Rs) have been shown to mediate cognitive-enhancing and neuroprotective effects in the central nervous system. However, little is known about their physiological roles on central neurotransmission, especially at the presynaptic level. Using purified synaptosomal preparations and immunofluorescence techniques, here we show for the first time that GLP-1Rs are localized on mouse cortical and hippocampal synaptic boutons, in particular on glutamatergic and GABAergic nerve terminals. Their activation by the selective agonist exendin-4 (1-100 nM) was able to increase the release of either [ 3 H]d-aspartate or [ 3 H]GABA. These effects were abolished by 10 nM of the selective GLP1-R antagonist exendin-3 (9-39) and were prevented by the selective adenylyl cyclase inhibitor 2',5'-dideoxyadenosine (10 µM), indicating the involvement of classic GLP-1Rs coupled to G s protein stimulating cAMP synthesis. Our data demonstrate the existence and activity of presynaptic receptors for GLP-1 that could represent additional mechanisms by which this neurohormone exerts its effects in the CNS. © 2017 BioFactors, 44(2):148-157, 2018., (© 2017 International Union of Biochemistry and Molecular Biology.)

Background: Since antidopaminergic drugs are pharmacological agents employed in the management of gastrointestinal motor disorders at all ages, we investigated whether the enteric dopaminergic system may undergo developmental changes after birth., Methods: Intestinal mechanical activity was examined in vitro as changes in isometric tension., Results: In 2-d-old (P2) mice, dopamine induced a contractile effect, decreasing in intensity with age, replaced, at the weaning (day 20), by a relaxant response. Both responses were tetrodotoxin (TTX)-insensitive. In P2, dopaminergic contraction was inhibited by D1-like receptor antagonist and mimicked by D1-like receptor agonist. In 90-d-old (P90) mice, the relaxation was reduced by both D1- and D2-like receptor antagonists, and mimicked by D1- and D2-like receptor agonists. In P2, contraction was antagonized by phospholipase C inhibitor, while in P90 relaxation was antagonized by adenylyl cyclase inhibitor and potentiated by phospholipase C inhibitor. The presence of dopamine receptors was assessed by immunofluorescence. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed a significant increase in D1, D2, and D3 receptor expression in proximal intestine with the age., Conclusion: In mouse small intestine, the response to dopamine undergoes developmental changes shifting from contraction to relaxation at weaning, as the consequence of D2-like receptor recruitment and increased expression of D1 receptors.

The objectives of this study were firstly to determine whether the stimulatory function of equine growth hormone (eGH) on equine oocyte maturation in vitro is mediated via cyclic adenosine monophosphate (cAMP); and secondly if the addition of eGH in vitro influences oocyte nuclear maturation and if this effect is removed when GH inhibitors are added to the culture. Cumulus-oocyte complexes (COCs) were recovered from follicles <25 mm in diameter and randomly allocated as follows: (i) control (no additives); and (ii) 400 ng/ml of eGH. A specific inhibitor against cyclic AMP-dependent protein kinase (H-89; 10-9, 10-11 or 10-15 M concentration) and a specific adenylate cyclase inhibitor, 2',3'-dideoxyadenosine (DDA; 10-8, 10-10 or 10-14 M concentration) were used to observe whether they could block the eGH effect. After 30 h of in vitro maturation at 38.5°C with 5% CO2 in air, oocytes were stained with 10 μg/ml of Hoechst to evaluate nuclear status. More mature oocytes (P < 0.05) were detected when COCs were incubated with eGH (29 of 84; 34.5%) than in the control group (18 of 82; 21.9%). The H-89 inhibitor used at a concentration of 10-9 M (4 of 29; 13.8%) decreased (P < 0.05) the number of oocytes reaching nuclear maturation when compared with eGH (11 of 29; 38%). The DDA inhibitor at a concentration of 10-8 M (2 of 27; 7.4%) also reduced (P < 0.05) the number of oocytes reaching maturity when compared with the eGH group (9 of 30; 30%). Results from the present study show that H-89 and DDA can be used in vitro to block the eGH effect on equine oocyte maturation.

The basolateral amygdala (BLA) plays crucial roles in stimulus value coding, as well as drug and alcohol dependence. Ethanol alters synaptic transmission in the BLA, while endocannabinoids (eCBs) produce presynaptic depression at BLA synapses. Recent studies suggest interactions between ethanol and eCBs that have important consequences for alcohol drinking behavior. To determine how ethanol and eCBs interact in the BLA, we examined the physiology and pharmacology of GABAergic synapses onto BLA pyramidal neurons in neurons from young rats. Application of ethanol at concentrations relevant to intoxication increased, in both young and adult animals, the frequency of spontaneous and miniature GABAergic inhibitory postsynaptic currents, indicating a presynaptic site of ethanol action. Ethanol did not potentiate sIPSCs during inhibition of adenylyl cyclase while still exerting its effect during inhibition of protein kinase A. Activation of type 1 cannabinoid receptors (CB1) in the BLA inhibited GABAergic transmission via an apparent presynaptic mechanism, and prevented ethanol potentiation. Surprisingly, ethanol potentiation was also prevented by CB1 antagonists/inverse agonists. Brief depolarization of BLA pyramidal neurons suppressed GABAergic transmission (depolarization-induced suppression of inhibition [DSI]), an effect previously shown to be mediated by postsynaptic eCB release and presynaptic CB1 activation. A CB1-mediated suppression of GABAergic transmission was also produced by combined afferent stimulation at 0.1 Hz (LFS), and postsynaptic loading with the eCB arachidonoyl ethanolamide (AEA). Both DSI and LFS-induced synaptic depression were prevented by ethanol. Our findings indicate antagonistic interactions between ethanol and eCB/CB1 modulation at GABAergic BLA synapses that may contribute to eCB roles in ethanol seeking and drinking., (Published by Elsevier Inc.)

The role of cAMP- and IKK-2-dependent pathways in stimulation of the growth capacity of mesenchymal progenitor cells with alkaloid songorine was studied in vitro. Inhibitors of adenylate cyclase and IKK-2 were shown to abolish the increase in proliferative activity of progenitor cells. Moreover, blockade of the inhibitory kinase complex was accompanied by a decrease in the intensity of progenitor cell differentiation.

Voltage-gated potassium channels (Kv) are important regulators of membrane potential in vascular smooth muscle cells, which is integral to controlling intracellular Ca2+ concentration and regulating vascular tone. Previous work indicates that Kv channels can be modulated by receptor-driven alterations of cyclic AMP-dependent protein kinase (PKA) activity. Here, we demonstrate that Kv channel activity is maintained by tonic activity of PKA. Whole-cell recording was used to assess the effect of manipulating PKA signalling on Kv and ATP-dependent K+ channels of rat mesenteric artery smooth muscle cells. Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of Kv currents. Tonic PKA-mediated activation of Kv appears maximal as application of isoprenaline (a β-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance Kv currents. We also show that this modulation of Kv by PKA can be reversed by protein phosphatase 2B/calcineurin (PP2B). PKA-dependent inhibition of Kv by KT5720 can be abrogated by pre-treatment with the PP2B inhibitor cyclosporin A, or inclusion of a PP2B auto-inhibitory peptide in the pipette solution. Finally, we demonstrate that tonic PKA-mediated modulation of Kv requires intact caveolae. Pre-treatment of the cells with methyl-β-cyclodextrin to deplete cellular cholesterol, or adding caveolin-scaffolding domain peptide to the pipette solution to disrupt caveolae-dependent signalling each attenuated PKA-mediated modulation of the Kv current. These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on Kv channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone.

