Deletion Of NO-sensitive Guanylyl Cyclase Research Articles

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.

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

Phosphodiesterase 3A expression and activity in the murine vasculature is influenced by NO-sensitive guanylyl cyclase.

Phosphodiesterase 3 (PDE3) exists in two isoforms (PDE3A and PDE3B) and is known to act as cGMP-inhibited cAMP-degrading PDE. Therefore, PDE3 may likely be involved in the interaction between the two second messenger pathways. NO-sensitive guanylyl cyclase (NO-GC) is the most important cytosolic generator of cGMP. Here, we investigated the effect of NO-GC deletion on PDE3A-mediated signaling in animals lacking NO-GC either globally (GCKO) or specifically in smooth muscle cells (SMC-GCKO). PDE3A expression is detected in murine aortic smooth muscle, platelets, and heart tissue. Expression and activity of PDE3A in aortae from GCKO and SMC-GCKO mice was reduced by approx. 50% compared to that in control animals. PDE3A downregulation can be linked to the reduction in NO-GC and is not an effect of the increased blood pressure levels resulting from NO-GC deletion. Despite the different PDE3A expression levels, smooth muscle relaxation induced by forskolin to stimulate cAMP signaling was similar in all genotypes. Basal and forskolin-stimulated cAMP levels in aortic tissue were not different between KO and control strains. However, the potency of milrinone, a selective inhibitor of PDE3A, to induce relaxation was higher in aortae from GCKO and SMC-GCKO than that in aorta from control animals. These data were corroborated by the effect of milrinone in vivo, which led to an increase in systolic blood pressure in both KO strains but not in control mice. We conclude that NO-GC modulates PDE3A expression and activity in SMC in vivo conceivably to preserve functional cAMP signaling.

Modulation of cGMP/cAMP crosstalk on the level of PDE3 in animals lacking NO-sensitive guanylyl cyclase

NO-sensitive guanylyl cyclase (NO-GC) is accepted to be the major receptor for the signaling molecule NO. Deletion of NO-GC in mice leads to disturbed NO/cGMP signaling. As a result, these mice show abolished NO-dependent relaxation of smooth muscle-containing tissues in the cardiovascular and gastrointestinal systems. Mice with general deletion (GCKO) suffer from increased blood pressure, reduced bleeding time, erectile dysfunction and die prematurely due to gastrointestinal dysmotility. Mice lacking NO-GC specifically in smooth muscle cells (SMC) show an elevated blood pressure similar to that seen in GCKO animals. Crosstalk between cGMP and cAMP signaling has been reported to occur in many different cells. Phosphodiesterase 3 (PDE3) is thought to be one of the prominent mediators of this crosstalk. Through its inhibitory action on PDE3, cGMP may regulate cAMP levels and cAMP-mediated signaling. Here, we reasoned that, in animals deficient in NO-GC, lack of NO-induced cGMP synthesis might affect cGMP/cAMP crosstalk on the level of PDE3. Smooth muscle relaxation induced by forskolin to stimulate adenylyl cyclase was similar in WT, GCKO and SMC-GCKO aortae. RIA-based detection of basal cAMP levels in aortic tissue from these mice did not reveal differences. Similarly, FRET measurements showed that PGE1-induced cAMP synthesis in isolated smooth muscle cells was unaffected by NO-GC deletion. However, aortae from GCKO and SMC-GCKO animals were more sensitive to milrinone, a specific inhibitor of PDE3, as the concentration curve for relaxation was shifted to the left. Analysis of PDE3 expression in aorta showed an approximately 50% reduction in PDE3 protein and specific enzyme activity. Similar reduction was detected in platelets but not in heart tissue of GCKO animals. Induction of SMC-specific NO-GC deletion by tamoxifen led to a parallel decline of NO-GC and PDE3 expression. Both proteins were strongly reduced already 5 days after the last tamoxifen injection. In contrast, systolic blood pressure in these animals started to rise only after 10 days and was maximal after 50 days. These data indicate that cGMP, not the increase in blood pressure, is responsible for the expression regulation of PDE3. We conclude that cGMP modulates PDE3 expression in SMC in order to keep up functional cAMP signaling.

Cell-specific impact of nitric oxide-dependent guanylyl cyclase on arteriogenesis and angiogenesis in mice.

Nitric oxide (NO) acts as essential regulator of vasculogenesis and angiogenesis and is critical for arteriogenesis. Whether NO's effects in vivo are mediated through NO-sensitive guanylyl cyclase (NO-GC) and thus by cGMP-dependent mechanisms has been only poorly addressed. Mice lacking NO-GC globally or specifically in smooth muscle cells (SMC) or endothelial cells (EC) were subjected to two established models for arteriogenesis and angiogenesis, namely hindlimb ischemia and oxygen-induced retinopathy. Our data clearly show the involvement of NO-GC in the recovery of blood flow after hindlimb ischemia, and this effect could be attributed to NO-GC in SMC. In the retina, global deletion of NO-GC led to reduced oxygen-induced vessel loss and hypoxia-induced capillary regrowth, whereas pathological neovascularization was increased. These effects were also seen in mice with SMC-specific NO-GC deletion but not in animals lacking NO-GC in EC. Intriguingly, NO-GC was found to be strongly expressed in retinal pericytes. Our data prove the involvement of NO-GC in growth and plasticity of hindlimb and retinal vasculature after ischemic/hypoxic insult.

Correlation of cellular expression with function of NO‐sensitive guanylyl cyclase in the murine lower urinary tract

The action of nitric oxide (NO) to stimulate NO-sensitive guanylyl cyclase (NO-GC), followed by production of cGMP, and eventually to cause smooth muscle relaxation is well known. In the lower urinary tract (LUT), in contrast to the cardiovascular system and the gastrointestinal tract, specific localization in combination with function of NO-GC has not been investigated to date. Consequently, little is known about the mechanisms regulating relaxation of the lower urinary tract in general and the role of NO-GC-expressing cells in particular. To study the distribution and function of NO-GC in the murine lower urinary tract, we used internal urethral sphincter and bladder detrusor from global (GCKO) and smooth muscle cell-specific (SM-GCKO) NO-GC knock-out mice for immunohistochemical analyses and organ bath experiments. In urethral sphincter, NO-GC-positive immunofluorescence was confined to smooth muscle cells (SMCs). Deletion of NO-GC in SMCs abolished NO-induced relaxation. In bladder detrusor, exposure to NO did not cause relaxation although immunohistochemistry uncovered the existence of NO-GC in the tissue. In contrast to the urethral sphincter, expression of NO-GC in bladder detrusor was limited to platelet-derived growth factor receptor α (PDGFRα)-positive interstitial cells. In conclusion, NO-GC found in SMCs of the urethral sphincter mediates NO-induced relaxation; bladder detrusor is unique as NO-GC is not expressed in SMCs and, thus, NO does not induce relaxation. Nevertheless, NO-GC expression was found in PDGFRα-positive interstitial cells of the murine bladder with an as yet unknown function. Further investigation is needed to clarify the role of NO-GC in the detrusor.

Cell type-specific knock out models to unravel NO/cGMP signaling in smooth muscle

NO-sensitive guanylyl cyclase (NO-GC) is accepted to be the major receptor for the signaling molecule NO. Deletion of NO-GC in mice leads to disturbed NO/cGMP signaling. As a result, these mice show abolished NO-dependent relaxation of smooth muscle-containing tissues in the cardiovascular and gastrointestinal systems. Mice with general deletion suffer from increased blood pressure, reduced bleeding time, erectile dysfunction and die prematurely due to gastrointestinal dysmotility. Several of the phenotypical changes caused by NO-GC deficiency are due to increased smooth muscle tone. Therefore, our work concentrated on NO/cGMP-mediated regulation of smooth muscle contraction/relaxation in various tissues. These include smooth muscle from blood vessels, gut, corpus cavernosum and lower urinary tract. NO-induced relaxation of all NO-GC-containing smooth muscle tissues was influenced by the deletion of the enzyme. To our surprise, the degree of smooth muscle dysfunction was not homogeneous: In contrast to vascular or urethral smooth muscle, gastric fundus and other GI muscles revealed an only a partially reduced NO-induced relaxation. Detrusor muscle of the bladder was unresponsive towards NO. Closer examination of the tissues showed variable expression of NO-GC in the different smooth muscle cells. Importantly, in addition to smooth muscle cells, many tissues showed NO-GC expression in other cell types such as endothelial cells, interstitial cells of Cajal and fibroblast-like cells. We have generated various cell-specific KO strains using the inducible Cre-lox-system. In the GI tract we were able to show a dual mechanism of NO-induced relaxation via interstitial cell of Cajal and smooth muscle cells. In addition, we identified a third type of NO-GC-expressing cell, the fibroblast-like cell, whose function is still enigmatic. In penile corpus cavernosum, strong NO-GC expression was found in smooth muscle cells and, surprisingly, also in endothelial cells. The function of NO-GC in the endothelium is currently being investigated. Further studies using double or triple KO mutants will hopefully allow advising cell-specific functions of NO/cGMP signaling in murine smooth muscle.

