NO-sensitive Guanylyl Cyclase Research Articles

Role of the NO-GC/cGMP signaling pathway in platelet biomechanics

Cyclic guanosine monophosphate (cGMP) is a second messenger produced by the NO-sensitive guanylyl cyclase (NO-GC). The NO-GC/cGMP pathway in platelets has been extensively studied. However, its role in regulating the biomechanical properties of platelets has not yet been addressed and remains unknown. We therefore investigated the stiffness of living platelets after treatment with the NO-GC stimulator riociguat or the NO-GC activator cinaciguat using scanning ion conductance microscopy (SICM). Stimulation of human and murine platelets with cGMP-modulating drugs decreased cellular stiffness and downregulated P-selectin, a marker for platelet activation. We also quantified changes in platelet shape using deep learning-based platelet morphometry, finding that platelets become more circular upon treatment with cGMP-modulating drugs. To test for clinical applicability of NO-GC stimulators in the context of increased thrombogenicity risk, we investigated the effect of riociguat on platelets from human immunodeficiency virus (HIV)-positive patients taking abacavir sulfate (ABC)-containing regimens. Our results corroborate a functional role of the NO-GC/cGMP pathway in platelet biomechanics, indicating that biomechanical properties such as stiffness or shape could be used as novel biomarkers in clinical research.

Anti-Fibrotic and Anti-Inflammatory Role of NO-Sensitive Guanylyl Cyclase in Murine Lung.

Pulmonary fibrosis is a chronic and progressive disease with limited therapeutic options. Nitric oxide (NO) is suggested to reduce the progression of pulmonary fibrosis via NO-sensitive guanylyl cyclase (NO-GC). The exact effects of NO-GC during pulmonary fibrosis are still elusive. Here, we used a NO-GC knockout mouse (GCKO) and examined fibrosis and inflammation after bleomycin treatment. Compared to wildtype (WT), GCKO mice showed an increased fibrotic reaction, as myofibroblast occurrence (p = 0.0007), collagen content (p = 0.0006), and mortality (p = 0.0009) were significantly increased. After fibrosis induction, lymphocyte accumulations were observed in the lungs of GCKO but not in WT littermates. In addition, the total number of immune cells, specifically lymphocytes (p = <0.0001) and neutrophils (p = 0.0047), were significantly higher in the bronchoalveolar lavage fluid (BALF) of GCKO animals compared to WT, indicating an increased inflammatory response in the absence of NO-GC. The pronounced fibrotic response in GCKO mice was paralleled by significantly increased levels of transforming growth factor β (TGFβ) in BALF (p = 0.0207), which correlated with the total number of immune cells. Taken together, our data show the effect of NO-GC deletion in the pathology of lung fibrosis and the effect on immune cells in BALF. In summary, our results show that NO-GC has anti-inflammatory and anti-fibrotic properties in the murine lung, very likely by attenuating TGFβ-mediated effects.

NO-sensitive guanylyl cyclase discriminates pericyte-derived interstitial from intra-alveolar myofibroblasts in murine pulmonary fibrosis

BackgroundThe origin of αSMA-positive myofibroblasts, key players within organ fibrosis, is still not fully elucidated. Pericytes have been discussed as myofibroblast progenitors in several organs including the lung.MethodsUsing tamoxifen-inducible PDGFRβ-tdTomato mice (PDGFRβ-CreERT2; R26tdTomato) lineage of lung pericytes was traced. To induce lung fibrosis, a single orotracheal dose of bleomycin was given. Lung tissue was investigated by immunofluorescence analyses, hydroxyproline collagen assay and RT-qPCR.ResultsLineage tracing combined with immunofluorescence for nitric oxide-sensitive guanylyl cyclase (NO-GC) as marker for PDGFRβ-positive pericytes allows differentiating two types of αSMA-expressing myofibroblasts in murine pulmonary fibrosis: (1) interstitial myofibroblasts that localize in the alveolar wall, derive from PDGFRβ+ pericytes, express NO-GC and produce collagen 1. (2) intra-alveolar myofibroblasts which do not derive from pericytes (but express PDGFRβ de novo after injury), are negative for NO-GC, have a large multipolar shape and appear to spread over several alveoli within the injured areas. Moreover, NO-GC expression is reduced during fibrosis, i.e., after pericyte-to-myofibroblast transition.ConclusionIn summary, αSMA/PDGFRβ-positive myofibroblasts should not be addressed as a homogeneous target cell type within pulmonary fibrosis.

