Nitric oxide, C-type natriuretic peptide and cGMP as regulators of endochondral ossification

Coordinated proliferation and differentiation of growth plate chondrocytes is required for endochondral bone growth, but the mechanisms and pathways that control these processes are not completely understood. Recent data demonstrate important roles for nitric oxide (NO) and C-type natriuretic peptide (CNP) in the regulation of cartilage development. Both NO and CNP stimulate the synthesis of cGMP and thus the activation of common downstream pathways. One of these downstream mediators, cGMP-dependent kinase II (cGKII), has itself been shown to be essential for normal endochondral bone formation. This review summarizes our knowledge of the roles and mechanisms of NO, CNP and cGKII signaling in cartilage and endochondral bone development.     

A heretical view on the role of NO and cGMP in vascular proliferative diseases

Endogenous nitric oxide (NO), and possibly NO-releasing drugs, can both inhibit and promote vascular proliferative disorders, such as atherosclerosis and restenosis. The cell types and signaling pathways that mediate these opposing effects are controversial. It is widely assumed that the NO-mediated synthesis of the second messenger cGMP and the activation of cGMP-dependent protein kinase type I (cGKI) inhibits the proliferation of vascular smooth muscle cells and, thus, vascular remodeling. However, recent data from transgenic mouse models challenge this view. Here, we propose that cGMP signaling through cGKI might promote vasculoproliferative processes and their clinical complications. This new concept has important implications for the use of cGMP-elevating drugs in humans and might help to identify novel therapeutic strategies for vascular proliferative diseases.     

Defective smooth muscle regulation in cGMP kinase I-deficient mice.

Regulation of smooth muscle contractility is essential for many important biological processes such as tissue perfusion, cardiovascular haemostasis and gastrointestinal motility. While an increase in calcium initiates smooth muscle contraction, relaxation can be induced by cGMP or cAMP. cGMP-dependent protein kinase I (cGKI) has been suggested as a major mediator of the relaxant effects of both nucleotides. To study the biological role of cGKI and its postulated cross-activation by cAMP, we inactivated the gene coding for cGKI in mice. Loss of cGKI abolishes nitric oxide (NO)/cGMP-dependent relaxation of smooth muscle, resulting in severe vascular and intestinal dysfunctions. However, cGKI-deficient smooth muscle responded normally to cAMP, indicating that cAMP and cGMP signal via independent pathways, with cGKI being the specific mediator of the NO/cGMP effects in murine smooth muscle.     

Atrial natriuretic peptide-initiated cGMP pathways regulate vasodilator-stimulated phosphoprotein phosphorylation and angiogenesis in vascular endothelium

Nitric oxide (NO)- and atrial natriuretic peptide (ANP)-initiated cGMP signaling cascades are important in the maintenance of cardiovascular homeostasis. The molecular signaling mechanisms downstream of cGMP are not well understood, however. We have used small interfering RNA (siRNA) approaches to specifically knock down a series of signaling proteins in bovine aortic endothelial cells, and we have combined biochemical analyses with physiological assays to investigate cGMP-mediated signal transduction pathways. Activation of particulate guanylate cyclase (GC-A) by ANP leads to a substantial, dose-dependent, rapid, and sustained increase in intracellular cGMP. In contrast, stimulation of soluble guanylate cyclase by NO yields only a weak and transient increase in cGMP. ANP-induced cGMP production is selectively suppressed by siRNA-mediated knockdown of GC-A. ANP greatly enhances the phosphorylation at Ser-239 of the vasodilator-stimulated phosphoprotein (VASP), a major substrate of cGMP-dependent protein kinase (PKG) that significantly influences actin dynamics. Moreover, the ANP-induced phosphorylation of VASP at Ser-239 is accompanied by increased actin stress fiber formation and enhanced endothelial tube formation. siRNA-mediated knockdown of GC-A, VASP, or PKG abolishes ANP-induced VASP Ser-239 phosphorylation, stress fiber formation, and endothelial tube formation. We have demonstrated similar findings in human umbilical vein endothelial cells, where ANP substantially enhances intracellular cGMP content, phosphorylation of VASP at Ser-239, and endothelial tube formation. Taken together, our findings suggest that ANP-mediated cGMP signal transduction pathways regulate PKG phosphorylation of VASP Ser-239 in endothelial cells, resulting in reorganization of the actin cytoskeleton and enhancement of angiogenesis.     

