RESUMO
Cyclic GMP (cGMP) is an important regulator in eukaryotes, and cGMP-dependent protein kinase (PKG) plays a key role in perceiving cellular cGMP in diverse physiological processes in animals. However, the molecular identity, property, and function of PKG in plants remain elusive. In this study, we have identified PKG from plants and characterized its role in mediating the gibberellin (GA) response in rice (Oryza sativa). PKGs from plants are structurally unique with an additional type 2C protein phosphatase domain. Rice PKG possesses both protein kinase and phosphatase activities, and cGMP stimulates its kinase activity but inhibits its phosphatase activity. One of PKG's targets is GAMYB, a transcription factor in GA signaling, and the dual activities of PKG catalyze the reversible phosphorylation of GAMYB at Ser6 and modulate the nucleocytoplasmic distribution of GAMYB in response to GA. Loss of PKG impeded the nuclear localization of GAMYB and abolished GAMYB function in the GA response, leading to defects in GA-induced seed germination, internode elongation, and pollen viability. In addition to GAMYB, PKG has multiple potential targets and thus has broad effects, particularly in the salt stress response.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Giberelinas/metabolismo , Oryza/metabolismo , Estresse Salino/genética , Fatores de Transcrição/metabolismo , Núcleo Celular/metabolismo , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Germinação/efeitos dos fármacos , Giberelinas/farmacologia , Mutação , Oryza/efeitos dos fármacos , Oryza/enzimologia , Oryza/genética , Fosforilação/efeitos dos fármacos , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/crescimento & desenvolvimento , Pólen/metabolismo , Regiões Promotoras Genéticas , Sementes/genética , Sementes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/genéticaRESUMO
Panax ginseng C. A. Meyer has been widely used in skin care. Our previous study showed that the phenolic acids in ginseng root extract (GRE) impart inhibitory effects on melanogenesis. In this study, we found that as the most abundant component of phenolic acids in GRE, vanillic acid decreased tyrosinase activity and melanin levels with or without α-MSH stimulation and suppressed the expression of microphthalmia-associated transcription factor (MITF) and melanogenic enzymes in B16F10 cells. Furthermore, vanillic acid downregulated NOS activity, nitric oxide (NO) content, cGMP level, guanylate cyclase (GC) and protein kinase G (PKG) activity, and the phosphorylation of cAMP-response element-binding protein (CREB), whereas arbutin had no effect on the NO/PKG pathway. These findings indicate that vanillic acid in GRE suppressed melanogenesis by inhibiting the NO/PKG signaling pathways. This study provides a potential mechanism underlying the inhibitory effect of ginseng on melanogenesis.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Melaninas/antagonistas & inibidores , Óxido Nítrico/antagonistas & inibidores , Panax/química , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Transdução de Sinais/efeitos dos fármacos , Ácido Vanílico/farmacologia , Animais , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Oxirredutases Intramoleculares/metabolismo , Melaninas/biossíntese , Melaninas/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Fator de Transcrição Associado à Microftalmia/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Oxirredutases/metabolismo , Regulação para Cima/efeitos dos fármacos , alfa-MSH/farmacologiaRESUMO
We have previously observed an increased of angiotensin II (ANG II) type 1 receptor (AT(1)R) with enhanced AT(1)R-mediated sympathetic outflow and concomitant downregulation of neuronal nitric oxide (NO) synthase (nNOS) with reduced NO-mediated inhibition from the paraventricular nucleus (PVN) in rats with heart failure. To test the hypothesis that NO exerts an inhibitory effect on AT(1)R expression in the PVN, we used primary cultured hypothalamic cells of neonatal rats and neuronal cell line NG108-15 as in vitro models. In hypothalamic primary culture, NO donor sodium nitroprusside (SNP) induced dose-dependent decreases in mRNA and protein of AT(1)R (10(-5) M SNP, AT(1)R protein was 10 ± 2% of control level) while NOS inhibitor N(G)-monomethyl-l-arginine (l-NMMA) induced dose-dependent increases in mRNA and protein levels of AT(1)R (10(-5) M l-NMMA, AT(1)R protein was 148 ± 8% of control level). Similar effects of SNP and l-NMMA