Compartmentalization of NO signaling cascade in skeletal muscles

Skeletal muscle functions regulated by NO are now firmly established. However, the literature on the compartmentalization of NO signaling in myocytes is highly controversial. To address this issue, we examined localization of enzymes engaged in L-arginine-NO-cGMP signaling in the rat quadriceps muscle. Employing immunocytochemical labeling complemented with tyramide signal amplification and electron microscopy, we found NO synthase expressed not only in the sarcolemma, but also along contractile fibers, in the sarcoplasmic reticulum and mitochondria. The expression pattern of NO synthase in myocytes showed striking parallels with the enzymes engaged in L-arginine-NO-cGMP signaling (arginase, phosphodiesterase, and soluble guanylyl cyclase). Our findings are indicative of an autocrine fashion of NO signaling in skeletal muscles at both cellular and subcellular levels, and challenge the notion that the NO generation is restricted to the sarcolemma.     

Nitric oxide modulates Gi-protein expression and adenylyl cyclase signaling in vascular smooth muscle cells

We have previously shown that treatment of rats with the nitric oxide (NO) synthase inhibitor N6-nitro-L-arginine methyl ester for 4 weeks resulted in the augmentation of blood pressure and enhanced levels of Gialpha proteins. The present studies were undertaken to investigate if NO can modulate the expression of Gi proteins and associated adenylyl cyclase signaling. A10 vascular smooth muscle cells (VSMC) and primary cultured cells from aorta of Sprague-Dawley rats were used for these studies. The cells were treated with S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) for 24 h and the expression of Gialpha proteins was determined by immunobloting techniques. Adenylyl cyclase activity was determined by measuring [32P]cAMP formation for [alpha-32P]ATP. Treatment of cells with SNAP (100 microM) or SNP (0.5 mM) decreased the expression of Gialpha-2 and Gialpha-3 by about 25-40% without affecting the levels of Gsalpha proteins. The decreased expression of Gialpha proteins was reflected in decreased Gi functions (receptor-independent and -dependent) as demonstrated by decreased or attenuated forskolin-stimulated adenylyl cyclase activity by GTPgammaS and inhibition of adenylyl cyclase activity by angiotensin II and C-ANP4-23, a ring-deleted analog of atrial natriuretic peptide (ANP) that specifically interacts with natriuretic peptide receptor-C (NPR-C) in SNAP-treated cells. The SNAP-induced decreased expression of Gialpha-2 and Gialpha-3 proteins was not blocked by 1H[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase, or KT5823, an inhibitor of protein kinase G, but was restored toward control levels by uric acid, a scavenger of peroxynitrite and Mn(111)tetralis (benzoic acid porphyrin) MnTBAP, a peroxynitrite scavenger and a superoxide dismutase mimetic agent that inhibits the production of peroxynitrite, suggesting that NO-mediated decreased expression of Gialpha protein was cGMP-independent and may be attributed to increased levels of peroxynitrite. In addition, Gsalpha-mediated stimulation of adenylyl cyclase by GTPgammaS, isoproterenol, and forskolin was significantly augmented in SNAP-treated cells. These results indicate that NO decreased the expression of Gialpha protein and associated functions in VSMC by cGMP-independent mechanisms. From these studies, it can be suggested that NO-induced decreased levels of Gi proteins and resultant increased levels of cAMP may be an additional mechanism through which NO regulates blood pressure.     

Paracrine role of soluble guanylate cyclase and type III nitric oxide synthase in ovine fetal pulmonary circulation: a double labeling immunohistochemical study

Endothelial nitric oxide synthase (eNOS) or NOS-III in the endothelium catalyzes production of nitric oxide (NO). Nitric oxide diffuses freely into vascular smooth muscle, where it activates soluble guanylate cyclase (sGC) to produce guanosine 3',5'-cyclic monophosphate (cGMP) and causes vasorelaxation. The NO/cGMP pathway is an important signaling pathway in the control of perinatal pulmonary circulation. An exact colocalization of NOS-III in the pulmonary endothelium and sGC in the vascular smooth muscle was demonstrated using a double immunolabeling technique. The sGC immunoreactivity was higher in resistant pulmonary vessels and veins than in conduit arteries, whereas NOS-III immunoreactivity was higher in conduit arteries than in veins. These results demonstrated anatomically in situ a paracrine role of NOS-III and sGC in the regulation of fetal pulmonary circulation and suggested a heterogeneous distribution of NOS-III and sGC within fetal ovine pulmonary vasculature. Our results provided an anatomic basis that supported previous functional studies on perinatal control of pulmonary circulation.     