Nitric oxide signaling participates in norepinephrine-induced activity of neuronal intracellular survival pathways

Much evidence has gathered that nitric oxide (NO) signaling, via cGMP-dependent mechanisms, may activate pro-survival pathways in hippocampal neurons and inhibit apoptosis. Past research has revealed that the enhancement of monoaminergic neurotransmission via exercise or treatment with antidepressant medications leads to an enhanced expression of brain-derived neurotrophic factor (BDNF). In isolated hippocampal neurons, norepinephrine (NE) application also increases the immunoreactivity of BDNF and several pro-survival signaling molecules. The data herein support the possibility that NO signaling plays an important role in enhancing neurotrophin expression and activation of the pro-survival phosphatidylinositol 3' kinase (PI-3K) pathway stimulated by NE. In isolated hippocampal neurons, the NO donor, sodium nitroprusside, increases BDNF, PI-3K, and phospho-ERK1 immunoreactivity. Specific inhibitors of the NO system suggest that NE-induced increases in hippocampal BDNF and the PI-3K pathway, but not stimulation of the MAPK pathway, depend upon NO signaling. In addition, inhibiting cGMP suggest that the effects of NE on BDNF immunoreactivity and Akt phosphorylation are also cGMP-dependent. Finally, the application of l-NAME to hippocampal neurons increases cell death. This is the first study of its kind demonstrating the involvement of NE-induced pro-survival signaling in three distinct signaling pathways: PI-3K, MAPK, and NO/cGMP. Possible mechanisms are discussed in light of the results.     

S-Nitrosothiols—NO donors regulating cardiovascular cell proliferation: Insight into intracellular pathway alterations

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) activates signaling pathways responsible for smooth muscle cell relaxation, leading to vasodilation and thus plays an important role in controlling vascular homeostasis, thrombosis and inflammation.Recent studies indicate that S-nitrosothiols produced in vivo as well as synthetic ones might be important reservoirs of NO. Based on a broad range of NO functions within the living organisms, this review highlights the impact of S-nitrosothiols on cardiovascular cell cycle. The cell membrane transport and the decomposition patterns responsible of S-nitrosothiols actions are presented. The effects of NO delivery through S-nitrosothiols have a significant potential in cardiovascular diseases with various underlying causes. The challenges related to their application in the pharmacotherapy of patients with various cardiovascular diseases are also discussed.     

Involvement of cyclic GMP and protein kinase G in the regulation of apoptosis and survival in neural cells

Our current understanding of nitric oxide (NO), cyclic GMP (cGMP) and protein kinase G (PKG) signaling pathways in the nervous systems has its origins in the early studies conducted on vascular tissues during the late 1970s and early to mid-1980s. The pioneering research into the NO/cGMP/PKG pathway in blood vessels conducted by the laboratories of Drs. Ferid Murad, Louis Ignarro and Robert Furchgott ultimately led to the awarding of the 1998 Nobel Prize in Physiology or Medicine to these three scientists. On the basis of further pioneering studies by Drs. John Garthwaite, Solomon Snyder, Steven Vincent and many other neuroscientists during the late 1980s and throughout the 1990s, it became recognized that NO serves as a neurotransmitter/neuromodulator in the central and peripheral nervous systems and that certain neural cells possess a cGMP signaling pathway similar to that in vascular smooth muscle cells. Although NO (at high concentrations) is toxic and thought to participate in neuronal cell death during stroke and neurodegenerative diseases (e.g. amyotrophic lateral sclerosis, Alzheimer's disease, HIV dementia and Parkinson's disease), recent evidence suggests that NO at low physiological concentrations can act as an antiapoptotic/prosurvival factor in certain neural cells (e.g. PC12 cells, motor neurons and neurons of dorsal root ganglia, hippocampus and sympathetic nerves). The antiapoptotic effects of NO are mediated, in part, by cGMP and a downstream target protein, PKG. Other cGMP-elevating factors (e.g. atrial and brain natriuretic peptides) and direct PKG activator (e.g. 8-bromo-cGMP) also have antiapoptotic effects which have been quantified by the new capillary electrophoresis with laser-induced fluorescence detector technology. Inhibition of soluble guanylyl cyclase and lowering of basal cGMP levels cause apoptosis in unstressed neural cells (NG108-15 and N1E-115 cells). The cGMP/PKG pathway appears to play an essential role in preventing activation of a proapoptotic pathway, thus promoting neural cell survival.     