The aim of this work was to study the participation of membrane adenylyl cyclase in heparin-induced capacitation in cryopreserved bovine spermatozoa. Sperm suspensions were incubated in Tyrode's albumin lactate pyruvate medium in the presence of heparin (10 IU ml(-1) ) or forskolin (1-75 μm), a well-known membrane adenylyl cyclase activator. The participation of membrane adenylyl cyclase was confirmed using a specific inhibitor, 2',5'-dideoxyadenosine (6-25 μm). Spermatozoa capacitated with forskolin (25 μm) were incubated with bovine follicular fluid to evaluate their ability to undergo acrosome reaction. Capacitation percentages were determined by the fluorescence technique with chlortetracycline, and true acrosome reaction was determined by trypan blue and differential interferential contrast. The forskolin concentrations employed had no effect on progressive motility or sperm viability. Capacitation values induced by 25-μm forskolin treatment (27.80 ± 2.59%) were significantly higher respect to the control (4.80 ± 1.30%). The inhibitor 2',5'-dideoxyadenosine prevented forskolin-induced capacitation and significantly diminished capacitation induced by heparin. Follicular fluid induced physiological acrosome reaction in spermatozoa previously capacitated with 25-μm forskolin (P < 0.05). Forskolin acts as a capacitation inducer and involves the participation of membrane adenylyl cyclase as part of the intracellular mechanisms that lead to capacitation in cryopreserved bovine spermatozoa., (© 2014 Blackwell Verlag GmbH.)

Diadenosine polyphosphates have been shown to inhibit neutrophil apoptosis, but mechanisms of the antiapoptotic effect are not known. Diadenosine diphosphate (Ap2A) is the simplest naturally occurring diadenosine polyphosphate, and its effect on neutrophil apoptosis has not previously been investigated. Here we report that Ap2A delays spontaneous apoptosis of human neutrophils, and the effect is reversed by the adenosine A2A receptor antagonists SCH442416 and ZM241385. Ap2A induced an elevation of intracellular cAMP and the elevation was blocked by the adenosine A2A receptor antagonists. The antiapoptotic effect of Ap2A was abrogated by 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase, and Rp-8-Br-cAMPS, an inhibitor of type I cAMP-dependent protein kinase A (PKA). Together, these results demonstrate that Ap2A delays neutrophil apoptosis via the adenosine A2A receptor and cAMP/PKA signaling axis.

Astrocytic glycogen, the only storage form of glucose in the brain, has been shown to play a fundamental role in supporting learning and memory, an effect achieved by providing metabolic support for neurons. We have examined the interplay between glycogenolysis and the bioenergetics of astrocytic Ca(2+) homeostasis, by analyzing interdependency of glycogen and store-operated Ca(2+) entry (SOCE), a mechanism in cellular signaling that maintains high endoplasmatic reticulum (ER) Ca(2+) concentration and thus provides the basis for store-dependent Ca(2+) signaling. We stimulated SOCE in primary cultures of murine cerebellar and cortical astrocytes, and determined glycogen content to investigate the effects of SOCE on glycogen metabolism. By blocking glycogenolysis, we tested energetic dependency of SOCE-related Ca(2+) dynamics on glycogenolytic ATP. Our results show that SOCE triggers astrocytic glycogenolysis. Upon inhibition of adenylate cyclase with 2',5'-dideoxyadenosine, glycogen content was no longer significantly different from that in unstimulated control cells, indicating that SOCE triggers astrocytic glycogenolysis in a cAMP-dependent manner. When glycogenolysis was inhibited in cortical astrocytes by 1,4-dideoxy-1,4-imino-D-arabinitol, the amount of Ca(2+) loaded into ER via sarco/endoplasmic reticulum Ca(2)-ATPase (SERCA) was reduced, which suggests that SERCA pumps preferentially metabolize glycogenolytic ATP. Our study demonstrates SOCE as a novel pathway in stimulating astrocytic glycogenolysis. We also provide first evidence for a new functional role of brain glycogen, in providing local ATP to SERCA, thus establishing the bioenergetic basis for astrocytic Ca(2+) signaling. This mechanism could offer a novel explanation for the impact of glycogen on learning and memory., (Copyright © 2014 Wiley Periodicals, Inc.)

Dopamine and adenosine both regulate transport of sodium chloride in the renal tubules in mammals. We have studied the effect of dopamine and adenosine on spontaneous activity of contractile vacuole of Amoeba proteous. Both substances stimulated contractile vacuole. The effect of dopamine was suppressed by D2 receptor antagonist, haloperidol, but not by D1 antagonist, SCH 39166. Adenylate cyclase inhibitor, 2.5-dideoxyadenosine, suppressed the effect of dopamine, but not of adenosine. Inhibitor of protein kinase C, staurosporine, in contrast, blocked the effect of adenosine, but not dopamine. Notably, dopamine opposed effect of adenosine and vice versa. These results suggest that similar effects of dopamine and adenosine could be mediated by different intracellulare mechanisms.

We studied the role of cAMP- and IKK-2-mediated pathways in the realization of growth potential of mesenchymal progenitor cells in vitro. It had been found that adenylate cyclase inhibitor 2',5'-dideoxyadenosine had no effect on the proliferation and differentiation of fibroblastic CFU. A decrease in differentiation rate of progenitor cells was observed after the treatment with specific IKK-2 blocker inhibitor-kinase complex.

We evaluated the efficacy, potency, and selectivity of the three most commonly used adenylate cyclase (AC) inhibitors in a battery of cell lines constructed to study signaling via three discrete cAMP sensors identified in neuroendocrine cells. SQ22,536 [9-(tetrahydrofuryl)-adenine] and 2',5'-dideoxyadenosine (ddAd) are effective and potent AC inhibitors in HEK293 cells expressing a cAMP response element (CRE) reporter gene, and MDL-12,330A [cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine hydrochloride] is not. Neuroscreen-1 (NS-1) cells were used to assess the specificity of the most potent AC inhibitor, SQ22,536, to block downstream cAMP signaling to phosphorylate CREB (via PKA); to activate Rap1 (via Epac); and to activate ERK signaling leading to neuritogenesis (via the newly described neuritogenic cAMP sensor NCS). SQ22,536 failed to inhibit the effects of cAMP analogs 8-Br-cAMP and 8-CPT-2'-O-Me-cAMP on PKA-mediated CREB activation/phosphorylation and Epac-mediated Rap1 activation, indicating that it does not inhibit these cAMP pathways beyond the level of AC. On the other hand, SQ22,536, but not ddAd, inhibited the effects of cAMP analogs 8-Br-cAMP and 8-CPT-cAMP on ERK phosphorylation and neuritogenesis, indicating that it acts not only as an AC blocker, but also as an inhibitor of the NCS. The observed off-target actions of SQ22,536 are specific to cAMP signaling: SQ22,536 does not block the actions of compounds not related to cAMP signaling, including ERK induction by PMA, and ERK activation and neuritogenesis induced by NGF. These data led us to indicate a second target for SQ22,536 that should be considered when interpreting its effects in whole cell and in vivo experiments.

