9-(tetrahydro-2-furyl)-adenine and 8-bromoadenosine-3--5--cyclic-monophosphorothioate

Patients with atopic dermatitis (AD) have significantly reduced plasma cAMP levels, and the cAMP level is correlated with the immunopathogenesis of AD. The production of thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) in keratinocytes is significantly enhanced in patients with AD. In the present study, we investigated the in vitro effects of the adenylyl cyclase-cAMP system on IFN-gamma and TNF-alpha-stimulated production of TARC and MDC in human HaCaT keratinocytes. Both forskolin (a direct activator of adenylyl cyclase) and dibutyryl-cAMP (DBcAMP, a permeable analog of cAMP) suppressed production of TARC and MDC in parallel with the activation of NF-kappaB in IFN-gamma and TNF-alpha-stimulated HaCaT cells. Moreover, inhibition of NF-kappaB suppressed TARC and MDC production induced by IFN-gamma plus TNF-alpha. However, dideoxyforskolin, a forskolin derivative that does not activate cAMP, failed to suppress the secretion of these chemokines. An inhibitor of p38 MAPK suppressed the production of TARC and MDC in parallel to the activation of NF-kappaB in HaCaT cells. Of note, the IFN-gamma plus TNF-alpha-stimulated activation of p38 MAPK was suppressed following incubation with forskolin or DBcAMP alone. These results indicate that the adenylyl cyclase-cAMP system has an inhibitory role in IFN-gamma plus TNF-alpha-stimulated production of TARC and MDC in HaCaT keratinocytes by inhibiting NF-kappaB activation through p38 MAPK pathway, implying that the adenylyl cyclase-cAMP system could be a candidate therapeutic target of Th2-skewed skin inflammation such as AD.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenine; Adenylyl Cyclases; Bucladesine; Chemokine CCL17; Chemokine CCL22; Colforsin; Cyclic AMP; Enzyme Activation; Humans; Interferon-gamma; Keratinocytes; Models, Immunological; NF-kappa B; p38 Mitogen-Activated Protein Kinases; STAT1 Transcription Factor; Thionucleotides; Tumor Necrosis Factor-alpha

Adrenomedullin (ADM), a 52-amino acid peptide, elicits differential cardiovascular responses when it is administered systemically or directly to the brain. We evaluated in the present study the hypothesis that ADM may modulate baroreceptor reflex (BRR) response through an ADM receptor-mediated cAMP/ protein kinase A (PKA)-dependent mechanism in the nucleus tractus solitarii (NTS), the terminal site for primary baroreceptor afferents, using Sprague-Dawley rats. Our immunoblot and immunohistochemical results showed that the two component proteins of the ADM(1) receptor complex, calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP)-2, were uniformly distributed and highly co-localized in the NTS. Site-specific microinjection of ADM (0.02-0.2pmol) unilaterally into the NTS significantly increased BRR response and sensitivity in a time- and dose-related manner, without affecting arterial pressure and heart rate. The BRR enhancing effect of ADM was also temporally correlated with an up-regulation of PKA(beta), the active form of PKA and an increase in PKA activity. In addition, the ADM-evoked BRR enhancement or PKA activation was abolished by co-microinjection with a selective ADM(1) receptor antagonist, ADM(22-52), an adenylyl cyclase inhibitor, SQ22536, or a PKA inhibitor, Rp-8-bromo-cAMP. These results suggest that ADM enhances BRR via activation of a cAMP/PKA-dependent mechanism by acting site-specifically on ADM(1) receptors in NTS.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenine; Adrenomedullin; Analysis of Variance; Animals; Baroreflex; Blood Pressure; Bronchodilator Agents; Calcitonin Receptor-Like Protein; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Heart Rate; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Peptide Fragments; Rats; Rats, Sprague-Dawley; Receptor Activity-Modifying Proteins; Receptors, Calcitonin; Signal Transduction; Solitary Nucleus; Thionucleotides; Time Factors

The aim of the present study was to investigate the involvement of adenosine 3',5'-cyclic monophosphate (cAMP) cascade in the acute impairment of contraction by 17beta-estradiol in porcine coronary arteries, and to elucidate the signaling pathway leading to the activation of this cascade by the hormone. Isometric tension was recorded in isolated rings of porcine coronary arteries. The contraction to U46619 was reduced significantly following 30 min incubation with 1 nM 17beta-estradiol or 1 nM isoproterenol. There was no additive effect when 17beta-estradiol and isoproterenol were administered together. The effect of 17beta-estradiol was mimicked by both the cyclic AMP analogue 8-Br-cAMP and the guanosine 3',5'-cyclic monophosphate (cyclic GMP) analogue 8-Br-cGMP. In rings with and without endothelium, the modulatory effect of 17beta-estradiol was abolished by the adenylyl cyclase inhibitor, SQ 22536, but was unaffected by the guanylyl cyclase inhibitor, ODQ. Both the cAMP antagonist Rp-8-Br-cAMPS and the cGMP antagonist inhibitor Rp-8-Br-cGMPS inhibited the effect of 17beta-estradiol. The effect of 17beta-estradiol was unaffected by the protein kinase A inhibitor, KT5720, but was abolished by the protein kinase G (PKG) inhibitor, KT5823, which also abolished the effect of isoproterenol. These data support our earlier findings that 17beta-estradiol (1 nM) acutely impairs contractile responses of porcine coronary arteries in vitro. This acute effect of 17beta-estradiol involves cAMP in vascular smooth muscles and the activation of PKG.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 8-Bromo Cyclic Adenosine Monophosphate; Adenine; Adenylyl Cyclase Inhibitors; Animals; Carbazoles; Coronary Vessels; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Drug Interactions; Estradiol; Indoles; Isometric Contraction; Isoproterenol; Muscle, Smooth, Vascular; Swine; Thionucleotides; Time Factors

