cyclic-gmp and oxophenylarsine

It has been pharmacologically suggested that 3',5'-cyclic guanosine-monophosphate (cGMP) mediates indolyl-3-butyric acid (IBA)-induced stomatal opening. In Arabidopsis thaliana (L.) Heynh., such investigations compared the wild type (Columbia and Ws ecotypes) to mutants knockout for either GTP-binding protein (G protein) α subunit 1 (gpa1-4), putative G protein-coupled receptor 1 (gcr1-5), calcineurin B-like isoform 1 (cbl1) or 9 (cbl9), or the NADPH oxidases AtrbohD and AtrbohF (atrbohD/F). Stomatal opening to IBA or the permeant cGMP analogue, 8-bromo-cGMP (8-Br-cGMP) was abolished in the atrbohD/F mutant. The IBA response was fully or partially suppressed, respectively, in the gcr1-5 mutant, or the gpa1-4 and cbl1 mutants. In the cbl9 mutant, the response to IBA or 8-Br-cGMP, respectively, was partially or fully suppressed. Phenylarsine oxide (PAO) affected the IBA response, which the cbl1 mutant overlapped or the gpa1-4 and cbl9 mutants increased up to 100% inhibition. 6-anilino-5,8-quinolinedione, mas17, the (Rp)-diastereomer of 8-bromo-3',5'-cyclic guanosine monophosphorothioate (Rp-8-Br-cGMPS), nicotinamide, ruthenium red (RRed), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), cyclosporine A (CsA) and FK506 converged to affect the IBA response, which the gpa1-4 and cbl9 mutants overlapped or the cbl1 mutant and PAO increased up to 100% inhibition. Rp-8-Br-cGMPS, nicotinamide, RRed, BAPTA, CsA or FK506 paralled the cbl9 and atrbohD/F mutants to abolish the 8-Br-cGMP response. Based on so far revealed features of these mutants and pharmacological compounds, these results confirmed cGMP as a Ca(2+)-mobilizing second messenger for apoplastic auxin whose perception and transduction would implicate a seven-transmembrane receptor - G protein - guanylyl cyclase unit at the guard cell plasma membrane.

Topics: Arabidopsis; Arsenicals; Calcium; Cyclic GMP; Guanylate Cyclase; Indoleacetic Acids; Indoles; Mutation; Plant Stomata; Second Messenger Systems; Signal Transduction

S-nitrosothiol compounds are important mediators of NO signalling and can give rise to various redox derivatives of NO: nitrosonium cation (NO+), nitroxyl anion (NO-) and NO* radical. Several enzymes and transporters have been implicated in the intracellular delivery of NO from S-nitrosothiols. In the present study we have investigated the role of GPx (glutathione peroxidase), the L-AT (L-amino acid transporter) system and PDI (protein disulfide-isomerase) in the delivery of NO redox derivatives into human platelets. Washed human platelets were treated with inhibitors of GPx, L-AT and PDI prior to exposure to donors of NO redox derivatives (S-nitrosoglutathione, Angeli's salt and diethylamine NONOate). Rapid delivery of NO-related signalling into platelets was monitored by cGMP accumulation and DAF-FM (4-amino-5-methylamino-2'7'-difluorofluorescein) fluorescence. All NO redox donors produced both a cGMP response and DAF-FM fluorescence in target platelets. NO delivery was blocked by inhibition of PDI in a dose-dependent manner. In contrast, inhibition of GPx and L-AT had only a minimal effect on NO-related signalling.PDI activity is therefore required for the rapid delivery into platelets of NO-related signals from donors of all NO redox derivatives. GPx and the L-AT system appeared to be unimportant in rapid NO signalling by the compounds used in the present study. This does not, however, exclude a possible role during exposure of cells to other S-nitrosothiol compounds, such as S-nitrosocysteine. These results further highlight the importance of PDI in mediating the action of a wide range of NO-related signals.

Topics: Amino Acid Transport System L; Arsenicals; Bacitracin; Biological Transport; Blood Platelets; Cyclic GMP; Enzyme Inhibitors; Glutathione Peroxidase; Guanylate Cyclase; Humans; Nitric Oxide; Oxidation-Reduction; Protein Disulfide-Isomerases; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase

Agonists elevate the cytosolic calcium concentration in human platelets via a receptor-operated mechanism, involving both Ca(2+) release from intracellular stores and subsequent Ca(2+) entry, which can be inhibited by platelet inhibitors, such as prostaglandin E(1) and nitroprusside which elevate cAMP and cGMP, respectively. In the present study we investigated the mechanisms by which cAMP and cGMP modulate store-mediated Ca(2+) entry. Both prostaglandin E(1) and sodium nitroprusside inhibited thapsigargin-evoked store-mediated Ca(2+) entry and actin polymerization. However, addition of these agents after induction of store-mediated Ca(2+) entry did not affect either Ca(2+) entry or actin polymerization. Furthermore, prostaglandin E(1) and sodium nitroprusside dramatically inhibited the tyrosine phosphorylation induced by depletion of the internal Ca(2+) stores or agonist stimulation without affecting the activation of Ras or the Ras-activated phosphatidylinositol 3-kinase or extracellular signal-related kinase (ERK) pathways. Inhibition of cyclic nucleotide-dependent protein kinases prevented inhibition of agonist-evoked Ca(2+) release but it did not have any effect on the inhibition of Ca(2+) entry or actin polymerization. Phenylarsine oxide and vanadate, inhibitors of protein-tyrosine phosphatases prevented the inhibitory effects of the cGMP and cAMP elevating agents on Ca(2+) entry and actin polymerization. These results suggest that Ca(2+) entry in human platelets is directly down-regulated by cGMP and cAMP by a mechanism involving the inhibition of cytoskeletal reorganization via the activation of protein tyrosine phosphatases.

