kt-5720 and 9-(tetrahydro-2-furyl)-adenine

This study investigated the expression of voltage-gated K⁺ (K

Topics: Action Potentials; Adenine; Carbazoles; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Humans; Myofibroblasts; Nitric Oxide; Oxadiazoles; Potassium Channels, Voltage-Gated; Pyrroles; Quinoxalines; S-Nitroso-N-Acetylpenicillamine

The direct ghrelin (Ghr) involvement in cardiovascular (CV) system homeostasis has been suggested by the expression of its receptor in CV tissues and by evidence that ghrelin mediates CV activities in animals and in humans. Moreover, low Ghr plasma levels have been reported in pathological conditions characterized by high cardiovascular risk. In the present study, we investigated Ghr effect on proliferation of human aortic endothelial cell (HAEC) and involved transduction pathways. Our results indicate that ghrelin elicited proliferation in a dose-dependent manner (EC(50) about of 5nmol/L) in cultured HAEC, and that this effect was inhibited by the receptor antagonist (D-Lys3)-GHRP-6. Western blot experiments documented an activation of external receptor activated kinases (ERK1/2) and Akt in a dose-dependent fashion, as well as involvement of the cAMP pathway in ERK1/2 phosphorylation. Experiments conducted with appropriate pharmacological inhibitors to investigate Ghr-induced HAEC proliferation confirmed the involvement of ERK1/2 and I3P/Akt pathways, as well as the role of AMP cyclase/PKA pathway in ERK1/2 phosphorylation. Our results indicate that Ghr promotes HAEC proliferation, and thus may be a protective factor against vascular damage. The low ghrelin serum levels reported in insulin-resistant states may not be able to effectively counteract endothelial cell injury.

Topics: Adenine; Androstadienes; Aorta; Carbazoles; Cell Proliferation; Cells, Cultured; Endothelium, Vascular; Enzyme Activation; Flavonoids; Ghrelin; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrroles; Wortmannin

Prostacyclin is a short-lived metabolite of arachidonic acid that is produced by several cells in the lung and prominently by endothelial cells. It increases intracellular cAMP levels activating downstream signaling thus regulating vascular mesenchymal cell functions. The alveolar wall contains a rich capillary network as well as a population of mesenchymal cells, i.e., fibroblasts. The current study evaluated the hypothesis that prostacyclin may mediate signaling between endothelial and mesenchymal cells in the alveolar wall by assessing the ability of prostacyclin analogs to modulate fibroblast release of VEGF. To accomplish this study, human lung fibroblasts were cultured in routine culture on plastic support and in three-dimensional collagen gels with or without three prostacyclin analogs, carbaprostacyclin, iloprost, and beraprost, and the production of VEGF was evaluated by ELISA and quantitative real-time PCR. Iloprost and beraprost significantly stimulated VEGF mRNA levels and protein release in a concentration-dependent manner. These effects were blocked by the adenylate cyclase inhibitor SQ-22536 and by the protein kinase A (PKA) inhibitor KT-5720 and were reproduced by a direct PKA activator but not by an activator of exchange protein directly activated by cAMP (Epac), indicating that cAMP-activated PKA signaling mediated the effect. Since VEGF serves to maintain the pulmonary microvasculature, the current study suggests that prostacyclin is part of a bidirectional signaling network between the mesenchymal and vascular cells of the alveolar wall. Prostacyclin analogs, therefore, have the potential to modulate the maintenance of the pulmonary microcirculation by driving the production of VEGF from lung fibroblasts.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Adult; Carbazoles; Cell Culture Techniques; Cells, Cultured; Epoprostenol; Fibroblasts; Humans; Iloprost; Lung; Prostaglandins I; Pyrroles; RNA, Messenger; Stimulation, Chemical; Vascular Endothelial Growth Factor A

Ghrelin, the endogenous ligand for the GH secretagogue receptor, is produced by the oxyntic cells of the stomach and is involved in the regulation of energy balance. However, an increasing number of direct ghrelin cardiovascular effects, and, among them, high ghrelin binding in atherosclerotic coronary arteries, are being reported. We investigated whether ghrelin affects migration of human aorta endothelial cells (HAEC). HAEC bound ghrelin in specific, saturable manner. Ghrelin, as such, did not affect HAEC migration, however it inhibited the angiotensin II-induced migration, and this effect was inhibited by the antagonist (D-Lys(3))-GHRP-6. In HAEC, ghrelin elicited increased intracellular concentration of cAMP that was involved in its effect on AngII-induced HAEC migration, as the AMP cyclase inhibitor SQ22.536 and PKA inhibitor KT5720, respectively, inhibited and blunted it. These findings suggest a role of ghrelin in the control of endothelial cell migration and its possible involvement in vascular changes present in disorders characterized by low plasma ghrelin.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Angiotensin II; Aorta; Carbazoles; Cell Movement; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Endothelium, Vascular; Ghrelin; Humans; Indoles; Oligopeptides; Peptide Hormones; Proto-Oncogene Proteins c-akt; Pyrroles