WED-536 - Deletion of no-sensitive guanylyl cyclase protects from metabolic obesity but not from fibrosis in murine non-alcoholic fatty liver disease

WED-536 - Deletion of no-sensitive guanylyl cyclase protects from metabolic obesity but not from fibrosis in murine non-alcoholic fatty liver disease

It takes two to tango: cardiac fibroblast-derived NO-induced cGMP enters cardiac myocytes and increases cAMP by inhibiting PDE3

The occurrence of NO/cGMP signalling in cardiac cells is a matter of debate. Recent measurements with a FRET-based cGMP indicator in isolated cardiac cells revealed NO-induced cGMP signals in cardiac fibroblasts while cardiomyocytes were devoid of these signals. In a fibroblast/myocyte co-culture model though, cGMP formed in fibroblasts in response to NO entered cardiomyocytes via gap junctions. Here, we demonstrate gap junction-mediated cGMP transfer from cardiac fibroblasts to myocytes in intact tissue. In living cardiac slices of mice with cardiomyocyte-specific expression of a FRET-based cGMP indicator (αMHC/cGi-500), NO-dependent cGMP signals were shown to occur in myocytes, to depend on gap junctions and to be degraded mainly by PDE3. Stimulation of NO-sensitive guanylyl cyclase enhanced Forskolin- and Isoproterenol-induced cAMP and phospholamban phosphorylation. Genetic inactivation of NO-GC in Tcf21-expressing cardiac fibroblasts abrogated the synergistic action of NO-GC stimulation on Iso-induced phospholamban phosphorylation, identifying fibroblasts as cGMP source and substantiating the necessity of cGMP-transfer to myocytes. In sum, NO-stimulated cGMP formed in cardiac fibroblasts enters cardiomyocytes in native tissue where it exerts an inhibitory effect on cAMP degradation by PDE3, thereby increasing cAMP and downstream effects in cardiomyocytes. Hence, enhancing β-receptor-induced contractile responses appears as one of NO/cGMP’s functions in the non-failing heart.

The differential roles of the two NO-GC isoforms in adjusting airway reactivity.

The enzyme, nitric oxide-sensitive guanylyl cyclase (NO-GC), is activated by binding NO to its prosthetic heme group and catalyzes the formation of cGMP. The NO-GC is primarily known to mediate vascular smooth muscle relaxation in the lung, and inhaled NO has been successfully used as a selective pulmonary vasodilator. In comparison, NO-GC's impact on the regulation of airway tone is less acknowledged and, most importantly, little is known about the issue that NO-GC signaling is accomplished by two isoforms: NO-GC1 and NO-GC2, implying the existence of distinct "cGMP pools." Herein, we investigated the functional role of the NO-GC isoforms in respiration by measuring lung function parameters of isoform-specific knockout (KO) mice using noninvasive and invasive techniques. Our data revealed the participation and ongoing influence of NO-GC1-derived cGMP in the regulation of airway tone by showing that respiratory resistance was enhanced in NO-GC1-KOs and increased more pronouncedly after the challenge with the bronchoconstrictor methacholine. The tissue resistance and stiffness of NO-GC1-KOs were also higher because of narrowed airways that cause tissue distortion. Contrariwise, NO-GC2-KOs displayed reduced tissue elasticity, elastic recoil, and airway reactivity to methacholine, which did not even increase in an ovalbumin model of asthma that induced hyperresponsiveness in NO-GC1-KOs. In addition, conscious NO-GC2-KOs showed a higher breathing rate with a shorter duration of inspiration and expiration time, which remained faster even in the presence of bronchoconstrictors that slow down breathing. Thus, we provide evidence of two distinct NO/cGMP pathways in airways, accomplished by either NO-GC1 or NO-GC2, adjusting differentially the airway reactivity.