on AT(1)R expression were also observed in NG108-15 cell line (10(-6) M SNP, AT(1)R protein was 30 ± 4% of control level while at the dose of 10(-6) M l-NMMA, AT(1)R protein was 171 ± 15% of the control level). Specific inhibition of nNOS, using antisense, caused an increase in AT(1)R expression while overexpression of nNOS, using adenoviral gene transfer (Ad.nNOS), caused an inhibition of AT(1)R expression in NG108 cells. Antisense nNOS transfection augmented the increase while Ad.nNOS infection blunted the increase in intracellular calcium concentration in response to ANG II treatment in NG108 cells. In addition, downregulation of AT(1)R mRNA as well as protein level in neuronal cell line in response to S-nitroso-N-acetyl pencillamine (SNAP) treatment was blocked by protein kinase G (PKG) inhibitor, while the peroxynitrite scavenger deforxamine had no effect. These results suggest that NO acts as an inhibitory regulator of AT(1)R expression and the activation of PKG is the required step in the regulation of AT(1)R gene expression via cGMP-dependent signaling pathway.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico/farmacologia , Receptor Tipo 1 de Angiotensina/biossíntese , Adenoviridae/genética , Angiotensina II/farmacologia , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , DNA Antissenso/genética , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Óxido Nítrico/farmacocinética , Doadores de Óxido Nítrico/farmacocinética , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/metabolismo , Nitroprussiato/farmacologia , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , S-Nitroso-N-Acetilpenicilamina/farmacologia , Transfecção/métodos , ômega-N-Metilarginina/farmacologiaRESUMO
Guanylate cyclases constitute a gene family of enzymes that synthesize the second messenger guanosine 3',5'-cyclic monophosphate (cGMP) and play important roles in diverse physiological functions. Here we report a novel, simple and highly sensitive method for measurement intracellular cGMP concentrations using a cAMP-responsive element (CRE) and cGMP-dependent protein kinase (cGK). Transient transfection of the CRE reporter plasmid, encoding a luciferase reporter gene under the control of a modified promoter containing a CRE, and a cGK expression vector into HEK293 cells followed by treatment with 8-bromo-cGMP showed a dose dependent increase in luciferase activity. Moreover, HEK293 cells expressing GC-A or GC-B natriuretic peptide receptors and harboring this reporter system responded to specific ligands in a dose dependent manner. Our results indicate that this reporter gene method enables high throughput screening of receptor-type GC selective agonists in the treatment of cardiovascular diseases and homeostatic dysfunctions.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/biossíntese , GMP Cíclico/análise , Receptores Acoplados a Guanilato Ciclase/agonistas , GMP Cíclico/análogos & derivados , GMP Cíclico/biossíntese , GMP Cíclico/genética , GMP Cíclico/farmacologia , Proteína Quinase Dependente de GMP Cíclico Tipo I , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/genética , Avaliação Pré-Clínica de Medicamentos , Genes Reporter/efeitos dos fármacos , Células HEK293 , Humanos , Elementos de Resposta , Transdução de SinaisRESUMO
BACKGROUND: Omega-3 polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid) from fish oil ameliorate cardiovascular diseases. However, little is known about the effects of ω-3 polyunsaturated fatty acids on cardiac fibrosis, a major cause of diastolic dysfunction and heart failure. The present study assessed the effects of ω-3 polyunsaturated fatty acids on cardiac fibrosis. METHODS AND RESULTS: We assessed left ventricular fibrosis and pathology in mice subjected to transverse aortic constriction after the consumption of a fish oil or a control diet. In control mice, 4 weeks of transverse aortic constriction induced significant cardiac dysfunction, cardiac fibrosis, and cardiac fibroblast activation (proliferation and transformation into myofibroblasts). Dietary supplementation with fish oil prevented transverse aortic constriction-induced cardiac dysfunction and cardiac fibrosis and blocked cardiac fibroblast activation. In heart tissue, transverse aortic constriction increased active transforming growth factor-ß1 levels and phosphorylation of Smad2. In isolated adult mouse cardiac fibroblasts, transforming growth factor-ß1 induced cardiac fibroblast transformation, proliferation, and