The role of arterial smooth muscle in vasorelaxation

The concept of endothelium-derived relaxing factor (EDRF) implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium in response to vasorelaxants such as acetylcholine (ACh) acts on the underlying vascular smooth muscle cells (VSMC) inducing vascular relaxation. The EDRF concept was derived from experiments on denuded blood vessel strips and, in frames of this concept, VSMC were regarded as passive recipients of NO from endothelial cells. However, it was later found that VSMC express NOS by themselves, but the principal question remained unanswered, is the NO generation by VSMC physiologically relevant? We hypothesized that the destruction of the vascular wall anatomical integrity by rubbing off the endothelial layer might increase vascular superoxides that, in turn, reduced the NO bioactivity as a relaxing factor. To test our hypothesis, we examined ACh-induced vasorelaxation under protection against oxidative stress and found that superoxide scavengers restored vasodilatory responses to ACh in endothelium-deprived blood vessels. These findings imply that VSMC can release NO in amounts sufficient to account for the vasorelaxatory response and challenge the concept of the obligatory role of endothelial cells in the relaxation of arterial smooth muscle.     

Neuron-glia communication via nitric oxide is essential in establishing antennal-lobe structure in Manduca sexta.

Nitric oxide synthase recently has been shown to be present in olfactory receptor cells throughout development of the adult antennal (olfactory) lobe of the brain of the moth Manduca sexta. Here, we investigate the possible involvement of nitric oxide (NO) in antennal-lobe morphogenesis. Inhibition of NO signaling with a NO synthase inhibitor or a NO scavenger early in development results in abnormal antennal lobes in which neuropil-associated glia fail to migrate. A more subtle effect is seen in the arborization of dendrites of a serotonin-immunoreactive neuron, which grow beyond their normal range. The effects of NO signaling in these types of cells do not appear to be mediated by activation of soluble guanylyl cyclase to produce cGMP, as these cells do not exhibit cGMP immunoreactivity following NO stimulation and are not affected by infusion of a soluble guanylyl cyclase inhibitor. Treatment with Novobiocin, which blocks ADP-ribosylation of proteins, results in a phenotype similar to those seen with blockade of NO signaling. Thus, axons of olfactory receptor cells appear to trigger glial cell migration and limit arborization of serotonin-immunoreactive neurons via NO signaling. The NO effect may be mediated in part by ADP-ribosylation of target cell proteins.     

Regulation of cGMP-dependent protein kinase expression by soluble guanylyl cyclase in vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) undergo many phenotypic changes when placed in culture. Several studies have shown that the levels of expression of soluble guanylyl cyclase (sGC) or cGMP-dependent protein kinase (PKG) are altered in cultured VSMC. In this study the mechanisms involved in the coordinated expression of sGC and PKG were examined. Pro-inflammatory cytokines that increase the expression of type II NO synthase (inducible NO synthase, or iNOS) decreased PKG expression in freshly isolated, non-passaged bovine aortic SMC. However, in several passaged VSMC lines (i.e. bovine aortic SMC, human aortic SMC, and A7r5 cells), PKG protein expression was not suppressed by cytokines or NO. sGC was highly expressed in non-passaged bovine aortic SMC but not in passaged cell lines. Restoration of expression of sGC to passaged bovine SMC using adenovirus encoding the alpha1 and beta1 subunits of sGC restored the capacity of the cells to increase cGMP in response to NO. Furthermore, treatment of these sGC-transduced cells with NO donors for 48 h resulted in decreased PKG protein expression. In contrast, passaged rat aortic SMC expressed high levels of NO-responsive sGC but demonstrated reduced expression of PKG. Adenovirus-mediated expression of the PKG catalytically active domain in rat aortic SMC caused a reduction in the expression of sGC in these cells. These results suggest that there is a mechanism for the coordinated expression of sGC and PKG in VSMC and that prolonged activation of sGC down-regulates PKG expression. Likewise, the loss of PKG expression appears to increase sGC expression. These effects may be an adaptive mechanism allowing growth and survival of VSMC in vitro.     

