Hepatic cyclic GMP formation is regulated by similar factors that modulate activation of purified hepatic soluble guanylate cyclase.

Background: We have shown that inhibition of cyclic GMP-phosphodiesterase 5A (PDE5A) by sildenafil (SIL) blunts cardiomyocyte β-adrenergic stimulation, but this effect depends on the activity of endothelial nitric oxide synthase (eNOS) to generate a specific pool of cyclic GMP. PDE5A normally localizes at Z-bands in myocytes, but localization is more diffuse in cells with eNOS chronically inhibited. Here, we tested whether the influence of eNOS on PDE5A localization and anti-adrenergic action depends upon cyclic GMP. Methods and Results: Mouse in vivo hemodynamics were assessed by pressure-volume analysis. Isoproterenol (ISO: 20 ng/kg/min, iv ) stimulated contractility was inhibited by SIL (100 μg/kg/min, iv ), however this did not occur in mice given N w -nitro-L-arginine methyl ester (L-NAME: 1 mg/mL in drinking water for 1 week) to inhibit NOS. Myocytes transfected with an adenoviral vector encoding a fusion protein (PDE5A-DSred) in vivo were subsequently isolated and examined for PDE5A/α-actinin localization. Normal cells showed strong co-localization, whereas L-NAME-treated cells had diffuse PDE5A distribution. If L-NAME was stopped for 1-wk washout, SIL regained anti-adrenergic activity, and PDE5A z-band localization was restored. If L-NAME was continued but combined with Bay 41– 8543 (BAY: 30 mg/kg/day, po ), a soluble guanylate cyclase (sGC) activator, both PDE5A localization and SIL anti-adrenergic action were also restored. Chronic L-NAME suppressed phosphorylation of vasodilator-stimulated protein (VASP), a marker of protein kinase G (PKG) activity, in hearts acutely exposed to ISO+SIL. After L-NAME washout or L-NAME+BAY, VASP phosphorylation with ISO+SIL was restored. Conclusion: NOS-dependent modulation of both PDE5A sarcomere localization and anti-adrenergic activity depends upon sGC-derived cyclic GMP, and is linked to PKG activation. This suggests sGC activators may have synergistic effects with PDE5A inhibitors.

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Nω-Nitro-L-arginine: a potent inhibitor of the L-arginine-dependent soluble guanylate cyclase activation pathway in LLC-PK1 cells

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y90-114 ◽

1990 ◽

Vol 68 (6) ◽

pp. 749-751 ◽

Cited By ~ 18

Author(s):

Kunio Ishii ◽

James F. Kerwin Jr. ◽

Ferid Murad

Keyword(s):

Guanylate Cyclase ◽

Cyclic Gmp ◽

Soluble Guanylate Cyclase ◽

Potent Inhibitor ◽

Specific Inhibitor ◽

Cell Types ◽

Porcine Kidney ◽

Relaxing Factor ◽

Endothelium Derived Relaxing Factor ◽

Guanylate Cyclase Activation

Oxytocin increased cyclic GMP levels in LLC-PK1 porcine kidney epithelial cells through activation of soluble guanylate cyclase. NG-Monomethyl-L-arginine and Nω-nitro-L-arginine inhibited oxytocin (10 μM) induced cyclic GMP accumulation with IC50 values of 2.3 μM and 140 nM, respectively, and the inhibition was prevented with L-arginine. Both inhibitors at 100 μM lowered the basal levels of cyclic GMP, but did not affect those induced by 1 μM sodium nitroprusside and 100 nM atrial natriuretic factor. These data support our hypothesis that an endothelium-derived relaxing factor-like substance is formed as the endogenous activator of soluble guanylate cyclase in an L-arginine-dependent fashion in various cell types. Nω-Nitro-L-arginine is 16 times more potent than NG-monomethyl-L-arginine as a specific inhibitor of this pathway in LLC-PK1 cells.Key words: porcine kidney epithelial (LLC-PK1) cells, cyclic GMP, Nω-nitro-L-arginine, NG-monomethyl-L-arginine, endothelium-derived relaxing factor.