Mechano-growth factor (MGF) is a product of alternative splicing of the insulin-like growth factor 1 (IGF-1) mRNA. MGF is known to stimulate myoblast proliferation and to protect neurons and cardiomyocytes from apoptosis. MGF expression is dramatically increased in response to mechanical stimuli and tissue damage. The mechanisms of induction of MGF expression are as yet imperfectly understood. There is certain evidence that some protein factors able to stimulate MGF synthesis in normal myoblasts are released from damaged muscle. This study was undertaken to explore the nature of these protein inductors of MGF expression and to investigate the mechanism of their action. We report here that myofibrillar fraction of skeletal muscle homogenate activated MGF expression in murine myoblasts and myotubes in culture. The expression of another splice form of IGF-1 gene, IGF-1Ea, was also stimulated by myofibrils. Three myofibrillar proteins able to stimulate MGF synthesis were isolated. These proteins were identified by MALDI and immunoblotting as myomesin, myosin-binding protein C, and titin. The activation of MGF expression was associated with the increase of cAMP level in the cells. Inhibitor of adenylyl cyclase dideoxyadenosine arrested stimulation of MGF synthesis by all three myofibrillar proteins.

Binding of specific microbial epitopes [MAMPs (microbe-associated molecular patterns)] to PRRs (pattern recognition receptors) and subsequent receptor kinase activation are key steps in plant innate immunity. One of the earliest detectable events after MAMP perception is a rapid and transient rise in cytosolic Ca2+ levels. In plants, knowledge about the signalling events leading to Ca2+ influx and on the molecular identity of the channels involved is scarce. We used a transgenic Arabidopsis thaliana line stably expressing the luminescent aequorin Ca2+ biosensor to monitor pharmacological interference with Ca2+ signatures following treatment with the bacterial peptide MAMPs flg22 and elf18, and the fungal carbohydrate MAMP chitin. Using a comprehensive set of compounds known to impede Ca2+-transport processes in plants and animals we found strong evidence for a prominent role of amino acid-controlled Ca2+ fluxes, probably through iGluR (ionotropic glutamate receptor)-like channels. Interference with amino acid-mediated Ca2+ fluxes modulates MAMP-triggered MAPK (mitogen-activated protein kinase) activity and affects MAMP-induced accumulation of defence gene transcripts. We conclude that the initiation of innate immune responses upon flg22, elf18 and chitin recognition involves apoplastic Ca2+ influx via iGluR-like channels.

Multiple pathways from three types of multiple receptor sites to three types of metabotropic signal transduction pathways were investigated in the whole cell-clamp experiments using isolated labellar sugar receptor neurons (cells) of the adult blowfly, Phormia regina. First, the concentration-response curves of three types of sweet taste components specialized to multiple receptor sites were obtained: sucrose for the pyranose sites (P-sites), fructose for the furanose sites (F-sites), and l-valine for the alkyl sites (R-sites). Next, the effects of inhibitors such as 2', 5'-dideoxyadenosine on adenylyl cyclase in the cAMP pathway, LY 83583 on guanylyl cyclase in the cGMP pathway, and U-73122 on phospholipase C in the IP₃ pathway were examined. The results showed that all of the inhibitors affected each specific target in the second-messenger transduction pathways. The obtained results verified that the P-site corresponded to the cAMP, the F-site to the cGMP, and the R-site to the IP₃ transduction pathway, and that these three signal pathways did not have crossing points., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We recently reported that HIV-1 resistant to 3'-azido-3'-deoxythymidine (AZT) is not cross-resistant to 3'-azido-2',3'-dideoxypurines. This finding suggested that the nucleoside base is a major determinant of HIV-1 resistance to nucleoside analogs. To further explore this hypothesis, we conducted in vitro selection experiments by serial passage of HIV-1(LAI) in MT-2 cells in increasing concentrations of 3'-azido-2',3'-dideoxyguanosine (3'-azido-ddG), 3'-azido-2',3'-dideoxycytidine (3'-azido-ddC), or 3'-azido-2',3'-dideoxyadenosine (3'-azido-ddA). 3'-Azido-ddG selected for virus that was 5.3-fold resistant to 3'-azido-ddG compared to wild-type HIV-1(LAI) passaged in the absence of drug. Population sequencing of the entire reverse transcriptase (RT) gene identified L74V, F77L, and L214F mutations in the polymerase domain and K476N and V518I mutations in the RNase H domain. However, when introduced into HIV-1 by site-directed mutagenesis, these 5 mutations only conferred ∼2.0-fold resistance. Single-genome sequencing analyses of the selected virus revealed a complex population of mutants that all contained L74V and L214F linked to other mutations, including ones not identified during population sequencing. Recombinant HIV-1 clones containing RT derived from single sequences exhibited 3.2- to 4.0-fold 3'-azido-ddG resistance. In contrast to 3'-azido-ddG, 3'-azido-ddC selected for the V75I mutation in HIV-1 RT that conferred 5.9-fold resistance, compared to the wild-type virus. Interestingly, we were unable to select HIV-1 that was resistant to 3'-azido-ddA, even at concentrations of 3'-azido-ddA that yielded high intracellular levels of 3'-azido-ddA-5'-triphosphate. Taken together, these findings show that the nucleoside base is a major determinant of HIV-1 resistance mechanisms that can be exploited in the design of novel nucleoside RT inhibitors.

VILIP-1 (visinin-like protein 1) is downregulated in various human squamous cell carcinoma (SCC). In a mouse skin SCC model VILIP-1 expression is reduced in aggressive tumor cells, accompanied by reduced cAMP levels. Overexpression of VILIP-1 in aggressive SCC cells led to enhanced cAMP production, in turn causing a reduction in invasive properties. Moreover, in primary neurons and neuronal tumor lines VILIP-1 enhanced cGMP signaling. Here, we set out to determine whether and how cAMP and cGMP signaling contribute to the VILIP-1 effect on enhanced SCC model cell migration, and thus most likely invasiveness in vivo. We found stronger increase in cGMP levels in aggressive, VILIP-1-negative SCC cells following stimulation of guanylyl cyclases NPR-A and -B with the natriuretic peptides ANP and CNP, respectively. Incubation with ANP or 8Br-cGMP to increase cGMP levels further enhanced the migration capacity of aggressive cells, whereas cell adhesion was unaffected. Increased cGMP was caused by elevated expression levels of NPR-A and -B. However, the expression level of VILIP-1 did not affect cGMP signaling and guanylyl cyclase expression in SCC. In contrast, VILIP-1 led to reduced migration of aggressive SCC cells depending on cAMP levels as shown by use of adenylyl cyclase (AC) inhibitor 2',3'-dideoxyadenosine. Involvement of cAMP-effectors PKA and EPAC play a role downstream of AC activation. VILIP-1-positive and -negative cells did not differ in mRNA expression of ACs, but an effect on enhanced protein expression and membrane localization of ACs was shown to underlie enhancement of cAMP production and, thus, reduction in cell migration by VILIP-1., (Copyright © 2010 Wiley-Liss, Inc.)