collagen synthesis. Eicosapentaenoic acid and docosahexaenoic acid increased cyclic GMP levels and blocked cardiac fibroblast transformation, proliferation, and collagen synthesis. Eicosapentaenoic acid and docosahexaenoic acid blocked phospho-Smad2/3 nuclear translocation. DT3, a protein kinase G inhibitor, blocked the antifibrotic effects of eicosapentaenoic acid and docosahexaenoic acid. Eicosapentaenoic acid and docosahexaenoic acid increased phosphorylated endothelial nitric oxide synthase and endothelial nitric oxide synthase protein levels and nitric oxide production. CONCLUSION: Omega-3 fatty acids prevent cardiac fibrosis and cardiac dysfunction by blocking transforming growth factor-ß1-induced phospho-Smad2/3 nuclear translocation through activation of the cyclic GMP/protein kinase G pathway in cardiac fibroblasts.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Suplementos Nutricionais , Ácidos Graxos Ômega-3/administração & dosagem , Ventrículos do Coração/patologia , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/prevenção & controle , Animais , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo III/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima , Remodelação Ventricular , Vitamina E/análogos & derivados , Vitamina E/metabolismoRESUMO
Previous research has found that hyperbaric oxygen (HBO(2)) produces an acute antinociceptive effect that is dependent on nitric oxide (NO). The present study was undertaken to determine whether HBO(2)-induced acute antinociception might involve a NO-cyclic GMP-protein kinase G-ATP-sensitive potassium (K(ATP)) channel pathway. Male NIH Swiss mice were subjected to a 5-min HBO(2) treatment (100% oxygen at 3.5 absolute atmospheres) and antinociception was assessed over the next 6 min still under HBO(2) using the acetic acid abdominal constriction test. Pretreatment with 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (carboxy-PTIO, an NO scavenger), 1H-[1,2,4]-oxadiazolo-[4,3-a]quinoxalin-1-one) (a soluble guanylyl cyclase-inhibitor, Rp-8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate (a protein kinase G-inhibitor) or glibenclamide (an ATP-sensitive potassium channel-inhibitor) all led to antagonism of the HBO(2)-induced acute antinociception in a dose-dependent manner. These findings suggest that HBO(2)-induced acute antinociception might be due to activation of a NO-cyclic GMP-protein kinase G-K(ATP) channel pathway.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Oxigenoterapia Hiperbárica , Canais KATP/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Óxido Nítrico/metabolismo , Dor/metabolismo , Ácido Acético , Animais , Benzoatos/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Glibureto/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Imidazóis/farmacologia , Canais KATP/antagonistas & inibidores , Masculino , Camundongos , Modelos Animais , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Guanilil Ciclase Solúvel , Resultado do TratamentoRESUMO
Recently studies have demonstrated that low doses of (Mn(+2)) in the form of manganese chloride can stimulate specific puberty-related hormones and advance signs of pubertal development in immature female and male rats. In the present study, we used an in vitro system to evaluate the ability of 0, 50, 250, and 500 microM doses of Mn(+2) to stimulate luteinizing hormone-releasing hormone (LHRH) secretion and to assess the hypothalamic mechanism of this action in adult male Sprague-Dawley rats. We demonstrated that Mn(+2) at 500 microM, but not the lower doses, increased LHRH release, nitric oxide (NO) synthase (NOS) activity, and the content of cyclic cGMP in the medial basal hypothalamus. Inhibition of NOS with a competitive inhibitor (Nomega-nitro-L-arginine methyl ester hydrochloride) prevented the Mn-induced increase in LHRH release. Additionally, methylene blue and KT5823, specific inhibitors of guanylyl cyclase and protein kinase G (PKG), respectively, also blocked the stimulatory effect of Mn(+2) on LHRH release. These in vitro studies demonstrated that the hypothalamic mechanism of Mn(+2) action in adult males is by activation of the NOS/NO system, resulting in increases in cGMP and PKG and thus the secretion of LHRH from the nerve terminals. These results indicate Mn(+2) can cause LHRH release in adult males, and this action is discussed in relation to age, gender, as well as mechanistic and functional differences between adult and immature animals.