PDGF regulates guanylate cyclase expression and cGMP signaling in vascular smooth muscle

The nitric oxide-cGMP (NO-cGMP) pathway is of outstanding importance for vascular homeostasis and has multiple beneficial effects in vascular disease. Neointimal hyperplasia after vascular injury is caused by increased proliferation and migration of vascular smooth muscle cells (VSMCs). However, the role of NO-cGMP signaling in human VSMCs in this process is still not fully understood. Here, we investigate the interaction between platelet derived growth factor (PDGF)-signaling, one of the major contributors to neointimal hyperplasia, and the cGMP pathway in vascular smooth muscle, focusing on NO-sensitive soluble guanylyl cyclase (sGC). We show that PDGF reduces sGC expression by activating PI3K and Rac1, which in turn alters Notch ligand signaling. These data are corroborated by gene expression analysis in human atheromas, as well as immunohistological analysis of diseased and injured arteries. Collectively, our data identify the crosstalk between PDGF and NO/sGC signaling pathway in human VSMCs as a potential target to tackle neointimal hyperplasia.

Hippocampal AMPA- and NMDA-induced cGMP signals are mainly generated by NO-GC2 and are under tight control by PDEs 1 and 2.

In the central nervous system, the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signalling cascade has an established role in fine-tuning of synaptic transmission. In the present study, we asked which isoform of NO-sensitive guanylyl cyclase, NO-GC1 or NO-GC2, is responsible for generation of N-methyl-d-aspartate (NMDA)- and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-induced cGMP signals and which of the phosphodiesterases (PDEs) is responsible for degradation. To this end, we performed live cell fluorescence measurements of primary hippocampal neurons isolated from NO-GC isoform-deficient mice. Although both isoforms contributed to the NMDA- and AMPA-induced cGMP signals, NO-GC2 clearly played the predominant role. Whereas under PDE-inhibiting conditions the cGMP levels elicited by both glutamatergic ligands were comparable, NMDA-induced cGMP signals were clearly higher than the AMPA-induced ones in the absence of PDE inhibitors. Thus, AMPA-induced cGMP signals are more tightly controlled by PDE-mediated degradation than NMDA-induced signals. In addition, these findings are compatible with the existence of at least two different pools of cGMP in both of which PDE1 and PDE2-known to be highly expressed in the hippocampus-are mainly responsible for cGMP degradation. The finding that distinct pools of cGMP are equipped with different amounts of PDEs highlights the importance of PDEs for the shape of NO-induced cGMP signals in the central nervous system.

Rise of cGMP by partial phosphodiesterase-3A degradation enhances cardioprotection during hypoxia

3′,5′-cyclic guanosine monophosphate (cGMP) is a druggable second messenger regulating cell growth and survival in a plethora of cells and disease states, many of which are associated with hypoxia. For example, in myocardial infarction and heart failure (HF), clinical use of cGMP-elevating drugs improves disease outcomes. Although they protect mice from ischemia/reperfusion (I/R) injury, the exact mechanism how cardiac cGMP signaling is regulated in response to hypoxia is still largely unknown. By monitoring real-time cGMP dynamics in murine and human cardiomyocytes using in vitro and in vivo models of hypoxia/reoxygenation (H/R) and I/R injury combined with biochemical methods, we show that hypoxia causes rapid but partial degradation of cGMP-hydrolyzing phosphodiesterase-3A (PDE3A) protein via the autophagosomal-lysosomal pathway. While increasing cGMP in hypoxia prevents cell death, partially reduced PDE3A does not change the pro-apoptotic second messenger 3′,5′-cyclic adenosine monophosphate (cAMP). However, it leads to significantly enhanced protective effects of clinically relevant activators of nitric oxide-sensitive guanylyl cyclase (NO-GC). Collectively, our mouse and human data unravel a new mechanism by which cardiac cGMP improves hypoxia-associated disease conditions.

Cyclic GMP-Dependent Regulation of Vascular Tone and Blood Pressure Involves Cysteine-Rich LIM-Only Protein 4 (CRP4).