Effect of JBP485 on obstructive jaundice is related to regulation of renal Oat1, Oat3 and Mrp2 expression in ANIT-treated rats

Pulmonary vascular endothelial nitric oxide (NO) synthase (eNOS)-derived NO is the major stimulant of cyclic guanosine 5'-monophosphate (cGMP) production and NO/cGMP-dependent vasorelaxation in the pulmonary circulation. We recently synthesized multiple peptides and reported that an eleven amino acid (SSWRRKRKESS) peptide (P1) but not scrambled P1 stimulated the catalytic activity but not expression of eNOS and causes NO/cGMP-dependent sustained vasorelaxation in isolated pulmonary artery (PA) segments and in lung perfusion models. Since cGMP levels can also be elevated by inhibition of phosphodiesterase type 5 (PDE-5), this study was designed to test the hypothesis that P1-mediated vesorelaxation is due to its unique dual action as NO-releasing PDE-5 inhibitor in the pulmonary circulation. Treatment of porcine PA endothelial cells (PAEC) with P1 caused time-dependent increase in intracellular NO release and inhibition of the catalytic activity of cGMP-specific PDE-5 but not PDE-5 protein expression leading to increased levels of cGMP. Acute hypoxia-induced PA vasoconstriction ex vivo and continuous telemetry monitoring of hypoxia (10% oxygen)-induced elevated PA pressure in freely moving rats were significantly restored by administration of P1. Chronic hypoxia (10% oxygen for 4 weeks)-induced alterations in PA perfusion pressure, right ventricular hypertrophy, and vascular remodeling were attenuated by P1 treatment. These results demonstrate the potential therapeutic effects of P1 to prevent and/or arrest the progression of hypoxia-induced PAH via NO/cGMP-dependent modulation of hemodynamic and vascular remodeling in the pulmonary circulation.     

Implication of microRNAs in atrial natriuretic peptide and nitric oxide signaling in vascular smooth muscle cells

MicroRNAs (miRs) are endogenous small RNA molecules that suppress gene expression by binding to complementary sequences in the 3' untranslated regions of their target genes. miRs have been implicated in many diseases, including heart failure, ischemic heart disease, hypertension, cardiac hypertrophy, and cancers. Nitric oxide (NO) and atrial natriuretic peptide (ANP) are potent vasorelaxants whose actions are mediated through receptor guanylyl cyclases and cGMP-dependent protein kinase. The present study examines miRs in signaling by ANP and NO in vascular smooth muscle cells. miR microarray analysis was performed on human vascular smooth muscle cells (HVSMC) treated with ANP (10 nM, 4 h) and S-nitroso-N-acetylpenicillamine (SNAP) (100 μM, 4 h), a NO donor. Twenty-two shared miRs were upregulated, and 21 shared miRs were downregulated, by both ANP and SNAP (P < 0.05). Expression levels of four miRs (miRs-21, -26b, -98, and -1826), which had the greatest change in expression, as determined by microarray analysis, were confirmed by quantitative RT-PCR. Rp-8-Br-PET-cGMPS, a cGMP-dependent protein kinase-specific inhibitor, blocked the regulation of these miRs by ANP and SNAP. 8-bromo-cGMP mimicked the effect of ANP and SNAP on their expression. miR-21 was shown to inhibit HVSMC contraction in collagen gel lattice contraction assays. We also identified by computational algorithms and confirmed by Western blot analysis new intracellular targets of miR-21, i.e., cofilin-2 and myosin phosphatase and Rho interacting protein. Transfection with pre-miR-21 contracted cells and ANP and SNAP blocked miR-21-induced HVSMC contraction. Transfection with anti-miR-21 inhibitor reduced contractility of HVSMC (P < 0.05). The present results implicate miRs in NO and ANP signaling in general and miR-21 in particular in cGMP signaling and vascular smooth muscle cell relaxation.     