Neuron–Glia Communication via Nitric Oxide Is Essential in Establishing Antennal-Lobe Structure in Manduca sexta

Nitric oxide synthase recently has been shown to be present in olfactory receptor cells throughout development of the adult antennal (olfactory) lobe of the brain of the moth Manduca sexta. Here, we investigate the possible involvement of nitric oxide (NO) in antennal-lobe morphogenesis. Inhibition of NO signaling with a NO synthase inhibitor or a NO scavenger early in development results in abnormal antennal lobes in which neuropil-associated glia fail to migrate. A more subtle effect is seen in the arborization of dendrites of a serotonin-immunoreactive neuron, which grow beyond their normal range. The effects of NO signaling in these types of cells do not appear to be mediated by activation of soluble guanylyl cyclase to produce cGMP, as these cells do not exhibit cGMP immunoreactivity following NO stimulation and are not affected by infusion of a soluble guanylyl cyclase inhibitor. Treatment with Novobiocin, which blocks ADP-ribosylation of proteins, results in a phenotype similar to those seen with blockade of NO signaling. Thus, axons of olfactory receptor cells appear to trigger glial cell migration and limit arborization of serotonin-immunoreactive neurons via NO signaling. The NO effect may be mediated in part by ADP-ribosylation of target cell proteins.     

Functional coupling of a Ca2+/calmodulin-dependent nitric oxide synthase and a soluble guanylyl cyclase in vertebrate photoreceptor cells.

Electrophysiological recordings on retinal rod cells, horizontal cells and on-bipolar cells indicate that exogenous nitric oxide (NO) has neuromodulatory effects in the vertebrate retina. We report here endogenous NO formation in mammalian photoreceptor cells. Photoreceptor NO synthase resembled the neuronal NOS type I from mammalian brain. NOS activity utilized the substrate L-arginine (Km = 4 microM) and the cofactors NADPH, FAD, FMN and tetrahydrobiopterin. The activity showed a complete dependence on the free calcium concentration ([Ca2+]) and was mediated by calmodulin. NO synthase activity was sufficient to activate an endogenous soluble guanylyl cyclase that copurified in photoreceptor preparations. This functional coupling was strictly controlled by the free [Ca2+] (EC50 = 0.84 microM). Activation of the soluble guanylyl cyclase by endogenous NO was up to 100% of the maximal activation of this enzyme observed with the exogenous NO donor compound sodium nitroprusside. This NO/cGMP pathway was predominantly localized in inner and not in outer segments of photoreceptors. Immunocytochemically, we localized NO synthase type I mainly in the ellipsoid region of the inner segments and a soluble guanylyl cyclase in cell bodies of cone photoreceptor cells. We conclude that in photoreceptors endogenous NO is functionally coupled to a soluble guanylyl cyclase and suggest that it has a neuromodulatory role in visual transduction and in synaptic transmission in the outer retina.     

Increased caveolae density and caveolin-1 expression accompany impaired NO-mediated vasorelaxation in diet-induced obesity

Diet-induced obesity induces changes in mechanisms that are essential for the regulation of normal artery function, and in particular the function of the vascular endothelium. Using a rodent model that reflects the characteristics of human dietary obesity, in the rat saphenous artery we have previously demonstrated that endothelium-dependent vasodilation shifts from an entirely nitric oxide (NO)-mediated mechanism to one involving upregulation of myoendothelial gap junctions and intermediate conductance calcium-activated potassium channel activity and expression. This study investigates the changes in NO-mediated mechanisms that accompany this shift. In saphenous arteries from controls fed a normal chow diet, acetylcholine-mediated endothelium-dependent vasodilation was blocked by NO synthase and soluble guanylyl cyclase inhibitors, but in equivalent arteries from obese animals sensitivity to these agents was reduced. The expression of endothelial NO synthase (eNOS) and caveolin-3 in rat saphenous arteries was unaffected by obesity, whilst that of caveolin-1 monomer and large oligomeric complexes of caveolins-1 and -2 were increased in membrane-enriched samples. The density of caveolae was increased at the membrane and cytoplasm of endothelial and smooth muscle cells of saphenous arteries from obese rats. Dissociation of eNOS from caveolin-1, as a prerequisite for activation of the enzyme, may be compromised and thereby impair NO-mediated vasodilation in the saphenous artery from diet-induced obese rats. Such altered signaling mechanisms in obesity-related vascular disease represent significant potential targets for therapeutic intervention.     