Comprehensive conformational analysis of the biologically active nucleoside 2',3'-didehydro-2',3'-dideoxyadenosine (d4A) has been performed at the MP2/6-311++G(d,p)//DFT B3LYP/6-31G(d,p) level of theory. The energetic, geometrical and polar characteristics of twenty one d4A conformers as well as their conformational equilibrium were investigated. The electron density topological analysis allowed us to establish that the d4A molecule is stabilized by eight types of intramolecular interactions: O5'H...N3, O5'H...C8, C8H...O5', C2'H...N3, C5'H1...N3, C5'H2...N3 Ta C8H...H1/2C5'. The obtained results of conformational analysis lead us to think that d4A may be a terminator of the DNA chain sythesis in the 5'-3' direction. Thus it can be inferred that d4A competes with canonical 2'-deoxyadenosine in binding an active site of the corresponding enzyme.

The existence of direct progestin actions on teleost sperm to stimulate hypermotility is not widely acknowledged because it has only been demonstrated in members of the family Sciaenidae. In the present study, progestin stimulation of sperm hypermotility was investigated in a non-sciaenid, southern flounder, and the potential role of membrane progestin receptor alpha (mPRα or Paqr7b) in mediating this action was examined. The major progestin produced in vitro by flounder testicular fragments co-migrated with 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) during thin-layer chromatography. Treatment of flounder sperm with 5 nM-100 nM 20β-S significantly increased sperm velocity in vitro, whereas 17,20β-dihydroxy-4-pregnen-3-one and other steroids were ineffective. A single class of high affinity (K(d) 22.95 nM), saturable, limited-capacity binding sites (B(max) 0.013 nM) specific for 20β-S was identified on sperm membranes. Treatment of sperm membranes with guanosine 5'-(3-O-thio)triphosphate reduced [(3)H]-20β-S binding, suggesting the 20β-S receptor couples to a G protein. The membrane adenylyl cyclase inhibitor 2',5'-dideoxyadenosine blocked 20β-S-induced sperm hypermotility, indicating 20β-S activates stimulatory G proteins. Finally, flounder paqr7b was cloned and characterized from testicular tissues. The Paqr7b protein is expressed on the midpiece of flounder sperm and is more abundant in individuals with high sperm motility than low motility donors. These findings suggest that 20β-S stimulates sperm hypermotility in flounder through activation of stimulatory G proteins, likely through Paqr7b. The finding that progestins directly stimulate sperm hypermotility in a flatfish, a highly derived species not belonging to the teleost family Sciaenidae, suggests this phenomenon is widespread among advanced fishes., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The effects of glucagon-like peptide 2 (GLP-2) on expression and activity of jejunal multidrug resistance-associated protein 2 (Mrp2; Abcc2) and glutathione transferase (GST) were evaluated. After GLP-2 treatment (12 μg/100 g b.wt. s.c., every 12 h, for 5 consecutive days), Mrp2 and the α class of GST proteins and their corresponding mRNAs were increased, suggesting a transcriptional regulation. Mrp2 was localized at the apical membrane of the enterocyte in control and GLP-2 groups, as detected by confocal immunofluorescence microscopy. As a functional assay, everted intestinal sacs were incubated in the presence of 1-chloro-2,4-dinitrobenzene in the mucosal compartment, and the glutathione-conjugated derivative, dinitrophenyl-S-glutathione (DNP-SG; model Mrp2 substrate), was detected in the same compartment by high-performance liquid chromatography. A significant increase in apical secretion of DNP-SG was detected in the GLP-2 group, consistent with simultaneous up-regulation of Mrp2 and GST. GLP-2 also promoted an increase in cAMP levels as detected in homogenates of intestinal mucosa. Treatment of rats with 2',3'-dideoxyadenosine (DDA), a specific inhibitor of adenylyl cyclase, abolished the increase in cAMP levels and Mrp2 protein promoted by GLP-2, suggesting cAMP as a mediator of Mrp2 modulation. Increased expression of Mrp2 and cAMP levels in response to GLP-2 occurred not only at the tip but also at the middle region of the villus, where constitutive expression of Mrp2 is normally low. In conclusion, our study suggests a role for GLP-2 in the prevention of cell toxicity of the intestinal mucosa by increasing Mrp2 chemical barrier function.

The nitric oxide (NO)-cGMP signaling system and cAMP system play critical roles in the formation of multiple-trial induced, protein synthesis-dependent long-term memory (LTM) in many vertebrates and invertebrates. The relationship between the NO-cGMP system and cAMP system, however, remains controversial. In honey bees, the two systems have been suggested to converge on protein kinase A (PKA), based on the finding in vitro that cGMP activates PKA when sub-optimal dose of cAMP is present. In crickets, however, we have suggested that NO-cGMP pathway operates on PKA via activation of adenylyl cyclase and production of cAMP for LTM formation. To resolve this issue, we compared the effect of multiple-trial conditioning against the effect of an externally applied cGMP analog for LTM formation in crickets, in the presence of sub-optimal dose of cAMP analog and in condition in which adenylyl cyclase was inhibited. The obtained results suggest that an externally applied cGMP analog activates PKA when sub-optimal dose of cAMP analog is present, as is suggested in honey bees, but cGMP produced by multiple-trial conditioning cannot activate PKA even when sub-optimal dose of cAMP analog is present, thus indicating that cGMP produced by multiple-trial conditioning is not accessible to PKA. We conclude that the NO-cGMP system stimulates the cAMP system for LTM formation. We propose that LTM is formed by an interplay of two classes of neurons, namely, NO-producing neurons regulating LTM formation and NO-receptive neurons that are more directly involved in the formation of long-term synaptic plasticity underlying LTM formation.

This paper reports the synthesis of novel 4'-hydrophobic pocket deoxythreosyl C-nucleosides. The key threose-like intermediates 9 and 14 were constructed from acyclic ketone derivatives, respectively. The antiviral activities of the synthesized compounds against the HIV-1, HSV-1, HSV-2, and HCMV viruses were evaluated. The 9-deaza-adenine derivatives 10 and 20 showed good anti-HIV activity without exhibiting significant cytotoxicity.