Assuntos
Cloretos/toxicidade , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores Etários , Animais , Carbazóis/farmacologia , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Feminino , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/metabolismo , Hemoglobinas/metabolismo , Hipotálamo/metabolismo , Técnicas In Vitro , Indóis/farmacologia , Masculino , Compostos de Manganês , Azul de Metileno/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Fatores SexuaisRESUMO
We have shown recently that Mn2+ stimulates gonadotropin secretion via an action at the hypothalamic level, and a diet supplemented with a low dose of the element is capable of advancing the time of female puberty. In this study, we used an in vitro approach to investigate the mechanism by which Mn2+ induces luteinizing hormone-releasing hormone (LHRH) secretion from prepubertal female rats. The medial basal hypothalamus from 30-day-old rats was incubated in Locke solution for 30 min to assess basal LHRH secretion, then incubated with buffer alone or buffer plus either a nitric oxide synthase (NOS) inhibitor (N-monomethyl-L-arginine (NMMA); 300 or 500 microM) or a soluble guanylyl cyclase (sGC) inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ); 100 or 250 microM) for another 30 min. Finally, the incubation continued for a further 30 min, but in the presence of MnCl2 (50 or 250 microM) to assess the effect of the blockers on stimulated LHRH secretion. Both 50 and 250 microM MnCl2 stimulated LHRH release (P < 0.05 and P < 0.01, respectively). The addition of 300-500 microM NMMA to the medium did not block Mn2+-stimulated release of LHRH, even with the higher dose of MnCl2. Furthermore, while 50, 100 and 250 microM MnCl2 all significantly induced LHRH release, the two lowest doses did not stimulate total nitrite released from the same tissue, an effect only observed with the highest dose. Taken together, these data suggest that Mn2+ is not an effective stimulator of NO. Conversely, inhibiting sGC with ODQ blocked the Mn2+-stimulated secretion of LHRH in a dose-dependent manner, indicating that GC is the site of action of Mn2+. Additionally, we showed that Mn2+ stimulated cGMP and LHRH from the same tissues, and that downstream blocking of protein kinase G formation with KT5823 (10 microM) inhibited Mn2+-induced LHRH release. These data demonstrate that the principal action of Mn2+ within the hypothalamus is to activate sGC directly and/or as a cofactor with available NO, hence generating cGMP and resulting in prepubertal LHRH release.
Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/metabolismo , Manganês/fisiologia , Transdução de Sinais/fisiologia , Animais , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Feminino , Guanilato Ciclase/antagonistas & inibidores , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Maturidade Sexual/fisiologiaRESUMO
Previous experiments demonstrated that intracerebroventricular infusion of the protein kinase G inhibitor KT5823 inhibits lordosis behavior in hormone-treated female rats. Present studies show that KT5823 attenuates lordosis in a dose-dependent manner when infused bilaterally into the ventromedial hypothalamus. Thus, activation of protein kinase G in the ventromedial hypothalamus is necessary for the expression of hormone-dependent lordosis behavior in female rats.
Assuntos
Alcaloides/administração & dosagem , Carbazóis , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos/administração & dosagem , Hipotálamo/efeitos dos fármacos , Hipotálamo/enzimologia , Indóis , Postura/fisiologia , Comportamento Sexual Animal/efeitos dos fármacos , Animais , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Feminino , Injeções Intraventriculares , Masculino , Atividade Motora/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
The present study examined the effect of hepatoma-associated antigen HAb18G (homologous to CD147) expression on the NO/cGMP-regulated Ca(2+) mobilization and metastatic process of human hepatoma cells. HAb18G/CD147 cDNA was transfected into human 7721 hepatoma cells to obtain a cell line stably expressing HAb18G/CD147, T7721, as demonstrated by Northern blot and immunocytochemical studies. 8-Bromo-cGMP (cGMP) inhibited the thapsigargin-induced Ca(2+) entry in a concentration-dependent manner in 7721 cells. The cGMP-induced inhibition was abolished by an inhibitor of protein kinase G, KT5823 (1 microm). However, expression of HAb18G/CD147 in T7721 cells decreased the inhibitory response to cGMP. A similar concentration-dependent inhibitory effect on the Ca(2+) entry was observed in 7721 cells in response to a NO donor, (+/-)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca(2+) entry was significantly reduced in HAb18G/CD147-expressing T7721 cells, indicating a role for HAb18G/CD147 in NO/cGMP-regulated Ca(2+) entry. Experiments investigating metastatic potentials demonstrated that HAb18G/CD147-expressing T7721 cells attached to the Matrigel-coated culture plates and invaded through Matrigel-coated permeable filters at the rate significantly greater than that observed in 7721 cells. Both the attachment and invasion rates could be suppressed by SNAP, and the inhibitory effect of SNAP could be reversed by NO inhibitor, N(G)-nitro-l-arginine methyl ester. The sensitivity of the attachment and invasion rates to cGMP was significantly reduced in T7721 cells as compared with 7721 cells when cells were pretreated with thapsigargin. The difference in the sensitivity between the two cells could be abolished by a Ca(2+) channel blocker, Ni(2+) (3 mm). These results suggest that HAb18G/CD147 enhances metastatic potentials in human hepatoma cells by disrupting the regulation of store-operated Ca(2+) entry by NO/cGMP.
Assuntos
Antígenos CD , Antígenos de Neoplasias , Antígenos de Superfície , Proteínas Aviárias , Proteínas Sanguíneas , Carbazóis , Carcinoma Hepatocelular/metabolismo , GMP Cíclico/análogos & derivados , Indóis , Neoplasias Hepáticas/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/fisiologia , Penicilamina/análogos & derivados , Alcaloides/farmacologia , Basigina , Northern Blotting , Cálcio/metabolismo , Adesão Celular , Linhagem Celular , Movimento Celular , Colágeno/química , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Inibidores Enzimáticos/farmacologia , Humanos , Imuno-Histoquímica , Laminina/química , NG-Nitroarginina Metil Éster/farmacologia , Metástase Neoplásica , Níquel/metabolismo , Óxido Nítrico/metabolismo , Penicilamina/farmacologia , Proteoglicanas/química , Transdução de Sinais , Tapsigargina/farmacologia , Fatores de Tempo , Transfecção , Células Tumorais CultivadasAssuntos
Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , GMP Cíclico/análogos & derivados , Óxido Nítrico/metabolismo , Transmissão Sináptica/fisiologia , Tálamo/fisiologia , Animais , Gatos , GMP Cíclico/fisiologia , Doadores de Óxido Nítrico/metabolismo , Ratos , Especificidade da Espécie , Tálamo/metabolismoRESUMO
Nitric oxide (NO) acts via soluble guanylyl cyclase to increase cyclic GMP (cGMP), which can regulate various targets including protein kinases. Western blotting showed that type II cGMP-dependent protein kinase (cGK II) is widely expressed in various brain regions, especially in the thalamus. In thalamic extracts, the phosphorylation of several proteins, including cGK II, was increased by exogenous NO or cGMP. In vivo pretreatment with a NO synthase inhibitor reduced the phosphorylation of cGK II, and this could be reversed by exogenous NO or cGMP. Conversely, brainstem electrical stimulation, which enhances thalamic NO release, caused a NO synthase-dependent increase in the phosphorylation of thalamic cGK II. These results indicate that endogenous NO regulates cGMP-dependent protein phosphorylation in the thalamus. The activation of cGKII by NO may play a role in thalamic mechanisms underlying arousal.