We examined the effects of acute hypoxia on vascular tone and coronary blood flow (CBF) in rabbit coronary arteries. In the pressurized arterial preparation of small arteries (<100 mum) and the Langendorff-perfused rabbit hearts, hypoxia induced coronary vasodilation and increased CBF in the presence of glibenclamide (K(ATP) channel blocker), Rp-8-Br-PET-cGMPs [cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor, Rp-cGMPs], and methionyl transfer RNA synthetase (MRS) 1334 (adenosine A(3) receptor inhibitor); these increases were inhibited by the inward rectifier K(+) (Kir) channel inhibitor, Ba(2+). These effects were blocked by the adenylyl cyclase inhibitor SQ 22536 and by the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) inhibitors Rp-8-CPT-cAMPs (Rp-cAMPs) and KT 5720. However, cGMP-dependent protein kinase was not involved in the hypoxia-induced increases of the vascular diameter and CBF. In summary, our results suggest that acute hypoxia can induce the opening of Kir channels in coronary artery that has small diameter (<100 mum) by activating the cAMP and PKA signalling pathway, which could contribute to vasodilation and, therefore, increased CBF.

Topics: Acute Disease; Adenine; Animals; Blood Pressure; Carbazoles; Coronary Circulation; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Female; Glyburide; Hypoxia; In Vitro Techniques; Indoles; Male; Potassium Channel Blockers; Potassium Channels, Inwardly Rectifying; Pyrroles; Rabbits; Signal Transduction; Thionucleotides; Vasodilation

It is well known that gonadal steroid hormones influence the level of plasma calcitonin (CT), but the mechanism by which progesterone affects CT secretion is not clear. Immortalized TT cells are a reliable model system for studying the endocrine function of human parafollicular cells. In the present study, the effects of progesterone on CT secretion were examined in TT cells. TT cells were incubated in medium containing vehicle (DMSO), progesterone or BSA-progesterone for 60 or 150 min, and then the levels of CT in the medium, progesterone receptors, cAMP accumulation and CT mRNA expression were measured. To study the correlation between progesterone effects and the cAMP-dependent protein kinase A (PKA) pathway, cell lysates or cells in 24-well plates were treated with either vehicle or progesterone plus RU486, SQ22536, KT5720, or 3-isobutyl-1-methylxanthine. Then, adenylyl cyclase and protein kinase A (PKA) activities were measured in the cell lysates, and the CT levels were measured in the medium from the 24-well plate. The activated cAMP response element binding protein (P-CREB) was also measured by immunofluorescence. Administration of 1 microM progesterone or 500 nM BSA-progesterone increased the secretion of CT by 381% and 100%, respectively. Progesterone receptors A and B were downregulated by progesterone treatment. The cAMP concentration, adenylyl cyclase and PKA activity, CT mRNA expression, and nuclear P-CREB concentrations all showed an increase after progesterone treatment. RU486, SQ22536 and KT5720 inhibited the progesterone-stimulated effects. These results suggest that a cAMP-dependent PKA pathway is involved in progesterone-stimulated effects on CT secretion from TT cells.

Topics: 1-Methyl-3-isobutylxanthine; Adenine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Blotting, Western; Calcitonin; Carbazoles; Cell Line, Tumor; Cell Nucleus; CREB-Binding Protein; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fluorescent Antibody Technique; Gene Expression; Humans; Indoles; Mifepristone; Progesterone; Progestins; Pyrroles; Receptors, Progesterone; RNA, Messenger; Signal Transduction; Time Factors