Glucagon-like peptide-1 (GLP-1) is a proglucagon-derived hormone with cellular protective actions. We hypothesized that GLP-1 would protect the endothelium from injury during inflammation. Our aims were to determine the: (1) effect of GLP-1 on basal microvascular permeability, (2) effect of GLP-1 on increased microvascular permeability induced by lipopolysaccaride (LPS), (3) involvement of the GLP-1 receptor in GLP-1 activity, and (4) involvement of the cAMP/PKA pathway in GLP-1 activity. Microvascular permeability (L(p)) of rat mesenteric post-capillary venules was measured in vivo. First, the effect of GLP-1 on basal L(p) was measured. Second, after systemic LPS injection, L(p) was measured after subsequent perfusion with GLP-1. Thirdly, L(p) was measured after LPS injection and perfusion with GLP-1+GLP-1 receptor antagonist. Lastly, L(p) was measured after LPS injection and perfusion with GLP-1+inhibitors of the cAMP/PKA pathway. Results are presented as mean area under the curve (AUC)+/-SEM. GLP-1 had no effect on L(p) (AUC: baseline=27+/-1.4, GLP-1=1+/-0.4, p=0.08). LPS increased L(p) two-fold (AUC: LPS=54+/-1.7, p<0.0001). GLP-1 reduced the LPS increase in L(p) by 75% (AUC: LPS+GLP-1=34+/-1.5, p<0.0001). GLP-1 antagonism reduced the effects of GLP-1 by 60% (AUC: LPS+GLP-1+antagonist=46+/-2.0, p<0.001). The cAMP synthesis inhibitor reduced the effects of GLP-1 by 60% (AUC: LPS+GLP-1+cAMP inhibitor=46+/-1.5, p<0.0001). The PKA inhibitor reduced the effects of GLP-1 by 100% (AUC: LPS+GLP-1+PKA inhibitor=56+/-1.5, p<0.0001). GLP-1 attenuates the increase in microvascular permeability induced by LPS. GLP-1 may protect the endothelium during inflammation, thus decreasing third-space fluid loss.

The hyperpolarization-activated cation current (I (h)) in rat pituitary lactotrophs (GH(3) cells) was characterized. Tramadol-induced block of this current was investigated. Effects of various related compounds on I (h) in GH(3) cells were also compared. Tramadol caused a time- and concentration-dependent reduction in the amplitude of I (h) with an IC(50) value of 13.6 microM. ZD7288 (30 microM), CsCl (2 mM), and propofol (30 microM) were effective in suppressing the amplitude of I (h). 2',5'-dideoxyadenosine (100 microM) suppressed I (h), while sp-cAMPS (100 microM) had no effect on it. Tramadol (10 microM) shifted the activation curve of I (h) to a more negative potential by approximately -20 mV, although no change in the slope factor was observed. Under current-clamp configuration, tramadol (10 microM) could reduce the firing frequency of action potentials. Intracellular Ca(2+) measurements revealed its ability to reduce spontaneous Ca(2+) oscillations in GH(3) cells. The results suggests that during cell exposure to tramadol used at clinically relevant concentration, the tramadol-mediated inhibition of I (h) could be direct and mediated via a non-opioid mechanism and would be one of the ionic mechanisms underlying reduced cell excitability.

Background: Urocortin II has previously been shown in anesthetized pigs to increase coronary blood flow through activation of the endothelial nitric oxide synthase (eNOS) pathway and involvement of the subtype 2 of corticotropin releasing factor receptors (CRFR2). However, little information has been available regarding the intracellular signalling through these receptors and leading to the release of nitric oxide (NO)., Aim: The present study was therefore planned to determine the mechanism involved in such signalling., Methods: In porcine aortic endothelial cells (PAE) the effects of urocortin II on NO production and ERK, Akt, p38 and eNOS phosphorylation were examined in absence or presence of the adenylyl cyclase agonist forskolin and antagonist 2'5' dideoxyadenosine, the Ca2+ ionophore A23187, the Ca2+-calmodulin-kinase inhibitor KN93, the CRFR2 blocker astressin 2B and of the protein kinases specific inhibitors UO126, wortmannin and SB203580., Results: Urocortin II caused a significant increase of NO production, which was amplified by forskolin and A23187 (P <0.05). All effects of urocortin II were abolished by l-NAME, 2'5' dideoxyadenosine, KN93, astressin 2B and by pre-treatment of cells with UO126, wortmannin and SB203580. Western Blot analysis confirmed the involvement of ERK, Akt and p38 in the eNOS activation., Conclusion: In PAE urocortin II interaction with CRFR2 caused a cAMP-dependent and Ca2+-related phoshorylation of ERK, Akt and p38 leading to eNOS activation., (2009 S. Karger AG, Basel.)

Progestin stimulation of sperm hypermotility remains poorly understood despite having been described in numerous vertebrate species. We show here that progestin stimulation of sperm hypermotility in a teleost, the Atlantic croaker (Micropogonias undulatus) is associated with activation of an olfactory G protein (Golf). Furthermore, we provide evidence that this progestin action is mediated by membrane progestin receptor-alpha (mPRalpha). Golf was identified in croaker sperm membranes and was specifically activated after treatment with the progestin 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S). Treatment of sperm membranes with 20beta-S caused an increase in cAMP production, which was blocked by pretreatment with cholera toxin and two membrane adenylyl cyclase inhibitors: 2',5'-dideoxyadenosine and SQ22536. Moreover, preincubation of croaker sperm with 2',5'-dideoxyadenosine and SQ22536 resulted in a significant inhibition of 20beta-S-stimulated hypermotility. Binding of [3H]20beta-S to sperm membranes was decreased after pretreatment with GTPgammaS but not pertussis toxin, suggesting the receptor is coupled to a pertussis toxin-insensitive G protein. Golf and mPRalpha were coexpressed on the sperm midpiece and flagella and were coimmunoprecipitated from sperm membranes. Finally, expression of mPRalpha protein on sperm increased after in vivo treatment with LHRH and was associated with increased induction of sperm motility by 20beta-S. These results suggest that 20beta-S activates mPRalpha in croaker sperm, which in turn activates Golf and membrane adenylyl cyclase to stimulate sperm hypermotility. Taken together these findings provide a plausible mechanism by which progestins stimulate sperm hypermotility in croaker and provide the first evidence of hormonal activation of Golf in any species.

In vivo, inhibition of fetal hemoglobin (HbF) expression in humans around the time of birth causes the clinical manifestation of sickle cell and beta-thalassemia syndromes. Inhibition of HbF among cultured cells was recently described by the adenosine derivative molecule named SQ22536. Here, a primary cell culture model was utilized to further explore the inhibition of HbF by adenosine derivative molecules. SQ22536 demonstrated down-regulation of growth and HbF expression among erythroblasts cultured from fetal and adult human blood. The effects upon HbF were noted in a majority of cells, and quantitative PCR analysis demonstrated a transcriptional mechanism. Screening assays demonstrated that two additional molecules named 5'-deoxy adenosine and 2',3'-dideoxy adenosine had effects on HbF comparable to SQ22536. Other adenosine derivative molecules, adenosine receptor binding ligands, and cAMP-signaling regulators failed to inhibit HbF in matched cultures. These results suggest that structurally related ribofuranose-substituted adenosine analogues act through an unknown mechanism to inhibit HbF expression in fetal and adult human erythroblasts.