Defects in cGMP-PKG pathway contribute to impaired NO-dependent responses in hepatic stellate cells upon activation

NO antagonizes hepatic stellate cell (HSC) contraction, although activated HSC in cirrhosis demonstrate impaired responses to NO. Decreased NO responses in activated HSC and mechanisms by which NO affects activated HSC remain incompletely understood. In normal rat HSC, the NO donor diethylamine NONOate (DEAN) significantly increased cGMP production and reduced serum-induced contraction by 25%. The guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) abolished 50% of DEAN effects, whereas the cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) reiterated half the observed DEAN response, suggesting both cGMP-dependent protein kinase G (PKG)-dependent and -independent mechanisms of NO-mediated antagonism of normal HSC contraction. However, NO donors did not increase cGMP production from in vivo activated HSC from bile duct-ligated rats and showed alterations in intracellular Ca(2+) accumulation suggesting defective cGMP-dependent effector pathways. The LX-2 cell line also demonstrated lack of cGMP generation in response to NO and a lack of effect of ODQ and 8-BrcGMP in modulating the NO response. However, cGMP-independent effects in response to NO were maintained in LX-2 and were associated with S-nitrosylation of proteins, an effect reiterated in primary HSC. Adenovirus-based overexpression of PKG significantly attenuated contraction of LX-2 by 25% in response to 8-BrcGMP. In summary, these studies demonstrate that NO affects HSC through cGMP-dependent and -independent pathways. The HSC activation process is associated with maintenance of cGMP-independent actions of NO but defects in cGMP-PKG-dependent NO signaling that are improved by PKG gene delivery in LX-2 cells. Activating targets downstream from NO-cGMP in activated HSC may represent a novel therapeutic target for portal hypertension.     

Mathematical modeling of the nitric oxide/cGMP pathway in the vascular smooth muscle cell

The nitric oxide (NO)/cGMP pathway in the vascular smooth muscle cell (VSMC) is an important cellular signaling system for the regulation of VSMC relaxation. We present a mathematical model to investigate the underlying mechanisms of this pathway. The model describes the flow of NO-driven signal transduction: NO activation of soluble guanylate cyclase (sGC), sGC- and phosphodiesterase-catalyzed cGMP production and degradation, cGMP-mediated regulation of protein targets including the Ca2+-activated K+ (KCa) channel, and the myosin contractile system. Model simulations reproduce major NO/cGMP-induced VSMC relaxation effects, including intracellular Ca2+ concentration reduction and Ca2+ desensitization of myosin phosphorylation and force generation. Using the model, we examine several testable principles. 1) Rapid sGC desensitization is caused by end-product cGMP feedback inhibition; a large fraction of the steady-state sGC population is in an inactivated intermediate state, and cGMP production is limited well below maximum. 2) NO activates the K(Ca) channel with both cGMP-dependent and -independent mechanisms; moderate NO concentration affects the K(Ca) via the cGMP-dependent pathway, whereas higher NO concentration is accommodated by a cGMP-independent mechanism. 3) Chronic NO synthase inhibition may cause underexpressions of K+ channels including inward rectifier and K(Ca) channels. 4) Ca2+ desensitization of the contractile system is distinguished from Ca2+ sensitivity of myosin phosphorylation. The model integrates these interactions among the heterogeneous components of the NO signaling system and can serve as a general modeling framework for studying NO-mediated VSMC relaxation under various physiological and pathological conditions. New data can be readily incorporated into this framework for interpretation and possible modification and improvement of the model.     