Vascular distribution of nitric oxide synthase and vasodilation in the Australian lungfish, Neoceratodus forsteri

The presence of nitric oxide synthase (NOS) and role of nitric oxide (NO) in vascular regulation was investigated in the Australian lungfish, Neoceratodus forsteri. No evidence was found for NOS in the endothelium of large and small blood vessels following processing for NADPH-diaphorase histochemistry. However, both NADPH-diaphorase histochemistry and neural NOS immunohistochemistry demonstrated a sparse network of nitrergic nerves in the dorsal aorta, hepatic artery, and branchial arteries, but there were no nitrergic nerves in small blood vessels in tissues. In contrast, nitrergic nerves were found in non-vascular tissues of the lung, gut and kidney. Dual-wire myography was used to determine if NO signalling occurred in the branchial artery of N. forsteri. Both SNP and SIN-1 had no effect on the pre-constricted branchial artery, but the particulate guanylyl cyclase (GC) activator, C-type natriuretic peptide, always caused vasodilation. Nicotine mediated a dilation that was not inhibited by the soluble GC inhibitor, ODQ, or the NOS inhibitor, L-NNA, but was blocked by the cyclooxygenase inhibitor, indomethacin. These data suggest that NO control of the branchial artery is lacking, but that prostaglandins could be endothelial relaxing factors in the vasculature of lungfish.     

Free nitric oxide diffusion in the bronchial microcirculation

Theoretical mass transfer rates and concentration distributions were determined for transient diffusion of free nitric oxide (NO) generated in vivo from vascular endothelial cells. Our analytical framework is typical of the bronchial circulation in the human pulmonary system but is applicable to the microvascular circulation in general. We characterized mass transfer rates in terms of the fractional mass flux across a boundary relative to the total endothelial NO production rate. NO concentration in the tissue surrounding blood vessels was expressed in terms of fractional soluble guanylate cyclase (sGC) activity. Our results suggest that endothelium-derived free NO is capable of vascular smooth muscle dilation despite its rapid consumption by hemoglobin in blood. An optimal blood vessel radius of 20 microm was estimated for NO signaling. We hypothesize intermittent generation of endothelial NO as a possible mechanism for sGC activation in vascular smooth muscle. This mechanism enhances the efficacy of NO-modulated vascular smooth muscle dilation while minimizing NO losses to blood and surrounding tissue.     

Superoxide determines nitric oxide uptake rate by vascular smooth muscle cells

Nitric oxide (NO) is generated in endothelial cells, which diffuses to vascular smooth muscle cells (SMCs), activates soluble guanylyl cyclase, and leads to blood vessel dilation. However, this scenario does not explain how SMCs are capable of competing with erythrocytic hemoglobin for NO in vivo. Here, we have developed a competition experiment to determine the NO uptake rate by SMCs and demonstrated that the SMC-NO uptake rate is positively dependent on intracellular superoxide levels. In addition, the superoxide-elicited NO influx is able to enhance cGMP production in SMCs. Our findings imply that vascular SMCs, in vivo, may use superoxide to compete with erythrocytic hemoglobin for NO and obtain the NO bioactivity.     

Immortalization of human melanocytes does not alter the de novo properties of nitric oxide to induce cell detachment from extracellular matrix components via cGMP

Nitric oxide (NO) is an important mediator in many (patho)physiological processes including inflammation and skin cancer. A key transducer in NO signaling is the soluble guanylyl cyclase (sGC) that catalyzes the formation of guanosine 3',5'-cyclic monophosphate (cGMP). The basic mechanism of NO-cGMP signaling in melanocytic cells is, however, not well elucidated. A setback for such studies is the limited availability of patient-derived melanocytes. Here, we report that immortalized human normal and vitiliginous cell lines generated via cell transfection with human papilloma virus 16 genes E6 and E7 express NO synthase and guanylyl cyclase isoforms and the multidrug resistance-associated proteins 4 and 5 as selective cGMP exporters. Donors of NO (e.g., the NONOate (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate (PAPA-NO) and reactive nitrogen oxygen species (RNOS) like 3-morpholino-sydnonimine (SIN-1) as a donor of peroxynitrite as well as YC-1 as a NO-independent sGC stimulator increased intracellular cGMP levels in immortalized melanocytes (up to eightfold over controls), indicating the expression of functional sGC in these cells. PAPA-NO and SIN-1 also reduced the attachment of immortalized melanocytes to extracellular matrix (ECM) components like fibronectin which was dependent on cellular melanin content and cGMP. Such effects on melanoma cells were positively related to metastatic potential and were cGMP independent. Intriguingly, nonpigmented metastatic melanoma cells were more sensitive to exogenous sources of RNOS than of NO. Thus, immortalized melanocytes can be used as a tool for further research on differences in cell signaling between the different melanocytic lineages in particular towards impairment of cell-ECM adhesion by NO or RNOS, which may be important in metastasis and vitiligo pathogenesis.     

Stimulatory roles of nitric-oxide synthase 3 and guanylyl cyclase in platelet activation

Nitric oxide (NO) stimulates soluble guanylyl cyclase and, thus, enhances cyclic guanosine monophosphate (cGMP) levels. It is a currently prevailing concept that NO inhibits platelet activation. This concept, however, does not fully explain why platelet agonists stimulate NO production. Here we show that a major platelet NO synthase (NOS) isoform, NOS3, plays a stimulatory role in platelet secretion and aggregation induced by low doses of platelet agonists. Furthermore, we show that NOS3 promotes thrombosis in vivo. The stimulatory role of NOS is mediated by soluble guanylyl cyclase and results from a cGMP-dependent stimulation of platelet granule secretion. These findings delineate a novel signaling pathway in which agonists sequentially activate NOS3, elevate cGMP, and induce platelet secretion and aggregation. Our data also suggest that NO plays a biphasic role in platelet activation, a stimulatory role at low NO concentrations and an inhibitory role at high NO concentrations.     

Can erythrocytes release biologically active NO?

Under physiological conditions, endothelial cells and the endothelial nitric oxide (NO) synthase (eNOS) are the main source of NO in the cardiovascular system. However, several other cell types have also been implicated in the NO-dependent regulation of cell function, including erythrocytes. NO derived from red blood cells has been proposed to regulate erythrocyte membrane fluidity, inhibit platelet activation and induce vasodilation in hypoxic areas, but these proposals are highly controversial. In the current issue of Cell Communication and Signaling, an elegant study by Gambaryan et al., assayed NO production by erythrocytes by monitoring the activation of the platelet intracellular NO receptor, soluble guanylyl cyclase, and its downstream kinase protein kinase G. After systematically testing different combinations of erythrocyte/platelet suspensions, the authors found no evidence for platelet soluble guanylyl cyclase/protein kinase G activation by erythrocytes and conclude that erythrocytes do not release biologically active NO to inhibit platelet activation.     

Responses of vascular smooth muscle cell to extracellular matrix degradation.