The cysteine-rich LIM-only protein 4 (CRP4), a LIM-domain and zinc finger containing adapter protein, has been implicated as a downstream effector of the second messenger 3′,5′-cyclic guanosine monophosphate (cGMP) pathway in multiple cell types, including vascular smooth muscle cells (VSMCs). VSMCs and nitric oxide (NO)-induced cGMP signaling through cGMP-dependent protein kinase type I (cGKI) play fundamental roles in the physiological regulation of vascular tone and arterial blood pressure (BP). However, it remains unclear whether the vasorelaxant actions attributed to the NO/cGMP axis require CRP4. This study uses mice with a targeted deletion of the CRP4 gene (CRP4 KO) to elucidate whether cGMP-elevating agents, which are well known for their vasorelaxant properties, affect vessel tone, and thus, BP through CRP4. Cinaciguat, a NO- and heme-independent activator of the NO-sensitive (soluble) guanylyl cyclase (NO-GC) and NO-releasing agents, relaxed both CRP4-proficient and -deficient aortic ring segments pre-contracted with prostaglandin F2α. However, the magnitude of relaxation was slightly, but significantly, increased in vessels lacking CRP4. Accordingly, CRP4 KO mice presented with hypotonia at baseline, as well as a greater drop in systolic BP in response to the acute administration of cinaciguat, sodium nitroprusside, and carbachol. Mechanistically, loss of CRP4 in VSMCs reduced the Ca2+-sensitivity of the contractile apparatus, possibly involving regulatory proteins, such as myosin phosphatase targeting subunit 1 (MYPT1) and the regulatory light chain of myosin (RLC). In conclusion, the present findings confirm that the adapter protein CRP4 interacts with the NO-GC/cGMP/cGKI pathway in the vasculature. CRP4 seems to be part of a negative feedback loop that eventually fine-tunes the NO-GC/cGMP axis in VSMCs to increase myofilament Ca2+ desensitization and thereby the maximal vasorelaxant effects attained by (selected) cGMP-elevating agents.

NO‐sensitive guanylyl cyclase in the lung

In the late 1960s, several labatories identified guanylyl cyclase (GC) as the cGMP-producing enzyme. Subsequently, two different types of GC were described that differed in their cellular localization. Primarily found in the cytosol, nitric oxide (NO)-sensitive guanylyl cyclase (NO-GC) acts as receptor for the signalling molecule NO, in contrast the membrane-bound isoenzyme is activated by natriuretic peptides. The lung compared with other tissues exhibits the highest expression of NO-GC. The enzyme has been purified from lung for biochemical analysis. Although expressed in smooth muscle cells (SMCs) and in pericytes, the function of NO-GC in lung, especially in pericytes, is still not fully elucidated. However, pharmacological compounds that target NO-GC are available and have been implemented for the therapy of pulmonary arterial hypertension. In addition, NO-GC has been suggested as drug target for the therapy of asthma, acute respiratory distress syndrome and pulmonary fibrosis. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Angiotensin II-Induced Cardiovascular Fibrosis Is Attenuated by NO-Sensitive Guanylyl Cyclase1.

In the NO/cGMP signaling cascade, relevant in the cardiovascular system, two NO-sensitive guanylyl cyclase (NO-GC) isoforms are responsible for NO-dependent cGMP generation. Here, the impact of the major NO-GC isoform, NO-GC1, on fibrosis development in the cardiovascular system was studied in NO-GC1-deficient mice treated with AngiotensinII (AngII), known to induce vascular and cardiac remodeling. Morphometric analysis of NO-GC1 KO’s aortae demonstrated an enhanced increase of perivascular area after AngII treatment accompanied by a higher aortic collagen1 mRNA content. Increased perivascular fibrosis also occurred in cardiac vessels of AngII-treated NO-GC1 KO mice. In line, AngII-induced interstitial fibrosis was 32% more pronounced in NO-GC1 KO than in WT myocardia associated with a higher cardiac Col1 and other fibrotic marker protein content. In sum, increased perivascular and cardiac interstitial fibrosis together with the enhanced collagen1 mRNA content in AngII-treated NO-GC1-deficient mice represent an exciting manifestation of antifibrotic properties of cGMP formed by NO-GC1, a finding with great pharmaco-therapeutic implications.