Adaptation of teleosts to very high salinity

Hydrogen sulfide (H(2)S), nitric oxide (NO) and nitrite (NO(2)(-)) are formed in vivo and are of crucial importance in the tissue response to hypoxia, particularly in the cardiovascular system, where these signaling molecules are involved in a multitude of processes including the regulation of vascular tone, cellular metabolic function and cytoprotection. This report summarizes current advances on the mechanisms by which these signaling pathways act and may have evolved in animals with different tolerance to hypoxia, as presented and discussed during the scientific sessions of the annual meeting of the Society for Experimental Biology in 2011 in Glasgow. It also highlights the need and potential for a comparative approach of study and collaborative effort to identify potential link(s) between the signaling pathways involving NO, nitrite and H(2)S in the whole-body responses to hypoxia.     

IRAG and novel PKG targeting in the cardiovascular system

Signaling by nitric oxide (NO) determines several cardiovascular functions including blood pressure regulation, cardiac and smooth muscle hypertrophy, and platelet function. NO stimulates the synthesis of cGMP by soluble guanylyl cyclases and thereby activates cGMP-dependent protein kinases (PKGs), mediating most of the cGMP functions. Hence, an elucidation of the PKG signaling cascade is essential for the understanding of the (patho)physiological aspects of NO. Several PKG signaling pathways were identified, meanwhile regulating the intracellular calcium concentration, mediating calcium desensitization or cytoskeletal rearrangement. During the last decade it emerged that the inositol trisphosphate receptor-associated cGMP-kinase substrate (IRAG), an endoplasmic reticulum-anchored 125-kDa membrane protein, is a main signal transducer of PKG activity in the cardiovascular system. IRAG interacts specifically in a trimeric complex with the PKG1β isoform and the inositol 1,4,5-trisphosphate receptor I and, upon phosphorylation, reduces the intracellular calcium release from the intracellular stores. IRAG motifs for phosphorylation and for targeting to PKG1β and 1,4,5-trisphosphate receptor I were identified by several approaches. The (patho)physiological functions for the regulation of smooth muscle contractility and the inhibition of platelet activation were perceived. In this review, the IRAG recognition, targeting, and function are summarized compared with PKG and several PKG substrates in the cardiovascular system.     

Heterogeneous nuclear ribonucleoprotein A1 is a novel cellular target of atrial natriuretic peptide signaling in renal epithelial cells

Two classes of guanylyl cyclases (GC) form intracellular cGMP. One is a receptor for atrial natriuretic peptide (ANP) and the other for nitric oxide (NO). The ANP receptor guanylyl cyclase (GC-A) is a membrane-bound, single subunit protein. Nitric oxide activated or soluble guanylyl cyclases (NOGC) are heme-containing heterodimers. These have been shown to be important in cGMP mediated regulation of arterial vascular resistance and renal sodium transport. Recent studies have shown that cGMP produced by both GCs is compartmentalized in the heart and vascular smooth muscle cells. To date, however, how intracellular cGMP generated by ANP and NO is compartmentalized and how it triggers specific downstream targets in kidney cells has not been investigated. Our studies show that intracellular cGMP formed by NO is targeted to cytosolic and cytoskeletal compartments whereas cGMP formed by ANP is restricted to nuclear and membrane compartments. We used two dimensional difference in gel electrophoresis and MALDI-TOF/TOF to identify distinct sub-cellular targets that are specific to ANP and NO signaling in HK-2 cells. A nucleocytoplasmic shuttling protein, heterogeneous nuclear ribonucleo protein A1 (hnRNP A1) is preferentially phosphorylated by ANP/cGMP/cGK signaling. ANP stimulation of HK-2 cells leads to increased cGK activity in the nucleus and translocation of cGK and hnRNP A1 to the nucleus. Phosphodiestaerase-5 (PDE-5 inhibitor) sildenafil augmented ANP-mediated effects on hnRNPA1 phosphorylation, translocation to nucleus and nuclear cGK activity. Our results suggest that cGMP generated by ANP and SNAP is differentially compartmentalized, localized but not global changes in cGMP, perhaps at different sub-cellular fractions of the cell, may more closely correlate with their effects by preferential phosphorylation of cellular targets.     