Cardiovascular response to NO donors in mice deficient in NO-GC

The NO/cGMP cascade is essential for control of the cardiovascular system and it is also involved in the pathophysiology of several cardiovascular diseases. One of the main vascular factors involved in the relaxation of blood vessels and regulation of blood pressure is nitric oxide (NO). Target of NO is the NO-sensitive guanylyl cyclase (NO-GC) which is expressed in different cell types in the cardiovascular system (e.g. smooth muscle cells, cardiomyocytes). The stimulation of the NO/cGMP cascade is used pharmacologically for the treatment of coronary heart disease; substances commonly used are compounds such as glycerol trinitrate (NTG) or isosorbide dinitrate which are thought to act by increasing NO concentration and activation of NO-GC. We have previously shown that complete knockout of NO-GC (GCKO) in mice led to an increase in systolic blood pressure (+30 mmHg) concomitant with an abolished NO responsiveness of vascular smooth muscle. Smooth muscle-specific KO mice for NO-GC (SM-GCKO) showed an increase in systolic blood pressure identical to that seen in general GCKO mice. In order to find out the relative contribution of different cell types expressing NO-GC in the cardiovascular system, we here used catheter measurements to compare the effect of different NO donors (NTG and DEA-NO) on blood pressure and heart rate in vivo. In WT mice, both NTG and the radical NO donor DEA-NO reduced systolic blood pressure and increased heart rate indicating the contribution of the baroreceptor reflex. The deletion of NO-GC in mice (GCKO) abrogated both effects. We are currently investigating mice lacking NO-GC in smooth muscle cells, endothelial cells and cardiomyocytes.

Cell-Specific Deletion of Nitric Oxide–Sensitive Guanylyl Cyclase Reveals a Dual Pathway for Nitrergic Neuromuscular Transmission in the Murine Fundus

It is not clear how nitric oxide (NO) released from enteric neurons relaxes gastrointestinal (GI) smooth muscle. In analogy to the vascular system, NO might directly induce relaxation of smooth muscle cells (SMCs) by acting on its receptor, NO-sensitive guanylyl cyclase (NO-GC). Alternatively, intermediate cells,such as the interstitial cells of Cajal (ICCs), might detect nitrergic signals to indirectly regulate smooth muscle tone, and thereby regulate the motor function of the GI tract. We investigated the role of ICCs and SMCs in nitrergic relaxation using mice with cell-specific disruption of the gene encoding the β1 subunit of NO-GC (GUCY1B3). We created mice that lack NO-GC specifically in SMCs (SM-guanylyl cyclase knockout [GCKO]), ICCs (ICC-GCKO), or both (SM/ICC-GCKO). We investigated the effects of exogenous and endogenous NO on murine fundus using isometric force studies. Total gut transit time was measured to monitor the functional consequences of NO-GC deletion on GI motility invivo. NO-GC is expressed in ICC and SMC. Deletion of the NO receptor from SMCs incompletely reduced NO-induced fundus relaxation, which was hardly affected after ICC-specific deletion. Gut transit time did not change in SM-GCKO or ICC-GCKO mice compared with control mice. However, nitrergic relaxation was not observed in SM/ICC-GCKO mice, which had increased gut transit time compared with controls. In mice, NO-GC is the only NO receptor to relax the fundus; deletion of NO-GC from the combination of SMCs and ICCs blocks nitrergic signaling. Therefore, ICCs and SMCs jointly mediate the relaxant effect of enteric NO.

Preserved fertility despite erectile dysfunction in mice lacking the nitric oxide receptor