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Atrial natriuretic factor and sodium nitroprusside increase cyclic GMP in cultured rat lung fibroblasts by activating different forms of guanylate cyclase

Biochemical Journal ◽

10.1042/bj2440069 ◽

1987 ◽

Vol 244 (1) ◽

pp. 69-74 ◽

Cited By ~ 54

Author(s):

D C Leitman ◽

V L Agnost ◽

J J Tuan ◽

J W Andresen ◽

F Murad

Keyword(s):

Sodium Nitroprusside ◽

Guanylate Cyclase ◽

Cyclic Gmp ◽

Atrial Natriuretic Factor ◽

Soluble Guanylate Cyclase ◽

Fold Increase ◽

Lung Fibroblasts ◽

Rat Lung ◽

Natriuretic Factor ◽

Stimulation Of

We used cultured rat lung fibroblasts to evaluate the role of particulate and soluble guanylate cyclase in the atrial natriuretic factor (ANF)-induced stimulation of cyclic GMP. ANF receptors were identified by binding of 125I-ANF to confluent cells at 37 degrees C. Specific ANF binding was rapid and saturable with increasing concentrations of ANF. The equilibrium dissociation constant (KD) was 0.66 +/- 0.077 nM and the Bmax. was 216 +/- 33 fmol bound/10(6) cells, which corresponds to 130,000 +/- 20,000 sites/cell. The molecular characteristics of ANF binding sites were examined by affinity cross-linking of 125I-ANF to intact cells with disuccinimidyl suberate. ANF specifically labelled two sites with molecular sizes of 66 and 130 kDa, which we have identified in other cultured cells. ANF and sodium nitroprusside produced a time- and concentration-dependent increase in intracellular cyclic GMP. An increase in cyclic GMP by ANF was detected at 1 nM, and at 100 nM an approx. 100-fold increase in cyclic GMP was observed. Nitroprusside stimulated cyclic GMP at 10 nM and at 1 mM a 500-600-fold increase in cyclic GMP occurred. The simultaneous addition of 100 nM-ANF and 10 microM-nitroprusside to cells resulted in cyclic GMP levels that were additive. ANF increased the activity of particulate guanylate cyclase by about 10-fold, but had no effect on soluble guanylate cyclase. In contrast, nitroprusside did not alter the activity of particulate guanylate cyclase, but increased the activity of soluble guanylate cyclase by 17-fold. These results demonstrate that rat lung fibroblasts contain ANF receptors and suggest that the ANF-induced stimulation of cyclic GMP is mediated entirely by particulate guanylate cyclase.

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Endothelin-1 Stimulates Cyclic GMP Formation in Porcine Kidney Epithelial Cells via Activation of the L-Arginine-Dependent Soluble Guanylate Cyclase Pathway

Journal of Cardiovascular Pharmacology ◽

10.1097/00005344-199100177-00071 ◽

1991 ◽

Vol 17 ◽

pp. S246-250 ◽

Cited By ~ 4

Author(s):

Kunio Ishii ◽

Timothy D. Warner ◽

Hong Sheng ◽

Ferid Murad

Keyword(s):

Epithelial Cells ◽

Guanylate Cyclase ◽

Cyclic Gmp ◽

Soluble Guanylate Cyclase ◽

Porcine Kidney ◽

Endothelin 1 ◽

Kidney Epithelial Cells

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Soluble guanylate cyclase and nitric oxide synthase in synaptosomal fractions of bovine retina

Visual Neuroscience ◽

10.1017/s0952523898155098 ◽

1998 ◽

Vol 15 (5) ◽

pp. 867-873 ◽

Cited By ~ 4

Author(s):