Amino acids and gaseous signaling

Gases, such as nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (H₂S), and sulfur dioxide (SO₂) are known toxic pollutants in the air. However, they are now recognized as important signaling molecules synthesized in animals and humans from arginine, glycine (heme), and cysteine, respectively. At physiological levels, NO, CO, and SO₂ activate guanylyl cyclase to generate cGMP which elicits a variety of responses (including relaxation of vascular smooth muscle cells, hemodynamics, neurotransmission, and cell metabolism) via cGMP-dependent protein kinases. H₂S is also a crucial regulator of both neurological function and endothelium-dependent relaxation through cGMP-independent mechanisms involving stimulation of membrane K_ATP channels and intracellular cAMP signaling. Additionally, NO, CO, and H₂S confer cytoprotective and immunomodulatory effects. Moreover, NH₃ is a major product of amino acid catabolism and profoundly affects the function of neurons and the vasculature through glutamine-dependent inhibition of NO synthesis. Emerging evidence shows that amino acids are not only precursors for these endogenous gases, but are also regulators of their production in a cell-specific manner. Thus, recent advances on gaseous signaling have greatly expanded our basic knowledge of amino acid biochemistry and nutrition. These exciting discoveries will aid in the design of new nutritional and pharmacological means to prevent and treat major health problems related to developmental biology and nutrient metabolism, including intrauterine growth restriction, preterm birth, aging, neurological disorders, cancer, obesity, diabetes, and cardiovascular disease.     

The function of NO-sensitive guanylyl cyclase: What we can learn from genetic mouse models

The signaling molecule nitric oxide (NO) acts as physiological activator of NO-sensitive guanylyl cyclase (NO-GC) in the cardiovascular, gastrointestinal and nervous systems. Two isoforms of NO-GC are known to exist on the protein level. The enzyme is a heterodimer consisting of an alpha (α₁ or α₂) and a beta subunit (β₁). Strategies for the genomic deletion of either subunit have been developed in the recent years. Removal of one of the two isoforms by deletion of one of the α subunits allowed the investigation of the specific functions of the respective isoform. The deletion of the β₁ subunit led to complete knock-out thus completely disrupting the NO/cGMP signaling cascade. The phenotypes of these KO mice have corroborated the already known physiological importance of the NO/cGMP cascade e.g. in the regulation of blood pressure, platelet inhibition, interneuronal communication; yet, they have also given hints to novel functions and mechanisms. In addition, mice lacking both NO-GC isoforms permitted the investigation of possible cGMP-independent signaling pathways of NO. As cell- and tissue-specific knock-out models are beginning to emerge, a more detailed analysis of the importance of the NO receptor in specific tissues will become possible.     

Regulator of G-protein signaling-2 mediates vascular smooth muscle relaxation and blood pressure

Nitric oxide (NO) inhibits vascular contraction by activating cGMP-dependent protein kinase I-alpha (PKGI-alpha), which causes dephosphorylation of myosin light chain (MLC) and vascular smooth muscle relaxation. Here we show that PKGI-alpha attenuates signaling by the thrombin receptor protease-activated receptor-1 (PAR-1) through direct activation of regulator of G-protein signaling-2 (RGS-2). NO donors and cGMP cause cGMP-mediated inhibition of PAR-1 and membrane localization of RGS-2. PKGI-alpha binds directly to and phosphorylates RGS-2, which significantly increases GTPase activity of G(q), terminating PAR-1 signaling. Disruption of the RGS-2-PKGI-alpha interaction reverses inhibition of PAR-1 signaling by nitrovasodilators and cGMP. Rgs2-/- mice develop marked hypertension, and their blood vessels show enhanced contraction and decreased cGMP-mediated relaxation. Thus, PKGI-alpha binds to, phosphorylates and activates RGS-2, attenuating receptor-mediated vascular contraction. Our study shows that RGS-2 is required for normal vascular function and blood pressure and is a new drug development target for hypertension.     