Moringa oleifera leaf extract enhances endothelial nitric oxide production leading to relaxation of resistance artery and lowering of arterial blood pressure

A mass of evidence has identified a promoting of nitric oxide (NO) production in endothelial cells using natural products as a potential strategy to prevent and treat hypertension. This study investigated whether the aqueous extract of Moringa oleifera leaves (MOE) could lower mean arterial pressure (MAP) and relax mesenteric arterial beds in rats via stimulating endothelium-derived NO production. Intravenous administration of MOE (1–30 mg/kg) caused a dose-dependent reduction in MAP in anesthetized rats. In rats pretreated with the NO-synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg, i.v.), the effect of MOE on MAP was significantly reduced. MOE (0.001–3 mg) induced relaxation in methoxamine (10 μM) pre-contracted mesenteric arterial beds, which was abolished by endothelium denudation. This endothelium-dependent vasorelaxation was reduced by L-NAME (100 μM) or the NO-sensitive guanylyl cyclase inhibitor, 1H- [1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (10 μM). In primary human pulmonary artery endothelial cells, MOE (3–30 μg/mL) induced NO production, which was inhibited by L-NAME (100 μM) pretreatment. These findings show that MOE stimulates the endothelium-derived NO release for driving its vasorelaxation to lower arterial blood pressure. These suggest the development of MOE as a natural antihypertensive supplement.

Cigarette smoke exposure has region-specific effects on GDAP1 expression in mouse hippocampus

Early detection markers for substance use disorders are urgently needed. Recently, an association between the methylation of Ganglioside-induced differentiation-associated protein 1 (GDAP1) and alcohol addiction was found in a US and German population. In this study, we investigate whether GDAP1 expression might be affected by cigarette smoke as well and thus might be a marker of substance addiction in general. 11 adult female C57BL/6 J mice (6 wildtype and 5 lacking the NO-sensitive guanylyl cyclase1 (NO-GC1 KO)) were exposed to cigarette smoke over a period of 5 weeks, their brains immunohistochemically stained and compared to 11 non exposed mice (5 WT and 6 KO). The deletion of NO-GC1 results in a complete loss of synaptic plasticity, therefore, addiction-related alterations might become more obvious. Co-staining of anti-GDAP1 and DAPI revealed protein in the stratum granulare of the hippocampus. Three randomized frames for dentate gyrus (DG) and three for Cornu Ammonis region 1 (CA1) were used to count GDAP1. Cigarette smoke exposure significantly influenced GDAP1 expression depending on the hippocampal region but was not influenced by guanyl cyclase. In conclusion, cigarette smoke exposure alone had an effect on GDAP1 amount in both regions. Therewith, GDAP1might be a biomarker for substance addiction in general.

Luteolin suppresses colonic smooth muscle motility via inhibiting L-type calcium channel currents in mice.

As a naturally occurring flavone, luteolin has received much attention due to its antioxidant, anti-inflammatory and anticancer functions. In the present study, we investigated the effect of luteolin on colonic motility and its mechanism using isometric muscle recording and the whole-cell patch-clamp technique in mice. Luteolin dose-dependently inhibited colonic smooth muscles motility and CMMC significantly. BayK8644, an L-type Ca2+ channel agonist, significantly attenuated the luteolin-induced inhibition. Moreover, the calcium currents recorded in colonic smooth muscle cells were dramatically inhibited by luteolin. However, no significant changes were found in the luteolin-induced inhibitory effect in the presence of TEA, a nonselective K+ channel blocker, glibenclamide, an ATP-dependent K+ channel blocker, and apamin, a small-conductance Ca2+-activated K+ channel blocker. Additionally, luteolin did not affect potassium currents. Furthermore, TTX, a Na+ channel blocker, L-NAME, an inhibitor of nitric oxide (NO) synthase, ODQ, an inhibitor of NO-sensitive guanylyl cyclase, and Ani9, a specific ANO1 channels blocker, had no effect on the luteolin-induced suppression. These results suggest that luteolin inhibited colonic smooth muscle motility by inhibiting L-type calcium channels in mice but not through potassium channels, the enteric nervous system (ENS), NO signaling pathways or ANO1 channels of interstitial cells of Cajal (ICCs).

Insight into the Mechanisms Underlying the Tracheorelaxant Properties of the Sideritis raeseri Extract.