Topics: Actins; Adenosine Diphosphate; Alprostadil; Arsenicals; Blood Platelets; Calcium; Carbazoles; Cyclic AMP; Cyclic GMP; Egtazic Acid; Enzyme Activation; Enzyme Inhibitors; Humans; In Vitro Techniques; Indoles; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nitroprusside; Phosphorylation; Platelet Activation; Platelet Aggregation Inhibitors; Protein Tyrosine Phosphatases; Pyrroles; Thapsigargin; Thrombin; Vanadates

The regulation of the reduced-folate transporter (RFT) by nitric oxide (NO) was analyzed in human retinal pigment epithelial (HRPE) cells. NO inhibited specifically and reversibly the uptake of N5-methyltetrahydrofolate by a cGMP-independent mechanism. The inhibition was associated with a decrease in substrate affinity. The NO-induced inhibition was prevented by antioxidants and NO scavengers. Agents capable of modifying thiol groups in proteins inhibited RFT, indicating that the likely mechanism of NO-induced inhibition is via modification of essential thiol groups in this protein. These studies suggest that NO produced during retinal disease may affect the function of RFT in adjacent RPE cells.

Topics: 4-Chloro-7-nitrobenzofurazan; 4-Chloromercuribenzenesulfonate; Antioxidants; Arsenicals; Carrier Proteins; Cells, Cultured; Cyclic GMP; Epithelial Cells; Folate Receptors, GPI-Anchored; Free Radical Scavengers; Humans; Imidazoles; Nitric Oxide; Nitric Oxide Donors; Nitrobenzenes; Oxidation-Reduction; Receptors, Cell Surface; Retinal Pigments; Sulfhydryl Compounds; Tetrahydrofolates; Tetranitromethane; Tyrosine

Ever since the identification of two distinct Ang II receptor subtypes, the function of the AT2 receptor has been a subject of debate. As opposed to the AT1 subtype, this receptor does not interact with G-proteins in most cell lines and tissues. We show here that, in intact PC12W cells which express only AT2 receptors, Ang II significantly decreases basal and atrial natriuretic peptide (ANP)-stimulated cGMP concentration. This effect is mimicked by the AT2 selective agonist CGP 42112, and is not prevented by the AT1 selective antagonist losartan, indicating that this is an AT2 receptor mediated response. The lack of effect of the phosphodiesterase (PDE) inhibitor IBMX shows that this mechanism does not involve PDE stimulation. This is confirmed by the finding that neither Ang II or CGP 42112 affect the Ca++/calmodulin dependent cGMP PDE activity. Furthermore Ang II and CGP 42112 have no effect on nitroprusside-stimulated cGMP levels in these cells, thus ruling out interactions between the AT2 receptor and soluble guanylate cyclase. These data indicate that the AT2 receptor mediated decrease of cGMP is due to the selective inhibition of particulate guanylate cyclase (pGC) activity. In an accompanying paper we report that interaction of Ang II with the AT2 receptor in the same cells results in the stimulation of phosphotyrosine phosphatase (PTPase) activity. Interestingly, the PTPase inhibitors sodium orthovanadate and phenylarsine oxyde, but not the Ser/Thr phosphatase inhibitor okadiac acid, inhibitthe Ang II and CGP 42112 induced decreases in cellular cGMP concentration. These findings suggest that stimulation of PTPase activity may be involved in the regulation of pGC activity via AT2 receptors.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-GMP Phosphodiesterases; Angiotensin II; Animals; Aorta; Arsenicals; Atrial Natriuretic Factor; Cell Membrane; Cells, Cultured; Cyclic GMP; GTP-Binding Proteins; Guanylate Cyclase; Muscle, Smooth, Vascular; Oligopeptides; PC12 Cells; Protein Tyrosine Phosphatases; Rats; Receptors, Angiotensin; Vanadates

A10 smooth muscle cells, derived from embryonic rat thoracic aorta, responded to the atrial natriuretic factor (ANF) with increased levels of cyclic GMP. These cells possess high-affinity (apparent Kd = 50 pM) plasma membrane receptors for ANF. Internalization of ANF at 37 degrees C was indicated by the following: approximately 25% of the 125I-ANF associated with the cells at elevated temperatures could not be dissociated from the surface of the cells, but could be released by permeabilization with saponin, and the amount of nondissociable ANF increased in the presence of chloroquine. In whole cells and in membranes, a single polypeptide of 60,000 Da was specifically labeled by a photoaffinity analog of 125I-ANF, as well as by crosslinking, and an IC50 of 80 pM for inhibition of the labeling by ANF was observed. The ANF receptor in A10 cells was distinguished from that in rabbit aorta by its high affinity for shorter and linear analogs of ANF, as well as by a different photolabeling pattern.

Topics: Animals; Arsenicals; Atrial Natriuretic Factor; Biological Transport; Cell Line; Cyclic GMP; Guanylate Cyclase; Membranes; Molecular Weight; Muscle, Smooth, Vascular; Rats; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface