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Impaired NO-cGMP signaling has been linked to several neurological disorders. NO-sensitive guanylyl cyclase (NO-GC), of which two isoforms&mdash, NO-GC1 and NO-GC2&mdash, are known, represents a promising drug target to increase cGMP in the brain. Drug-like small molecules have been discovered that work synergistically with NO to stimulate NO-GC activity. However, the effects of NO-GC stimulators in the brain are not well understood. In the present study, we used F&ouml, rster/fluorescence resonance energy transfer (FRET)-based real-time imaging of cGMP in acute brain slices and primary neurons of cGMP sensor mice to comparatively assess the activity of two structurally different NO-GC stimulators, IWP-051 and BAY 41-2272, in the cerebellum, striatum and hippocampus. BAY 41-2272 potentiated an elevation of cGMP induced by the NO donor DEA/NO in all tested brain regions. Interestingly, IWP-051 potentiated DEA/NO-induced cGMP increases in the cerebellum and striatum, but not in the hippocampal CA1 area or primary hippocampal neurons. The brain-region-selective activity of IWP-051 suggested that it might act in a NO-GC isoform-selective manner. Results of mRNA in situ hybridization indicated that the cerebellum and striatum express NO-GC1 and NO-GC2, while the hippocampal CA1 area expresses mainly NO-GC2. IWP-051-potentiated DEA/NO-induced cGMP signals in the striatum of NO-GC2 knockout mice but was ineffective in the striatum of NO-GC1 knockout mice. These results indicate that IWP-051 preferentially stimulates NO-GC1 signaling in brain slices. Interestingly, no evidence for an isoform-specific effect of IWP-051 was observed when the cGMP-forming activity of whole brain homogenates was measured. This apparent discrepancy suggests that the method and conditions of cGMP measurement can influence results with NO-GC stimulators. Nevertheless, it is clear that NO-GC stimulators enhance cGMP signaling in the brain and should be further developed for the treatment of neurological diseases.

Nitric oxide (NO) modulates renal blood flow (RBF) and kidney function and is involved in blood pressure (BP) regulation predominantly via stimulation of the NO-sensitive guanylyl cyclase (NO-GC), existing in two isoforms, NO-GC1 and NO-GC2. Here, we used isoform-specific knockout (KO) mice and investigated their contribution to renal hemodynamics under normotensive and angiotensin II-induced hypertensive conditions. Stimulation of the NO-GCs by S-nitrosoglutathione (GSNO) reduced BP in normotensive and hypertensive wildtype (WT) and NO-GC2-KO mice more efficiently than in NO-GC1-KO. NO-induced increase of RBF in normotensive mice did not differ between the genotypes, but the respective increase under hypertensive conditions was impaired in NO-GC1-KO. Similarly, inhibition of endogenous NO increased BP and reduced RBF to a lesser extent in NO-GC1-KO than in NO-GC2-KO. These findings indicate NO-GC1 as a target of NO to normalize RBF in hypertension. As these effects were not completely abolished in NO-GC1-KO and renal cyclic guanosine monophosphate (cGMP) levels were decreased in both NO-GC1-KO and NO-GC2-KO, the results suggest an additional contribution of NO-GC2. Hence, NO-GC1 plays a predominant role in the regulation of BP and RBF, especially in hypertension. However, renal NO-GC2 appears to compensate the loss of NO-GC1, and is able to regulate renal hemodynamics under physiological conditions.

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.

The role of nitric oxide (NO)/cGMP signaling in long-term potentiation (LTP) has been a lingering matter of debate. Within the cascade, the NO receptor guanylyl cyclase (GC), the cGMP-forming enzyme that is stimulated by NO, plays a key role. Two isoforms of GC (α2-GC, α1-GC) exist. To evaluate their contribution to synaptic plasticity, we analyzed knock-out mice lacking either one of the GC isoforms. We found that LTP induced in the visual cortex is NO dependent in the wild-type mice, absent in either of the GC isoform-deficient mice, and restored with application of a cGMP analog in both strains. The requirement of both NO receptor GCs for LTP indicates the existence of two distinct NO/cGMP-mediated pathways, which have to work in concert for expression of LTP.