ALEXANDER MARGULIS ◽

NIKOLAY POZDNYAKOV ◽

LOAN DANG ◽

ARI SITARAMAYYA

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Guanylate Cyclase ◽

Cyclic Gmp ◽

Soluble Guanylate Cyclase ◽

Cyclase Activity ◽

Inner Retina ◽

Synaptosomal Fraction ◽

Oxide Synthase ◽

Plexiform Layers

Cyclic GMP has been shown in recent years to directly activate ion channels in bipolar and ganglion cells, and to indirectly regulate coupling between horizontal cells, and between bipolar and amacrine cells. In all of these cases, the effects of cyclic GMP are mimicked by nitric oxide. An increase in calcium concentration stimulates the production of nitric oxide by neuronal and endothelial forms of nitric oxide synthase, which in turn activates soluble guanylate cyclases, enhancing the synthesis of cyclic GMP. Though some effects of nitric oxide do not involve cyclic GMP, the nitric oxide-cyclic GMP cascade is well recognized as a signaling mechanism in brain and other tissues. The widespread occurrence of nitric oxide/cyclic GMP-regulated ion channel activity in retinal neurons raises the possibility that nitric-oxide-sensitive soluble guanylate cyclases play an important role in cell–cell communication, and possibly, synaptic transmission. Immunohistochemical studies have indicated the presence of soluble guanylate cyclase in retinal synaptic layers, but such studies are not suitable for determination of the density or quantitative subcellular distribution of the enzyme. Microanalytical methods involving microdissection of frozen retina also showed the presence of cyclase activity in retinal plexiform layers but these methods did not permit distinction between nitric oxide-sensitive and insensitive cyclases. In this study, we fractionated retinal homogenate into the cytosolic and synaptosomal fractions and investigated the specific activity and distribution of soluble guanylate cyclase and nitric oxide synthase. The results show that both enzymes are present in the synaptosomal fractions derived from inner and outer plexiform layers. The synaptosomal fraction derived from inner retina was highly enriched in cyclase activity. Nitric oxide synthase activity was also higher in the inner than outer retinal synaptosomal fraction. The results suggest that the nitric oxide-cyclic GMP system is operational in both synaptic layers of retina and that it may play a more significant role in the inner retina.

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N-hydroxylamine is not an intermediate in the conversion of l-arginine to an activator of soluble guanylate cyclase in neuroblastoma N1E-115 cells

Biochemical Journal ◽

10.1042/bj2730547 ◽

1991 ◽

Vol 273 (3) ◽

pp. 547-552 ◽

Cited By ~ 6

Author(s):

S Pou ◽

W S Pou ◽

G M Rosen ◽

E E el-Fakahany

Keyword(s):

Guanylate Cyclase ◽

Cyclic Gmp ◽

Soluble Guanylate Cyclase ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Intact Cells ◽

Relaxing Factor ◽

Endothelium Derived Relaxing Factor ◽

Dose Dependent

This study evaluates the role of N-hydroxylamine (NH2OH) in activating soluble guanylate cyclase in the mouse neuroblastoma clone N1E-115. It has been proposed that NH2OH is a putative intermediate in the biochemical pathway for the generation of nitric oxide (NO)/endothelium-derived relaxing factor (EDRF) from L-arginine. NH2OH caused a time- and concentration-dependent increase in cyclic GMP formation in intact cells. This response was not dependent on Ca2+. In cytosol preparations the activation of guanylate cyclase by L-arginine was dose-dependent and required Ca2+ and NADPH. In contrast, NH2OH itself did not activate cytosolic guanylate cyclase but it inhibited the basal activity of this enzyme in a concentration-dependent manner. The formation of cyclic GMP in the cytosolic fractions in response to NH2OH required the addition of catalase and H2O2. On the other hand, catalase and/or H2O2 lead to a decrease in L-arginine-induced cyclic GMP formation. Furthermore, NH2OH inhibited L-arginine- and sodium nitroprusside-induced cyclic GMP formation in the cytosol. The inhibition of L-arginine-induced cyclic GMP formation in the cytosol by NH2OH was not reversed by the addition of superoxide dismutase. These data strongly suggest that NH2OH is not a putative intermediate in the metabolism of L-arginine to an activator of guanylate cyclase.

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