The purpose of the present study was to ascertain the role of adenylate (AC) versus guanylate cyclase (GC) signaling pathways in the internal anal sphincter (IAS) smooth muscle relaxation by beta(1)-, beta(2)-, and beta(3)-adrenoceptor (AR) activation by xamoterol, procaterol, and disodium 5-[(2R)-2-(3-chlorophenyl)-2-hydroxy-ethyl]amino)propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243), respectively. The above-mentioned agonists produced concentration-dependent relaxation of the smooth muscle strips. Both the selective G(i/o)alpha and G(s)alpha antagonists 8,8'-(carbonylbis(imino-3,1-phenylene))bis-(1,3,5-naphthalene trisulfonic acid) (NF 023) and 4,4',4",4"'-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF 449), respectively, inhibited the relaxation induced by procaterol. However, only NF 023 inhibited the relaxation induced by xamoterol and CL 316243. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one, a soluble GC inhibitor, significantly inhibited the relaxation induced by different agonists. In contrast, the selective AC inhibitor [9-(tetrahydro-2'-furyl)adenine] (SQ 22536) inhibited only the relaxation induced by procaterol. (9R,10S,12S)-2,3,9,10,11,12-Hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg: 3',2',1'-kl]pyrrolo[3,4-l][1,6]benzodiazocine-10-carboxylic acid, hexyl ester (KT 5720), a cAMP-dependent protein kinase inhibitor, attenuated the relaxation by procaterol, whereas (9S,10R,12R)-2,3,9,10,11,12, hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9.12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-I][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT 5823), a selective cGMP-dependent protein kinase (PKG) inhibitor, attenuated the relaxation induced by xamoterol and CL 316243. Xamoterol produced significant increase in cGMP levels, whereas only procaterol enhanced the cAMP levels. Western blot analysis confirmed the presence of beta(1), beta(2), and beta(3)-AR subtypes in the IAS. In summary, beta(2)-AR activates both G(s)alpha and G(i/o)alpha-protein subunits and induces relaxation in the rat IAS via both cAMP/cGMP pathways. In contrast, the beta(1)/beta(3)-ARs activation causes the smooth muscle relaxation via G(i/o)alpha-protein subunit/GC/GMP/PKG pathway. These studies are important for the understanding of intracellular mechanisms underlying IAS smooth muscle relaxation and in turn the pathophysiology of certain anorectal motility disorders.

Topics: Adenine; Adenylyl Cyclases; Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Animals; Blotting, Western; Carbazoles; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; GTP-Binding Proteins; Guanylate Cyclase; Indoles; Male; Muscle Relaxation; Muscle, Smooth; Oxadiazoles; Procaterol; Pyrroles; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta; Receptors, Adrenergic, beta-1; Receptors, Adrenergic, beta-2; Receptors, Adrenergic, beta-3; Suramin; Xamoterol

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites of cytochrome P450 monooxygenase, which are released from endothelial cells and dilate arteries. Dilation seems to be caused by activation of large-conductance Ca2+ activated K+ channels (BK(Ca)) leading to membrane hyperpolarization. Previous studies suggest that EETs activate BK(Ca) channels via ADP-ribosylation of the G protein Galphas with a subsequent membrane-delimited action on the channel [Circ Res 78:415-423, 1996; 80:877-884, 1997; 85:349-356, 1999]. The present study examined whether this pathway is present in human embryonic kidney (HEK) 293 cells when the BK(Ca) alpha-subunit (cslo-alpha) is expressed without the beta-subunit. 11,12-EET increased outward K+ current in whole-cell recordings of HEK293 cells. In cell-attached patches, 11,12-EET also increased the activity of cslo-alpha channels without affecting unitary conductance. This action was mimicked by cholera toxin. The ADP-ribosyltransferase inhibitors 3-aminobenzamide and m-iodobenxylguanidine blocked the stimulatory effect of 11,12-EET. In inside-out patches 11,12-EET was without effect on channel activity unless GTP was included in the bathing solution. GTP and GTPgammaS alone also activated cslo-alpha channels. Dialysis of cells with anti-Galphas antibody completely blocked the activation of cslo-alpha channels by 11,12-EET, whereas anti-Galphai/o and anti-Gbetagamma antibodies were without effect. The protein kinase A inhibitor KT5720 and the adenylate cyclase inhibitor SQ22536 did not reduce the stimulatory effect of 11,12-EET on cslo-alpha channels in cell-attached patches. These data suggest that EET leads to Galphas-dependent activation of the cslo-alpha subunits expressed in HEK293 cells and that the cslo-beta subunit is not required.

Topics: 8,11,14-Eicosatrienoic Acid; Adenine; ADP Ribose Transferases; Antibodies; Carbazoles; Cells, Cultured; Drug Interactions; Electrophysiology; Enzyme Inhibitors; Gene Expression Regulation; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Indoles; Kidney; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Large-Conductance Calcium-Activated Potassium Channel beta Subunits; Large-Conductance Calcium-Activated Potassium Channels; Potassium Channels; Potassium Channels, Calcium-Activated; Pyrroles