Hyperreactivity of airways to bronchoconstrictive agents is a common feature of reactive airway diseases. In addition to its established role on vascular smooth muscle tone, the NO/cGMP pathway is also expected to balance the contractile responses of airway smooth muscle. The NO-sensitive guanylyl cyclase (NO-GC) which forms cyclic GMP in response to NO holds a key position in this pathway and exists in two isoforms, NO-GC1 and NO-GC2, which both have been identified in bronchial and pulmonary blood vessels smooth muscle. Here we determined the contribution of the NO-GC isoforms to the regulation of airway resistance. Airway resistance was determined in a whole body plethysmography chamber in conscious mice deficient in either NO-GC1 or NO-GC2 in response to methacholine and serotonine inhalation. L-NAME was applied to NO-GC KOs to analyse the effect mediated by the remaining NO-GC isoform and to WT to inhibit both isoforms to see a possible synergistic or antagonistic action. The ganglionic blocker hexamethonium was use to differentiate bronchial and neuronal pathways. Preliminary results indicate that both NO-GC isoforms contribute to airway responsiveness.

β-Adrenergic agonists stimulate cardiac contractility and simultaneously blunt this response by coactivating NO synthase (NOS3) to enhance cGMP synthesis and activate protein kinase G (PKG-1). cGMP is also catabolically regulated by phosphodiesterase 5A (PDE5A). PDE5A inhibition by sildenafil (Viagra) increases cGMP and is used widely to treat erectile dysfunction; however, its role in the heart and its interaction with β-adrenergic and NOS3/cGMP stimulation is largely unknown. In nontransgenic (control) murine in vivo hearts and isolated myocytes, PDE5A inhibition (sildenafil) minimally altered rest function. However, when the hearts or isolated myocytes were stimulated with isoproterenol, PDE5A inhibition was associated with a suppression of contractility that was coupled to elevated cGMP and increased PKG-1 activity. In contrast, NOS3-null hearts or controls with NOS inhibited by N G -nitro- l -arginine methyl ester, or soluble guanylate cyclase (sGC) inhibited by 1 H -[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one, showed no effect of PDE5A inhibition on β-stimulated contractility or PKG-1 activation. This lack of response was not attributable to altered PDE5A gene or protein expression or in vitro PDE5A activity, but rather to an absence of sGC-generated cGMP specifically targeted to PDE5A catabolism and to a loss of PDE5A localization to z-bands. Re-expression of active NOS3 in NOS3-null hearts by adenoviral gene transfer restored PDE5A z-band localization and the antiadrenergic efficacy of PDE5A inhibition. These data support a novel regulatory role of PDE5A in hearts under adrenergic stimulation and highlight specific coupling of PDE5A catabolic regulation with NOS3-derived cGMP attributable to protein subcellular localization and targeted synthetic/catabolic coupling.

Background Alterations in vascular relaxation have been reported in various models of hypertension. With its tonic activity, the NO/cGMP signalling cascade plays an important role in the regulation of the vascular tone. In blood vessels, NO is continuously being produced by the endothelial NO synthases (eNOS) which leads to cGMP production by stimulating the NO-sensitive guanylyl cyclases (NO-GCs). In vascular smooth muscle cells, two NO-GC isoforms, NO-GC1 and NO-GC2, mediate relaxation, with NO-GC1 being responsible for approximately 90% of the NO-stimulated cGMP formation. Deletion of eNOS or both NO-GCs abrogates endothelium-dependent relaxation and causes substantial hypertension. However, deletion of only NO-GC1 does not lead to hypertension despite a 50% reduction of endothelium-dependent relaxation.