Recently, we have shown that various types of antidepressants, including selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine, have negative immunoregulatory effects. These antidepressants suppress the interferon-gamma (IFN-gamma)/interleukin-10 (IL-10) production ratio, which is of critical importance for the determination of the capacity of immunocytes to inhibit or activate monocytic/lymphocytic functions. Since cyclic adenosine monophosphate (cAMP) production is stimulated by some antidepressants, and since cAMP inhibits IFN-gamma and stimulates IL-10 production, we postulate that the negative immunoregulatory effects of antidepressants result from their effects on the cAMP-dependent protein kinase A (PKA) pathway. The aim of the present study was to determine whether the negative immunoregulatory effects of fluoxetine may be blocked by antagonists of the cAMP-dependent PKA pathway, such as, e.g., SQ 22536, an adenylate cyclase inhibitor, and Rp-8-Br-cAMPs (Rp-isomer of 8-bromo-adenosine-3',5'-monophosphorothioate), a PKA antagonist. To this end, diluted whole blood collected from 17 normal volunteers was incubated with fluoxetine (10(-6) and 10(-5) M), with or without SQ 22536 (10(-6) and 10(-4) M) and Rp-8-Br-cAMPs (10(-6) and 10(-4) M), afterwards, IFN-gamma, IL-10 and the tumor necrosis factor alpha (TNF-alpha) were determined. Fluoxetine, 10(-6) and 10(-5) M, significantly reduced the production of IFN-gamma and TNF-alpha, and significantly decreased the IFN-gamma/IL-10 production ratio. SQ 22536 and Rp-8-Br-cAMPs were unable to block the suppressant effects of fluoxetine on the IFN-gamma/IL-10 ratio. Rp-8-Br-cAMPs, 10(-4), but not 10(-6) M, normalized the fluoxetine-induced suppression of TNF-alpha production. It is concluded that the suppressant effect of fluoxetine on the IFN-gamma/IL-10 production ratio is probably not related to the induction of the cAMP-dependent PKA pathway, whereas the suppressant effect on TNF-alpha may be related to the induction of PKA. The obtained results suggest that increased activation of the PKA-dependent pathway may constitute an important molecular basis for some (suppression of TNF-alpha production), but not all (suppression of IFN-gamma production), negative immunoregulatory effects of fluoxetine.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenine; Adult; Cyclic AMP; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Female; Fluoxetine; Humans; Immunosuppressive Agents; In Vitro Techniques; Interferon-gamma; Interleukin-10; Lipopolysaccharides; Male; Protein Kinase Inhibitors; Signal Transduction; Thionucleotides; Tumor Necrosis Factor-alpha

Four corticotropin-releasing factor (CRF)-related peptides have been found in mammals and are known as CRF, urocortin, urocortin II, and urocortin III (also known as stresscopin). The three urocortins have considerably higher affinities for CRF receptor type 2 (CRF R2) than CRF, and urocortin II and urocortin III are highly selective for CRF R2. In the present study, the authors examined the hypothesis that urocortin II or urocortin III, in addition to urocortin, produces vasodilation as a candidate for natural ligands of CRF R2beta in rat thoracic aorta. Involvement of protein kinases on urocortin-induced vasodilation was also explored. The vasodilative effects of urocortin II and urocortin III were more potent than that of CRF, but less potent than that of urocortin. Urocortin II-induced vasodilation was significantly attenuated by a CRF R2-selective antagonist, antisauvagine-30. Both SQ22536, an adenylate cyclase inhibitor, and Rp-8-Br-cAMPS, a protein kinase A (PKA) inhibitor, were found to attenuate the urocortin II-induced vasodilation. SB203580, a p38 mitogen-activated protein (MAP) kinase inhibitor, also inhibited the effects of urocortin and urocortin II on vasodilation. Thus, urocortins contribute to vasodilation via p38 MAP kinase as well as PKA pathways.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Aorta, Thoracic; Corticotropin-Releasing Hormone; Cyclic AMP-Dependent Protein Kinases; Imidazoles; Mice; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Peptide Fragments; Pyridines; Rats; Receptors, Corticotropin-Releasing Hormone; Signal Transduction; Thionucleotides; Urocortins; Vasodilation