Nitric oxide (NO) and cGMP have been shown to be important mediators of penile erection. Erectile dysfunction may result from reduced or non-functional signal transduction within this cascade. There is, however, some inconsistency in the available data as mice lacking NO synthases (endothelial and neuronal nitric oxide synthase, or both) appear to be fertile whereas mice deficient in cGMP-dependent protein kinase I (PKGI) suffer from erectile dysfunction. To clarify this discrepancy we performed studies on mice lacking the NO receptor NO-sensitive guanylyl cyclase (NO-GC). In addition, we generated cell-specific NO-GC knockout (KO) lines to investigate the function of NO in individual cell types. NO-GC was specifically deleted in smooth muscle or endothelial cells (SM-guanylyl cyclase knockout (SM-GCKO) and EC-GCKO, respectively) and these KO lines were compared with total knockouts (GCKO) and wild-type animals. We investigated expression of NO-GC, NO-induced relaxation of corpus cavernosum smooth muscle and their ability to generate offspring. NO-GC-positive immunostaining was detected in smooth muscle and endothelial cells of murine corpus cavernosum but not in interstitial cells of Cajal. NO released from NO donors as well as from nitrergic neurons failed to relax precontracted corpus cavernosum from GCKO mice in organ bath experiments. Similar results were obtained in corpus cavernosum from SM-GCKO mice whereas deletion of NO-GC in endothelial cells did not affect relaxation. The lack of NO-induced relaxation in GCKO animals was not compensated for by guanosine 3,5-cyclic monophosphate (cGMP) signalling. To our surprise, GCKO males were fertile although their ability to produce offspring was decreased. Our data show that deletion of NO-GC specifically in smooth muscle cells abolishes NO-induced corpus cavernosum relaxation but does not lead to infertility.

Smooth Muscle–Specific Deletion of Nitric Oxide–Sensitive Guanylyl Cyclase Is Sufficient to Induce Hypertension in Mice

Arterial hypertension is one of the major diseases in industrial countries and a major cause of mortality. One of the main vascular factors responsible for the relaxation of blood vessels and regulation of blood pressure is nitric oxide (NO). NO acts predominantly via NO-sensitive guanylyl cyclase (NO-GC), which is made up of 2 different subunits (alpha and beta). Deletion of the beta(1) subunit leads to a global NO-GC knockout, and these mice are hypertensive. However, global deletion of NO-GC in mice does not allow identification of the cell/tissue type responsible for the elevated blood pressure. To determine the relative contribution of smooth muscle cells to the hypertension seen in NO-GC knockout mice, we generated smooth muscle-specific knockout mice for the beta(1) subunit of NO-GC using a tamoxifen-inducible system. Male mice were investigated because the Cre transgene used is located on the Y chromosome. Tamoxifen injection led to a rapid reduction of NO-GC expression in smooth muscle but did not affect that in other tissues. Parallel to a reduction in NO-induced cGMP accumulation, NO-induced relaxation of aortic smooth muscle was gradually lost after induction by tamoxifen. Concomitantly, these animals developed hypertension within 3 to 4 weeks. We generated a model in which the development of hypertension can be visualized over time by deletion of a single gene in smooth muscle cells. In sum, our data provide evidence that deletion of NO-GC solely in smooth muscle is sufficient to cause hypertension.

NO signalling in mice lacking the NO receptor guanylyl cyclase

NO-sensitive guanylyl cyclase (NO-GC) is accepted to be the major receptor for the signaling molecule NO. NO-GC catalyzes the production of the intracellular second messenger cGMP and thereby has a key regulatory function regarding various physiological processes. NO-GC is made up of one β subunit and one α subunit. On the protein level, there are two α subunits (α1 and α2) and one β subunit (β1) known, and thus two different GC isoforms exist (α1β1 and α2β1). In the enteric nervous system, non-adrenergic non-cholinergic (NANC) neurons control the motor function of the gastrointestinal tract. These NANC neurons express neuronal NO synthase. NO has been demonstrated to be an important physiological mediator of NANC relaxation of gastrointestinal smooth muscle, leading to relaxation via activation of NO-GC. In this study, we intended to clarify the role of NO-GC with regards to gastrointestinal (GI) motility. Immunohistochemical analysis revealed that GI smooth muscle expresses only minor amounts of NO-GC. Interestingly, strong expression was detected in the interstitial cells of Cajal (ICC). Using mice in which exon 10 of the β1 subunit is surrounded by loxP sites (floxed mice), we have generated mice lines that lack NO-GC either ubiquitously or specifically in smooth muscle. In isometric force studies we used endogenous NO production induced by electric field stimulation and pharmacological NO release using NO donors. Total gut transit time was measured to study the consequences of NO-GC deletion in vivo. General lack of NO-GC abolishes NO-dependent relaxation of GI smooth muscle. Neither physiological concentrations of NO using NO donors nor electric field stimulation at low frequencies led to relaxation of gastric fundus strips or sphincters. The whole gut transit time was increased approximately three times compared with WT. This dysfunction led to early mortality starting around day 18 after birth. Mortality was reduced by omission of dietary fiber. Surprisingly, in smooth muscle-specific knockout mice (SMKO) the NO-dependent relaxation was reduced, but still intact. Total gut transit time showed no difference and survival of SMKO animals was similar to that of WT mice. In conclusion, the NO receptor guanylyl cyclase in GI smooth muscle is dispensable for motility and survival.