Phosphodiesterase 5 Attenuates the Vasodilatory Response in Renovascular Hypertension

NO/cGMP signaling plays an important role in vascular relaxation and regulation of blood pressure. The key enzyme in the cascade, the NO-stimulated cGMP-forming guanylyl cyclase exists in two enzymatically indistinguishable isoforms (NO-GC1, NO-GC2) with NO-GC1 being the major NO-GC in the vasculature. Here, we studied the NO/cGMP pathway in renal resistance arteries of NO-GC1 KO mice and its role in renovascular hypertension induced by the 2-kidney-1-clip-operation (2K1C). In the NO-GC1 KOs, relaxation of renal vasculature as determined in isolated perfused kidneys was reduced in accordance with the marked reduction of cGMP-forming activity (80%). Noteworthy, increased eNOS-catalyzed NO formation was detected in kidneys of NO-GC1 KOs. Upon the 2K1C operation, NO-GC1 KO mice developed hypertension but the increase in blood pressures was not any higher than in WT. Conversely, operated WT mice showed a reduction of cGMP-dependent relaxation of renal vessels, which was not found in the NO-GC1 KOs. The reduced relaxation in operated WT mice was restored by sildenafil indicating that enhanced PDE5-catalyzed cGMP degradation most likely accounts for the attenuated vascular responsiveness. PDE5 activation depends on allosteric binding of cGMP. Because cGMP levels are lower, the 2K1C-induced vascular changes do not occur in the NO-GC1 KOs. In support of a higher PDE5 activity, sildenafil reduced blood pressure more efficiently in operated WT than NO-GC1 KO mice. All together our data suggest that within renovascular hypertension, cGMP-based PDE5 activation terminates NO/cGMP signaling thereby providing a new molecular basis for further pharmacological interventions.     

Nitric oxide attenuates endothelin-1-induced activation of ERK1/2, PKB, and Pyk2 in vascular smooth muscle cells by a cGMP-dependent pathway

Nitric oxide (NO), in addition to its vasodilator action, has also been shown to antagonize the mitogenic and hypertrophic responses of growth factors and vasoactive peptides such as endothelin-1 (ET-1) in vascular smooth muscle cells (VSMCs). However, the mechanism by which NO exerts its antimitogenic and antihypertrophic effect remains unknown. Therefore, the aim of this study was to determine whether NO generation would modify ET-1-induced signaling pathways involved in cellular growth, proliferation, and hypertrophy in A-10 VSMCs. Treatment of A-10 VSMCs with S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP), two NO donors, attenuated the ET-1-enhanced phosphorylation of several key components of growth-promoting and hypertrophic signaling pathways such as ERK1/2, PKB, and Pyk2. On the other hand, inhibition of the endogenous NO generation with N(G)-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, increased the ET-1-induced phosphorylation of these signaling components. Since NO mediates its effect principally through a cGMP-soluble guanylyl cyclase (sGC) pathway, we investigated the role of these molecules in NO action. 8-Bromoguanosine 3',5'-cyclic monophosphate, a nonmetabolizable and cell-permeant analog of cGMP, exhibited a effect similar to that of SNAP and SNP. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of sGC, reversed the inhibitory effect of NO on ET-1-induced responses. SNAP treatment also decreased the protein synthesis induced by ET-1. Together, these data demonstrate that NO, in a cGMP-dependent manner, attenuated ET-1-induced phosphorylation of ERK1/2, PKB, and Pyk2 and also antagonized the hypertrophic effects of ET-1. It may be suggested that NO-induced generation of cGMP contributes to the inhibition of ET-1-induced mitogenic and hypertrophic responses in VSMCs.