Vessels remodel to compensate for increases in blood flow/pressure. The chronic exposure of blood vessels to increased flow and circulatory redox-homocysteine may injure vascular endothelium and disrupt elastic laminae. In order to understand the role of extracellular matrix (ECM) degradation in vascular structure and function, we isolated human vascular smooth muscle cells (VSMC) from normal and injured coronary arteries. The apparently normal vessels were isolated from explanted human hearts. The vessels were injured by inserting a blade into the lumen of the vessel, which damages the inner elastic laminae in the vessel wall and polarizes the VSMC by producing a pseudopodial phenotypic shift in VSMC. This shift is characteristic of migratory, invasive, and contractile nature of VSMC. We measured extracellular matrix metalloproteinases (MMPs), tissue plasminogen activator (tPA), tissue inhibitor of metalloproteinase (TIMP), and collagen I expression in VSMC by specific substrate zymography and Northern blot analyses. The injured and elastin peptide, val-gly-val-ala-pro-gly, treated VSMC synthesized active MMPs and reduced expression of TIMP. The level of tPA and collagen type I was induced in the injured, invasive VSMC and in the val-gly-val-ala-pro-gly treated cells. To demonstrate the angiogenic role of elastin peptide to VSMC we performed in vitro organ culture with rings from normal coronary artery. After 3 days in culture the vascular rings in the collagen gel containing elastin peptide elaborated MMP activity and sprouted and grew. The results suggest that val-gly-val-ala-pro-gly peptide generated at the site of proteolysis during vascular injury may have angiogenic activity.     

Altered regulation of renal nitric oxide and atrial natriuretic peptide systems in angiotensin II-induced hypertension

The present study was aimed to determine whether there is an altered role of local nitric oxide (NO) and atrial natriuretic peptide (ANP) systems in the kidney in association with the angiotensin (Ang) II-induced hypertension. Male Sprague-Dawley rats were used. Ang II (100 ng·min?1·kg?1) was infused through entire time course. Thirteenth day after beginning the regimen, kidneys were taken. The protein expression of NO synthase (NOS) and nitrotyrosine was determined by semiquantitative immunoblotting. The mRNA expression of components of ANP system was determined by real-time polymerase chain reaction. The activities of soluble and particulate guanylyl cyclases were determined by the amount of cGMP generated in responses to sodium nitroprusside and ANP, respectively. There developed hypertension and decreased creatinine clearance in the experimental group. The protein expression of eNOS, nNOS and nitrotyrosine was increased in the cortex, while that of iNOS remained unaltered. The urinary excretion of NO increased in Ang II-induced hypertensive rats. The catalytic activity of soluble guanylyl cyclase was blunted in the glomerulus in Ang II-induced hypertensive rats. The mRNA expression of ANP was increased in Ang II-induced hypertensive rats. Neither the expression of NPR-A nor that of NPR-C was changed. The protein expression of neutral endopeptidase was decreased and the activity of particulate guanylyl cyclase was blunted in the glomerulus and papilla in Ang II-induced hypertensive rats. In conclusion, the synthesis of NO and ANP was increased in the kidney of Ang II-induced hypertension, while stimulated cGMP response was blunted. These results suggest desensitization of guanylyl cyclase in the kidney of Ang II-induced hypertensive rats, which may contribute to the associated renal vasoconstriction and hypertension.     

Paracrine neuroprotective effect of nitric oxide in the developing retina

The retina of newborn rats consists of the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL) containing amacrine cells and the neuroblastic layer (NBL). In retinal explants, the GCL enters cell death after sectioning of the optic nerve, whereas there is almost no cell death in the NBL. When protein synthesis is inhibited with anisomycin, cell death is blocked in the GCL and induced in the NBL. We tested the roles of nitric oxide (NO) on cell death in the retina in vitro. Either L-arginine, the substrate for NO synthase or the NO donor S:-nitroso-acetylpenicillamine (SNAP) blocked cell death induced by anisomycin in the NBL, but had no effect in the GCL. Sepiapterin, a precursor of the nitric oxide synthase (NOS)-cofactor tetrahydrobiopterin also had a protective effect against anisomycin. The use of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble form of guanylyl cyclase, showed that anti-apoptotic effect of SNAP is partially mediated by cGMP generated by activation of guanylyl cyclase. NADPH-diaphorase histochemistry stained cells only in the GCL and INL. Thus, the degenerative effect of anisomycin is observed within the NBL, whereas the localization of NOS is restricted to the GCL and INL. The protective effect of both the NO substrate and cofactor upon cell death induced by anisomycin in the NBL, indicates that NO produced by amacrine and ganglion cells is a paracrine modulator of cell death within the retinal tissue.