s-nitro-n-acetylpenicillamine and 8-bromoguanosino-3--5--cyclic-monophosphorothioate

Human serotonin transporter (hSERT) activity expressed in HeLa cells was stimulated by agents that release nitric oxide, stimulate soluble guanylyl cyclase, or activate cGMP-dependent protein kinase (PKG). This stimulation was blocked by a PKG inhibitor. A naturally occurring mutation, I425V, associated with obsessive-compulsive disorder and other neuropsychiatric disorders, activated hSERT and eliminated stimulation via the PKG pathway. Inhibitors of soluble guanylyl cyclase or PKG decreased activity of the I425V mutant, but not wild type, indicating that both wild-type and mutant transporters could exist in both high and low activity forms. Mutation of Thr-276 in the fifth transmembrane domain (TM5) to alanine or aspartate prevented activation of wild-type hSERT through the PKG pathway and also blocked the inhibition of I425V activity by inhibitors of the pathway. The accessibility of positions in TM5 near Thr-276 was modified in T276D, but not in I425V. These results are consistent with the hypothesis that PKG phosphorylates hSERT at Thr-276 and increases its activity by modifying the substrate permeation pathway formed, in part, by TM5. The effect of the I425V mutation may shift the balance of hSERT toward the phosphorylated form, possibly by interfering with the action of a phosphatase. However, association of hSERT with protein phosphatase 2A was not decreased in the I425V mutant.

Topics: Biotinylation; Cell Adhesion Molecules; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Activation; Enzyme Inhibitors; HeLa Cells; Humans; Immunoprecipitation; Microfilament Proteins; Mutagenesis, Site-Directed; Mutation; Nitric Oxide Donors; Penicillamine; Phosphoproteins; Phosphorylation; Protein Transport; Serotonin; Serotonin Plasma Membrane Transport Proteins; Thionucleotides

We investigated the role of the NO/cGMP system in the vasodilatory response to hypercapnia after cortical spreading depression (CSD) in barbiturate anesthetized rats in vivo. Regional cerebral blood flow (rCBF) was measured by laser Doppler flowmetry (LDF). Hypercapnia (arterial pCO2 50-60 mm Hg) increased rCBF by 2.8+/-1.0%/mm Hg (n = 34). Fifteen minutes after CSD, resting rCBF was reduced to 87%, and rCBF response to hypercapnia was abolished (p < 0.001, n = 28). Within 1 h after CSD, only little restoration of vascular reactivity occurred. Topical application of the NO-donors S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN1), or spermine/NO complex (Sperm/NO), or of the cell permeable guanosine 3',5'-cyclic monophosphate (cGMP) analogue 8-Br-cGMP reestablished resting rCBF to values measured before CSD, and reversed CSD-induced attenuation of the cerebrovascular response to hypercapnia. Restoration of resting rCBF to pre-CSD level by the NO-independent vasodilator papaverine had no effect on the attenuated hypercapnic response. In conclusion, we have shown that the compromised vascular reactivity to hypercapnia after CSD can be reversed to normal reactivity by restoration of the basal NO or cGMP concentration in the cortex, suggesting a reduction of the cerebrovascular NO or cGMP concentration following CSD.

Topics: Analysis of Variance; Animals; Cerebral Cortex; Cerebrovascular Circulation; Cerebrovascular Disorders; Cortical Spreading Depression; Cyclic GMP; Drug Interactions; Hypercapnia; Laser-Doppler Flowmetry; Male; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Penicillamine; Rats; Rats, Wistar; Spermine; Thionucleotides; Time Factors

Electrical stimulation of sensory neurons that innervate receptors on the tailfan of crayfish evokes a reflex response of motor neurons that produce movements of the blades of the tailfan, the uropods. We analyzed the modulatory effects of nitric oxide (NO) on the spike frequency of the reflex response. Bath application of L-arginine and SNAP, which elevate endogenous and exogenous NO levels, increased the frequency of the evoked response, whereas the application of L-NAME and PTIO, which reduce NO levels, decreased the frequency of the response. To determine through what pathway and target NO exerted these effects we bath applied ODQ, an inhibitor of soluble guanylyl cyclase (sGC), which decreased the frequency of response, and 8-br-cGMP, which increased the spike frequency of response. To provide further evidence that NO acts via sGC, we elevated NO levels with L-arginine while simultaneously inhibiting sGC with ODQ. This application reduced the response to control levels, indicating that NO in the terminal ganglion of crayfish acts via sGC to modulate cGMP levels, which in turn regulate the responses of the uropod motor neurons.

Topics: Analysis of Variance; Animals; Arginine; Astacoidea; Cyclic GMP; Cyclic N-Oxides; Drug Interactions; Electric Stimulation; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Female; Free Radical Scavengers; Imidazoles; In Vitro Techniques; Male; Muscles; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Oxadiazoles; Penicillamine; Picolines; Quinoxalines; Reflex; Thionucleotides

The mechanism(s) responsible for the persistent coexpression of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in the failing heart is unknown.. To determine whether NO was sufficient to provoke TNF-alpha biosynthesis, we examined the effects of an NO donor, S-nitroso-N-acetyl penicillamine (SNAP), in buffer-perfused Langendorff hearts. SNAP (1 micromol/L) treatment resulted in a time- and dose-dependent increase in myocardial TNF-alpha mRNA and protein biosynthesis in adult cat hearts. The effects of SNAP were completely abrogated by a NO quenching agent, 2-(4-carboxyphenyl)-4, 4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (C-PTIO), and mimicked by sodium nitroprusside. Electrophoretic mobility shift assays demonstrated that SNAP treatment led to the rapid induction of nuclear factor kappa-beta (NF-kappaB) but not AP-1. The importance of the cGMP pathway in terms of mediating NO-induced TNF-alpha biosynthesis was shown by studies that demonstrated that 8-bromo-cGMP mimicked the effects of SNAP and that the effects of SNAP could be completely abrogated using a cGMP antagonist, 1H-(1,2, 4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), or protein kinase G antagonist (Rp-8-Br-cGMPS). SNAP and 8-Br-cGMP were both sufficient to lead to the site-specific phosphorylation (serine 32) and degradation of IkappaBalpha in isolated cardiac myocytes. Finally, protein kinase G was sufficient to directly phosphorylate IkappaBalpha on serine 32, a critical step in the activation of NF-kappaB.. These studies show that NO provokes TNF-alpha biosynthesis through a cGMP-dependent pathway, which suggests that the coincident expression of TNF-alpha and NO may foster self-sustaining positive autocrine/paracrine feedback inflammatory circuits within the failing heart.

Topics: Animals; Benzoates; Cats; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; DNA-Binding Proteins; Electrophoresis; I-kappa B Proteins; Imidazoles; In Vitro Techniques; Myocardium; NF-kappa B; NF-KappaB Inhibitor alpha; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Oxadiazoles; Penicillamine; Phosphorylation; Protein Kinases; Quinoxalines; RNA, Messenger; Thionucleotides; Tumor Necrosis Factor-alpha