This work examines the effect of cyclic AMP (cAMP) on the in vitro barrier function of tubes of human dermal lymphatic microvascular endothelial cells (LECs). Under baseline conditions, the barrier function of LEC tubes was weak, with diffusional permeability coefficients to bovine serum albumin and 10 kDa dextran of 1.4(-0.6)(+0.9)x10(-6) cm/s and 1.7(-0.5)(+0.8)x10(-6) cm/s (geometric mean+/-95% CI), respectively, and 1.2+/-0.5 (mean+/-95% CI) focal leaks per mm. Exposure to low concentrations (3 microM) of a cell-permeant analog of cAMP did not alter the barrier function. Exposure to higher concentrations (80 and 400 microM) and/or the phosphodiesterase inhibitor Ro-20-1724 (20 microM) lowered permeabilities and the number of focal leaks, and increased the selectivity of the barrier. Decreased permeabilities were accompanied by an increase in continuous VE-cadherin staining at cell-cell borders. Exposure to 1 mM 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, did not increase permeabilities. LECs expressed the lymphatic-specific master transcription factor Prox-1, regardless of whether barrier function was weak or strong. Our results indicate that the permeability of LEC tubes in vitro responds to cAMP in a manner similar to that well-described for the permeability of blood microvessels.

Aim: To investigate the antiviral effect of beta-L-enantiomer of 2',3'-didehydro-2',3'-dideoxyadenosine (beta-L-D4A) on 2.2.15 cells transfected with the hepatitis B virus (HBV) genome., Methods: Lamivudine (3TC) as a positive control. Then, HBV DNA in treated 2.2.15 cells and the Hepatitis B surface antigen (HBsAg) in the culture supernatants were detected to determine the inhibitory effect of beta-L-D4A. At the same time, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to detect the survival ratio of 2.2.15 cells., Results: beta-L-D4A has a dose-dependent inhibitory effect on HBV DNA replication; this effect was apparent when the concentration was above 1 mol/L. When beta-L-D4A was at the highest concentration, 100 mol/L, the HBsAg inhibition ratio was above 50%. The Therapeutic index (TI) of beta-L-D4A was above 2.1., Conclusion: beta-L-D4A has a dose-dependent inhibitory effect on the replication of HBV DNA and the secretion of HBsAg at low toxicity.

We previously reported that PEGylated conjugated linoleic acid (PCLA) as a pro-drug treatment of cultures of 3T3-L1 cells containing differentiated adipocytes caused de-differentiation by downregulation of PPARgamma2-induced adipogenesis, and cell apoptosis induced by PCLA was lower than that induced by conjugated linoleic acid (CLA) owing to the biocompatible and hydrophilic properties of poly(ethylene glycol) (PEG). To further investigate our previous observations, the present study is designed to evaluate the lipolytic action of PCLA and its role in biochemical signaling pathways of 3T3-L1 cells when compared to the CLA itself. Although both CLA and PCLA stimulated lipolysis, our results indicated a sensitivity difference between CLA and PCLA treatment: a time-dependent effect on lipolysis and p-extracellular signal-related kinases (ERK) expression was observed for PCLA-treated, but not for CLA-treated cultures. Also, the induction by PCLA of mitogen-activated protein kinase kinase (MEK)/ERK mitogen-activated protein kinase (MAPK) activation was linked to secretion of adipo-cytokines, interleukin-6 (IL-6), and interleukin-8 (IL-8), in time-dependent manners. Interestingly, adenylyl cyclase inhibitor, 2', 5'-dideoxyadenosine (DDA), pre-treatment did not prevent PCLA-stimulated lipolysis. In fact, isoproterenol, but not PCLA, caused a significant increase in cyclic adenosine monophosphate (cAMP) levels, suggesting that the PCLA-induced lipolysis was not mediated in the conventional cAMP-dependent pathway and the cAMP was the intracellular mediator for isoproterenol-induced lipolysis. Overall, our findings provide support for a role for PCLA as a pro-drug in the regulation of metabolism in adipose tissue., ((c) 2007 Wiley-Liss, Inc.)

β-L-enantiomer of 2',3'-didehydro-2',3'-dideoxyadenosine-5'-triphosphate (β-L-D4A-TP) has previously been proven to inhibit the replication of viral DNA in the Hep G2 2.2.15 cells and in transgenic mouse harboring 1.3-fold-overlength genome of Hepatitis B virus (HBV). To study the inhibition mechanism of the nucleoside analog β-L-D4A-TP, a polymerase reaction in vitro with the recombinant HBV nucleocapsids was conducted to determine the exact mode of inhibition of the HBV replication by β-L-D4A-TP. The HBV viral DNA and viral DNA-polymerase complex formed in the polymerase reaction were assayed. The results of this study showed that β-L-D4A-TP inhibited the replication of HBV DNA by inactivating the reverse transcriptase (RT) activity in a concentration-dependent manner. The kinetics of β-L-D4A-TP inhibition of the RT activity was the result of an apparent competitive inhibition with dATP.

Aim: To investigate the safety of beta-L-D4A on DNA polymerase alpha., Methods: Ion exchange chromatography was used to separate DNA polymerase alpha from crude extract of human Hela cells. Detailed kinetic parameters were determined for beta-L-D4A against DNA polymerase alpha., Results: DNA polymerase alpha was purified with 4% yield and 31000 units/mg specific activity. The Michaelis constant (Km = 3.22 micromol/L), 50% inhibition concentration (IC50 = 178.49 micromol/L) and inhibition constant (Ki = 126 micromol/L) of beta-L-D4A were determined by kinetic analysis., Conclusion: beta-L-D4A is a more safe nucleoside for hepatitis B virus infection with a lower host toxicity.

Adiponectin is a protein secreted from adipocytes that exhibits salutary effects in the vascular endothelium by signaling mechanisms that are not well understood. In obesity-related disease states and type 2 diabetes, circulating substances, including tumor necrosis factor-alpha (TNFalpha) and high glucose, activate IkappaB kinase (IKK)beta and reduce the abundance of its substrate, inhibitor of kappaB (IkappaB)alpha, leading to nuclear translocation of the transcription factor NF-kappaB and stimulation of an inflammatory signaling cascade closely associated with endothelial dysfunction. The present study demonstrates that the globular domain of adiponectin (gAd) potently suppresses the activation of IKKbeta by either TNFalpha or high glucose in human umbilical vein endothelial cells and ameliorates the associated loss of IkappaBalpha protein. Interestingly, activation of AMP kinase was substantially more effective than cAMP signaling in suppressing high glucose-induced IKKbeta activity, whereas both pathways were comparably active in suppressing the TNFalpha-induced increase in IKKbeta. Both cAMP/protein kinase A signaling and activation of the AMP kinase pathway played a role in the suppression by gAd of TNFalpha- and high glucose-mediated IKKbeta activation. These findings support an important role for adiponectin in anti-inflammatory signaling in the endothelium and also imply that multiple pathways are involved in the cellular effects of adiponectin.

Mechanical stress is thought to regulate the expression of genes in the periodontal ligament (PDL) cells. Using a microarray approach, we recently identified a regulator of G-protein signaling 2 (RGS2) as an up-regulated gene in the PDL cells under compressive force. The RGS protein family is known to turn off G-protein signaling. G-protein signaling involves the production of cAMP, which is thought to be one of the biological mediators in response to mechanical stress. Here, we investigated the role of RGS2 in the PDL cells under mechanical stress. PDL cells derived from the ligament tissues of human premolar teeth were cultured in collagen gels and subjected to static compressive force. Compressive force application time-dependently enhanced RGS2 expression and intracellular cAMP levels. To examine the interrelationship between RGS2 and cAMP, the PDL cells were treated with 2',5'-dideoxyadenosine (DDA), an inhibitor of adenyl cyclase, or antisense S-oligonucleotide (S-ODN) to RGS2 under compressive force. DDA dose-dependently inhibited RGS2 stimulated by compressive force. Blockage of RGS2 by antisense S-ODN elevated the cAMP levels compared with controls. These results indicate that cAMP stimulates RGS2 expression, which in turn leads to a decrease in the cAMP production by inactivating the G-protein signaling in the mechanically stressed PDL cells., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

In addition to its well known stimulation of cAMP production, the human melanocortin type 4 (hMC4) receptor recently has been shown to mediate p44/42 MAPK activation. This finding opens new questions about the structural and signaling mechanisms that connect the receptor to this alternate cell signaling pathway. Point mutants in the hMC4 receptor that have been associated with obesity were constructed and transfected into HEK 293 cells. Functional analyses then were done to determine if these mutations would similarly impact cAMP formation and p44/42 MAPK signaling. Whereas a D90N mutation in the second transmembrane domain and a D298A mutation in the seventh transmembrane domain impaired both cAMP formation and p44/42 MAPK activation, a more conservative D298N mutation retained cAMP formation but abolished p44/42 MAPK activation. The D298N mutation identified, for the first time, differential structural requirements of the hMC4 receptor for activation of the cAMP and p44/42 MAPK pathways. Furthermore, functional characterizations of a series of chimeric receptors combining the hMC4 receptor and the hMC3 subtype, a receptor that does not couple to p44/42 MAPK activation despite stimulating adenylyl cyclase, indicate that the hMC4 cytoplasmic tail is a necessary structural element for p44/42 MAPK signaling. Subsequent investigation of the signaling requirements for p44/42 MAPK activation demonstrated that the adenylyl cyclase inhibitor 2', 5'-dideoxyadenosine blocked agonist-induced p44/42 MAPK activation, but the PKA inhibitor Rp cAMPS did not. Taken together, these data indicate that cAMP is required, but not sufficient for p44/42 MAPK activation and suggest structural elements required for hMC4 receptor signaling.

Trichinella spiralis infection causes hyperexcitability in enteric after-hyperpolarising (AH) sensory neurons that is mimicked by neural, immune or inflammatory mediators known to stimulate adenylyl cyclase (AC)/cyclic 3',5'-adenosine monophosphate (cAMP) signaling. The hypothesis was tested that ongoing modulation and sustained amplification in the AC/cAMP/phosphorylated cAMP related element binding protrein (pCREB) signaling pathway contributes to hyperexcitability and neuronal plasticity in gut sensory neurons after nematode infection. Electrophysiological, immunological, molecular biological or immunochemical studies were done in T. spiralis-infected guinea-pigs (8000 larvae or saline) after acute-inflammation (7 days) or 35 days p.i., after intestinal clearance. Acute-inflammation caused AH-cell hyperexcitability and elevated mucosal and neural tissue levels of myeloperoxidase, mast cell tryptase, prostaglandin E2, leukotrine B4, lipid peroxidation, nitric oxide and gelatinase; lower level inflammation persisted 35 days p.i. Acute exposure to blockers of AC, histamine, cyclooxygenase or leukotriene pathways suppressed AH-cell hyperexcitability in a reversible manner. Basal cAMP responses or those evoked by forskolin (FSK), Ro-20-1724, histamine or substance P in isolated myenteric ganglia were augmented after T. spiralis infection; up-regulation also occurred in AC expression and AC-immunoreactivity in calbindin (AH) neurons. The cAMP-dependent slow excitatory synaptic transmission-like responses to histamine (mast cell mediator) or substance P (neurotransmitter) acting via G-protein coupled receptors (GPCR) in AH neurons were augmented by up to 2.5-fold after T. spiralis infection. FSK, histamine, substance P or T. spiralis acute infection caused a 5- to 30-fold increase in cAMP-dependent nuclear CREB phosphorylation in isolated ganglia or calbindin (AH) neurons. AC and CREB phosphorylation remained elevated 35 days p.i.. Ongoing immune activation, AC up-regulation, enhanced phosphodiesterase IV activity and facilitation of the GPCR-AC/cAMP/pCREB signaling pathway contributes to T. spiralis-induced neuronal plasticity and AH-cell hyperexcitability. This may be relevant in gut nematode infections and inflammatory bowel diseases, and is a potential therapeutic target.

Background: Intravascular volume loss from ischemia-reperfusion (IR) injury is a major clinical concern. We hypothesize that angiotensin II decreases IR-mediated microvascular fluid leak via the angiotensin II type 2 receptor in a cAMP dependent manner. We therefore sought to determine hydraulic permeability after IR of venules treated with 1) angiotensin II, 2) angiotensin II and cAMP synthesis inhibitor, and 3) angiotensin II and an angiotensin II type 2 receptor antagonist., Methods: Rat mesenteric post-capillary venules were micro-cannulated to measure hydraulic permeability (L(p)). IR was achieved by placing animals in a 5% oxygen environment and preventing venular flow, after which blood flow was allowed to resume. L(p) was measured after IR and treatment with 1) angiotensin II (20 nm), 2) angiotensin II (20 nm) + cAMP synthesis inhibitor (DDA,10uM), and 3) angiotensin II (20 nm) + type 2 receptor antagonist (PD-123319, 300 mum) (n=6 in each group)., Results: Compared with the seven-fold increase in L(p) because of IR alone: 1) angiotensin II attenuated the seven-fold increase by 50% (P<0.005), 2) cAMP inhibition did not change the effect of angiotensin II on leak, and the type 2 receptor antagonist completely blocked the effects of angiotensin II on IR mediated leak., Conclusion: Treatment with angiotensin II attenuated increases in hydraulic permeability because of IR by 50%. Inhibition of cAMP synthesis did not change the effect of angiotensin II on IR mediated leak, and angiotensin II type 2 receptor inhibition completely blocked the effects of angiotensin II. These results indicate that angiotensin II decreases IR induced leak via the angiotensin II type 2 receptor in a cAMP independent manner. A better understanding of mediators that reduce intravascular fluid loss from IR-induced microvascular dysfunction may help clinicians treat uncontrolled fluid extravasation that occurs during shock and sepsis.

Telomerase adds telomeric DNA repeats to the ends of linear chromosomal DNA. 3'-Azido-3'-deoxythymidine 5'-triphosphate (AZTTP) is a known telomerase inhibitor. To obtain more selective and potent inhibitors that can be employed as tools for studying telomerase, we investigated the telomerase-inhibitory effects of purine nucleosides bearing a 3'-down azido group: 3'-azido-2',3'-dideoxyguanosine (AZddG) 5'-triphosphate (AZddGTP), 3'-azido-2',3'-dideoxy-6-thioguanosine (AZddSG) 5'-triphosphate (AZddSGTP), 3'-azido-2',3'-dideoxyadenosine (AZddA) 5'-triphosphate (AZddATP) and 3'-azido-2',3'-dideoxy-2-aminoadenosine (AZddAA) 5'-triphosphate (AZddAATP). Of these, AZddGTP showed the most potent inhibitory activity against HeLa cell telomerase. AZddGTP was significantly incorporated into the 3'-terminus of DNA by partially purified telomerase. However, AZddGTP did not exhibit significant inhibitory activity against DNA polymerases alpha and delta, suggesting that AZddGTP is a selective inhibitor of telomerase. We also investigated whether long-term treatment with these nucleosides could alter telomere length and growth rates of human HL60 cells in culture. Southern hybridization analysis of genomic DNA prepared from cells cultured in the presence of AZddG and AZddAA revealed reproducible telomere shortening.

Telomerase is thought to play an important role in the mechanism of tumor cell immortalization by maintenance of telomere length. To obtain information on the susceptibility of telomerase to nucleoside analogues, the effects of base-modified 3'-azido-2',3'-dideoxynucleoside triphosphates on the enzyme were investigated. It is suggested that the 2-amino group of the nucleotide purine nucleus is important for the inhibitory activity. Telomere shortening caused by long-term treatment with these nucleosides is also described.

Prostaglandin E(2) (PGE(2)) has been shown to have a strong cytoprotective effect, inhibiting apoptosis. In the present study, we evaluated whether PGE(2) has a protective effect on cigarette smoke extract (CSE)-induced apoptosis in human lung fibroblasts. Apoptosis was assessed by various methods, including DNA content analysis. CSE (15%-20%) led to apoptosis and induced imbalance in favor of pro- over anti-apoptotic protein expression and activated caspases. PGE(2) blocked CSE-induced apoptosis and modulated the balance of pro- and anti-apoptotic proteins and decreased the activation of caspases. This anti-apoptotic effect was mediated via EP(2) receptor activation as the EP(2) agonist butaprost mimicked PGE(2) activity and siRNA for the EP(2) receptor blocked it. An adenylyl cyclase inhibitor was found to abolish the PGE(2)-mediated cytoprotective effect. Correspondingly, c-AMP analogs blocked CSE-induced apoptosis. Consistently, the protein kinase A (PKA) inhibitor KT-5720 abolished PGE(2)-mediated protection. PGE(2) and butaprost phosphorylated Bad and KT-5720 blocked phosphorylation. These results suggest that PGE(2) inhibits CSE-induced apoptosis via EP(2) receptor activation and activation of PKA, which leads to an alteration in the balance between pro- and anti-apoptotic factors. Through such a mechanism, PGE(2) may alter survival of cells in the smoke-exposed lungs, thus affecting the pathogenesis of cigarette smoke-induced disease.

Treatment of primary keratinocytes (HEKAp) with trypsin led to the production and release of CXCL8. Production of CXCL8 was exquisitely sensitive to inhibition by co-treatment with the beta(2) agonist sabutamol (IC(50)=1.1 nM). The inhibitory effect of salbutamol was beta receptor-mediated since the effect was prevented by the beta antagonist sotalol. Salbutamol also elevated intracellular levels of cAMP (EC(50)=82 nM) but the relationship to the inhibition of CXCL8 secretion was not clear-cut since much higher concentrations of salbutamol were required to elevate total cellular cAMP than inhibit CXCL8 production. However, the effect of salbutamol is likely to be mediated by elevation of cAMP since forskolin, an adenylyl cyclase activator, mimicked the effects of salbutamol while the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine inhibited the effects of salbutamol. Potentiation of cAMP production by co-treatment with the phosphodiesterase type 4 inhibitor rolipram only marginally enhanced the inhibitory effect of salbutamol on CXCL8 production. Taken together, these data suggest that elevation of cAMP production is required for the inhibitory effect of salbutamol on CXCL8 production by keratinocytes and that low threshold levels of cAMP are sufficient to mediate this effect.

Although butyrate modulates proliferation and cytokine production by PBMC in some species, the role of butyrate as a regulator of immunocyte function in the pig has not been studied. Therefore, the primary objective of this study was to determine whether butyrate influences peripheral blood mononuclear cell (PBMC) proliferation, cytokine secretion and mRNA expression in the pig in vitro. We also sought to determine whether alterations in cytokine production attributable to butyrate were associated with changes in the expression of suppressor of cytokine signaling-3 (SOCS3). Porcine PBMC were isolated from venous blood and stimulated with concanavalin A (ConA) in the presence or absence of sodium butyrate at 0.2 or 2.0 mM. Butyrate at 2.0 mM suppressed (P<0.05) ConA-induced PBMC proliferation and led to a paradoxical increase (P<0.05) in IL-2 mRNA expression. The secretion and mRNA expression of interferon-gamma (IFN-gamma) by ConA-activated PBMC was increased (P<0.05) by butyrate at 2.0 mM. Exposing activated PBMC to butyrate at 2.0 mM decreased (P<0.05) the secretion of interleukin-10 (IL-10). In contrast, butyrate at 0.2 mM increased (P<0.05) both IL-10 secretion and mRNA expression. Activation of porcine PBMC with ConA increased (P<0.05) the expression of SOCS3 mRNA, and butyrate treatment further augmented (P<0.05) SOCS3 mRNA expression in a dose-dependent manner. Mechanistically, pretreatment with the adenyl cyclase inhibitor 2,5-dideoxyadenosine abolished (P<0.05) the inhibitory effect of 2.0 mM butyrate on IL-10 secretion, and partially reversed (P<0.05) the increase in IFN-gamma secretion induced by 2.0mM butyrate. These data indicate that the effect of butyrate on cytokine production by porcine PBMC is dose-dependent, and that butyrate increases the expression of SOCS3 in activated PBMC. In addition, we provide evidence that the effects of butyrate on IFN-gamma and IL-10 production are mediated in part via a cAMP-dependent mechanism.