The aim of this study was to investigate the exact mechanism of interaction between nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) as inhibitory non-adrenergic non-cholinergic (NANC) neurotransmitters in isolated smooth muscle cells and smooth muscle strips of the pig gastric fundus. In isolated smooth muscle cells, the maximal relaxant effect of VIP (10(-9) M) was inhibited by 94% by the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine (L-NA, 10(-4) M) and by 85% by the inducible NOS (iNOS)-selective inhibitor N-(3-(aminomethyl)-benzyl)acetamide (1400W; 10(-6) M). The relaxant effect of VIP was reduced by more than 70% by the guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ; 10(-6) M), the glucocorticoid dexamethasone (10(-5) M) and three protein kinase A inhibitors: (R)-p-cyclic adenosine-3', 5'-monophosphothioate ((R)-p-cAMPS; 10(-6) M), ¿(8R,9S, 11S)-(-)-9-hydroxy-9-n-hexylester-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a, g]cycloocta[cde]-trin-den-1-one¿ (KT5720; 10(-6) M) and N-(2-(p-bromo-cinnamylamino)ethyl))-5-isoquinoline sulfonamide dihydrochloride (H-89; 10(-5) M). In contrast, no influence of the NOS inhibitors, ODQ, dexamethasone, nor the protein kinase A inhibitors could be observed on the relaxant effect of VIP in smooth muscle strips. These data demonstrate that the experimental method completely changes the influence of NOS inhibitors on the relaxant effect of VIP in the pig gastric fundus. The isolation procedure of the smooth muscle cells might induce iNOS that can be activated by VIP.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Amidines; Animals; Arginine; Benzylamines; Carbazoles; Colforsin; Cyclic AMP; Dexamethasone; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gastric Fundus; In Vitro Techniques; Indoles; Isoquinolines; Muscle Relaxation; Muscle, Smooth; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Oxadiazoles; Protein Kinase Inhibitors; Pyrroles; Quinoxalines; Sulfonamides; Swine; Thionucleotides; Vasoactive Intestinal Peptide

This study was conducted to determine the mechanism of arachidonic acid (AA) release elicited by phenylephrine (PHE) stimulation of alpha adrenergic receptor (AR), and its modulation by cyclic adenosine 3',5'-monophosphate (cAMP) in Rat-1 fibroblasts (R-1Fs) transfected with the alpha-1A, alpha-1B or alpha-1D AR. PHE increased AA release and also caused a marked accumulation of cAMP in R-1Fs expressing the alpha-1 AR subtypes, but not in those transfected with vector alone. PHE also enhanced phospholipase D (PLD), but not phospholipase A2 (PLA2) activity. The increase in PHE-induced AA release, PLD activity and cAMP accumulation differed among the various alpha AR subtypes with: alpha-1A > alpha-1B > alpha-1D AR. The effect of PHE to increase AA release was attenuated by C2-ceramide, an inhibitor of PLD; propranolol, a phosphatidate phosphohydrolase inhibitor; and RHC-80267, a diacylglycerol lipase inhibitor in R-1Fs expressing the alpha-1A AR. Forskolin, which activates adenylyl cyclase, increased cAMP accumulation and inhibited PHE-induced AA release and PLD activity in alpha-1A-AR-expressing R-1Fs. 8-(4-chlorophenyl-thio)-cAMP, a nonhydrolyzable analog of cAMP, also attenuated the rise in AA release and PLD activity elicited by PHE in these cells. In contrast, SQ 22536, an adenylyl cyclase inhibitor, and KT 5720, a protein kinase A inhibitor, increased PHE-induced AA release and PLD activity in R-1Fs expressing the alpha-1A AR. These data suggest that the alpha-1A, alpha-1B and alpha-1D ARs are coupled to PLD activation and cAMP accumulation. Moreover, PHE promotes AA release in R-1Fs expressing the alpha-1A AR through PLD activation. Furthermore, cAMP generated by alpha-1A AR stimulation acts as an inhibitory modulator of PLD activity and AA release via protein kinase A.

Topics: Adenine; Adenylate Cyclase Toxin; Animals; Arachidonic Acid; Calcimycin; Carbazoles; Cell Line; Cholera Toxin; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Glycerides; GTP-Binding Proteins; Indoles; Lipase; Phenylephrine; Phospholipase D; Phospholipases A; Phospholipases A2; Prazosin; Pyrroles; Radioligand Assay; Rats; Receptors, Adrenergic, alpha-1; Transfection; Virulence Factors, Bordetella

The role of cAMP and cAMP-dependent protein kinase A (PKA) in endothelin (ET)-stimulated migration of human neutrophils was studied. Endothelins caused an increase in neutrophil migration when they were applied in low (nanomolar) concentrations; stimulation of migration was either predominantly chemokinetic (ET-1) or chemotactic (ET-2, ET-3). All endothelins, at concentrations which gave maximal stimulation of migration, caused an increase of cAMP level. Two inhibitors of adenylate cyclase, MDL-12330A and SQ-22536, completely inhibited migration activated by ET-1, ET-2 or ET-3, indicating that cAMP generation played a decisive role in endothelin-activated migration. The role of PKA in endothelin-activated migration was considered. Two specific antagonists of PKA strongly inhibited endothelin-activated migration. KT-5720, an inhibitor of PKA, also inhibited ET-activated migration, but only when electroporated cells were used. The results suggest that the effect of cAMP on endothelin-activated migration was mediated by PKA.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Carbazoles; Cell Movement; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Endothelin-1; Endothelin-2; Endothelin-3; Endothelins; Enzyme Inhibitors; Humans; Imines; Indoles; Neutrophils; Pyrroles; Thionucleotides