Background NO/cGMP-mediated signal transduction is involved in synaptic plasticity in various brain regions. NO effects are transduced by the NO receptor guanylyl cyclase (NO-GC) that exists in two isoforms, NO-GC1 and NO-GC2, with indistinguishable regulatory properties. Mice deficient in either NO-GC1 or NO-GC2 revealed that both NO-GC isoforms are required for LTP indicating the existence of two separated NO/cGMP pathways. Recently, we demonstrated a presynaptic role of NO/cGMP in facilitation of glutamate release and indentified eNOS and NO-GC1 as the participating enzymes. Yet, the involved cGMP-hydrolysing phosphodiesterases (PDE) remained unknown.

Background Hypertension, the leading risk factor for cardiovascular mortality has been associated with alterations in endotheliumand smooth muscle-dependent vascular relaxation. The NO/cGMP signaling cascade is one of the major pathways that mediate vascular relaxation. In this pathway, the NO receptor guanylyl cyclase (NO-GC) holds a key position by converting the NO signal into cGMP increases. Two isoforms of the heterodimeric NO receptor GC exist; both isoforms share an identical β subunit but differ in their respective α subunit (α1 or α2) and are referred to as NO-GC1 (so far α1β1heterodimer) and NO-GC2 (so far α2β1heterodimer), respectively. Knockout (KO) mice deficient in either one of the NO-GCs, NO-GC1 or NO-GC2, revealed that both NO-GCs are capable to mediate vascular relaxation. The NO-GC1 appears to be the major isoform, particularly in the aorta, where NO-GC1 represents approximately 90% of total NO-GC content. Deletion of the NO-GC1 resulted in reduced endothelium-dependent relaxation and reduced vasodilatory response to exogenous NO, which is mediated by the NO receptor GC2 in NO-GC1-deficient mice. Despite the low NO-GC2 content, NO-GC1 KO mice exhibit only a minute blood pressure increase.

Increased production of reactive oxygen species and failure of the antioxidant defense system are considered to play a central role in the pathogenesis of cardiovascular disease. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key master switch controlling the expression of antioxidant and protective enzymes, and was proposed to participate in protection of vascular and cardiac function. This study was undertaken to analyze cardiac and vascular phenotype of mice lacking Nrf2. We found that Nrf2 knock out (Nrf2 KO) mice have a left ventricular (LV) diastolic dysfunction, characterized by prolonged E wave deceleration time, relaxation time and total diastolic time, increased E/A ratio and myocardial performance index, as assessed by echocardiography. LV dysfunction in Nrf2 KO mice was associated with cardiac hypertrophy, and a downregulation of the sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) in the myocardium. Accordingly, cardiac relaxation was impaired, as demonstrated by decreased responses to β-adrenergic stimulation by isoproterenol ex vivo, and to the cardiac glycoside ouabain in vivo. Surprisingly, we found that vascular endothelial function and endothelial nitric oxide synthase (eNOS)-mediated vascular responses were fully preserved, blood pressure was decreased, and eNOS was upregulated in the aorta and the heart of Nrf2 KO mice. Taken together, these results show that LV dysfunction in Nrf2 KO mice is mainly associated with cardiac hypertrophy and downregulation of SERCA2a, and is independent from changes in coronary vascular function or systemic hemodynamics, which are preserved by a compensatory upregulation of eNOS. These data provide new insights into how Nrf2 expression/function impacts the cardiovascular system.

NO-sensitive guanylyl cyclases are the most important receptors for the signalling molecule NO. Activation of guanylyl cyclase by NO leads to the formation of the intracellular messenger cGMP. The enzyme is a heterodimer which consists of two subunits, α and β. Three different subunits exist in vivo: Two α subunits (α1 and α2) and one β subunit (β1). Functional enzymes are formed by heterodimerization. The two known guanylyl cyclase isoforms are composed of one of either α subunit and the β1 subunit (i.e. α1β1 and α2β1).