Sideritis raeseri Boiss. and Heldr. (Lamiaceae), known as “mountain tea,” is a native plant from the Mediterranean region, which is widely used in traditional medicine. This study evaluates the effects of the ethanol extract of Sideritis raeseri (SR) on airway smooth muscle activity and identifies the underlying mechanism. The S. raeseri extract (SRE) was extracted from air-dried parts of the shoot system of SR. The SRE (0.3–2 mg/mL) was tested in isolated rabbit tracheal rings, suspended in the organ bath, filled with Krebs solution, and bubbled with the carbogen mixture (95% O2/5% CO2) under a resting tension of 1 g in 37°C. In in vitro experiments, the SRE relaxed against acetylcholine-induced constriction in tracheal rings. Furthermore, SRE inhibited Ca2+-induced contractions in carbachol (CCh, 1 μM) as well as in the K+-depolarized trachea (80 mM). Our findings showed the NO/cGMP involvement in tracheorelaxant effects of SR. To this end, the effect of the SRE was potentiated by bradykinin (nitric oxide (NO) synthase activator, 100 nM), whereas it was inhibited by ODQ (inhibitor of NO-sensitive guanylyl cyclase, 10 μM) and L-NAME (NO synthase inhibitor, 100 μM), as well as indomethacin (cyclooxygenase inhibitor, 10 μM). These data suggest that the tracheorelaxant effect of the SRE is mediated at least partly by NO/cyclic guanosine monophosphate and cyclooxygenase-1-prostaglandin E2-dependent signaling. These findings indicate that the SRE may be used in various respiratory disorders.

The basal release of endothelium-derived catecholamines regulates the contractions of Chelonoidis carbonaria aorta caused by electrical-field stimulation.

ABSTRACTThe contractions of Chelonoidis carbonaria aortic rings induced by electrical field stimulation (EFS) are not inhibited by blockade of the voltage-gated sodium channels by tetrodotoxin but almost abolished by the α1/α2-adrenoceptor antagonist phentolamine. The objective of this study was to identify the mediator(s) responsible for the EFS-induced contractions of Chelonoidis carbonaria aortic rings. Each ring was suspended between two wire hooks and mounted in isolated 10 ml organ chambers filled with oxygenated and heated Krebs-Henseleit's solution. Dopamine, noradrenaline and adrenaline concentrations were analysed by liquid chromatography coupled to tandem mass spectrometry. The contractions caused by dopamine and EFS were done in absence and presence of the nitric oxide (NO) synthesis inhibitor L-NAME, the NO-sensitive guanylyl cyclase inhibitor ODQ, the D1-like receptor antagonist SCH-23390, the D2-like receptor antagonists risperidone, quetiapine, haloperidol, and the tyrosine hydroxylase inhibitors salsolinol and 3-iodo-L-tyrosine. Basal concentrations of dopamine, noradrenaline and adrenaline were detected in Krebs-Henseleit solution containing the aortic rings. The catecholamine concentrations were significantly reduced in endothelium-denuded aortic rings. L-NAME and ODQ significantly potentiated the dopamine-induced contractions. The D2-like receptor antagonists inhibited the EFS-induced contractions of the aortic rings treated with L-NAME, whereas SCH 23390 had no effect. Similar results were observed in the contractions induced by dopamine in L-NAME treated aortic rings. These results indicate that catecholamines released by endothelium regulate the EFS-induced contractions. This may constitute a suitable mechanism by which reptilia modulate specific organ blood flow distribution.This paper has an associated First Person interview with the first author of the article.

Dorsal hippocampus cholinergic and nitrergic neurotransmission modulates the cardiac baroreflex function in rats.

Hippocampus is a limbic structure involved in the baroreflex and chemoreflex control that receives extensive cholinergic input from basal forebrain. Hippocampal muscarinic receptors activation by acetylcholine might evoke nitric oxide synthesis, which is an important neuromodulator of cardiovascular responses. Thus, we hypothesize that cholinergic and nitrergic neurotransmission within the DH modulates the baroreflex and chemoreflex function. We have used vasoactive drugs (phenylephrine and sodium nitroprusside), and potassium cyanide infused peripherally to induce, respectively, baroreflex or chemoreflex responses in awake animals. Bilateral injection into the DH of the acetylcholinesterase inhibitor (neostigmine) reduced baroreflex responses. Meanwhile, the non-selective muscarinic receptor antagonist (atropine) or the M1-selective muscarinic receptor antagonist increased baroreflex responses (pirenzepine). Furthermore, the neuronal nitric oxide synthase inhibitor (N-propyl) or the intracellular NO scavenger (carboxy-PTIO) increased baroreflex responses, as well as the selective inhibitor of NO-sensitive guanylyl cyclase (ODQ), increased the baroreflex responses. Besides, bilateral administration of an ineffective dose of a neuronal nitric oxide synthase inhibitor abolished the reduction in the baroreflex responses evoked by an acetylcholinesterase inhibitor. On the other hand, we have demonstrated that hippocampal cholinergic neurotransmission did not influence the chemoreflex function. Taken together, our findings suggest that nNOS-derived nitric oxide in the DH participates in acetylcholine-evoked baroreflex responses.

AMPA Induces NO-Dependent cGMP Signals in Hippocampal and Cortical Neurons via L-Type Voltage-Gated Calcium Channels.

The nitric oxide (NO)/cGMP signaling cascade has an established role in synaptic plasticity. However, with conventional methods, the underlying cGMP signals were barely detectable. Here, we set out to confirm the well-known NMDA-induced cGMP increases, to test the impact of AMPA on those signals, and to identify the relevant phosphodiesterases (PDEs) using a more sensitive fluorescence resonance energy transfer (FRET)-based method. Therefore, a "knock-in" mouse was generated that expresses a FRET-based cGMP indicator (cGi-500) allowing detection of cGMP concentrations between 100nM and 3μM. Measurements were performed in cultured hippocampal and cortical neurons as well as acute hippocampal slices. In hippocampal and cortical neurons, NMDA elicited cGMP signals half as high as the ones elicited by exogenous NO. Interestingly, AMPA increased cGMP independently of NMDA receptors and dependent on NO synthase (NOS) activation. NMDA- and AMPA-induced cGMP signals were not additive indicating that both pathways converge on the level of NOS. Accordingly, the same PDEs, PDE1 and PDE2, were responsible for degradation of NMDA- as well as AMPA-induced cGMP signals. Mechanistically, AMPAR induced calcium influx through L-type voltage-gated calcium channels leading to NOS and finally NO-sensitive guanylyl cyclase activation. Our results demonstrate that in addition to NMDA also AMPA triggers endogenous NO formation and hence cGMP production.

Cell-specific effects of nitric oxide on the efficiency and frequency of long distance contractions in murine colon.

Nitric oxide (NO) mediates inhibitory neurotransmission and is a critical component of neuronal programs that generate propulsive contractions. NO acts via its receptor NO-sensitive guanylyl cyclase (NO-GC) which is expressed in smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Organ bath studies with colonic rings from NO-GC knockout mice (GCKO) have indicated NO-GC to modulate spontaneous contractions. The cell-specific effects of NO-GC on the dominant pan-colonic propulsive contraction, the long distance contractions (LDCs), of whole colon preparations have not yet been described. Contractions of whole colon preparations from wild type (WT), global, and cell-specific GCKO were recorded. After transformation into spatiotemporal maps, motility patterns were analyzed. Simultaneous perfusion of the colon enabled the correlation of outflow with LDCs to analyze contraction efficiency. Deletion of NO-GC in both ICC and SMC (ie, in GCKO and SMC/ICC-GCKO) caused loss of typical LDC activity and instead generated high-frequency LDC-like contractions with inefficient propulsive activity. Frequency was also increased in WT, SMC-GCKO, and ICC-GCKO colon in the presence of L-NAME to block neuronal NO synthase. LDC efficiency was dependent on NO-GC in SMC as it was reduced in GCKO, SMC-GCKO, and ICC/SMC-GCKO colon; LDC efficiency was decreased in all genotypes in the presence of L-NAME. NO/cGMP signaling is critical for normal peristaltic movements; as NO-GC in both SMC and ICC is essential, both cell types appear to work in synchrony. The efficiency of contractions to expel fluid is particularly influenced by NO-GC in SMC.