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Pericytes and endothelial cells (ECs) constitute the fundamental components of blood vessels. While the role of ECs in tumor angiogenesis and the tumor microenvironment is well appreciated, pericyte function in tumors remains underexplored. In this study, we used pericyte-specific deletion of the nitric oxide (NO) receptor, soluble guanylate cyclase (sGC), to investigate via single-cell RNA sequencing how pericytes influence the vascular niche and the tumor microenvironment. Our findings demonstrate that pericyte sGC deletion disrupts EC-pericyte interactions, impairing Notch-mediated intercellular communication and triggering extensive transcriptomic reprogramming in both pericytes and ECs. These changes further extended their influence to neighboring cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) through paracrine signaling, collectively suppressing tumor growth. Inhibition of pericyte sGC has minimal impact on quiescent vessels but significantly increases the vulnerability of angiogenic tumor vessels to conventional anti-angiogenic therapy. In conclusion, our findings elucidate the role of pericytes in shaping the tumor vascular niche and tumor microenvironment and support pericyte sGC targeting as a promising strategy for improving anti-angiogenic therapy for cancer treatment., (© 2024. The Author(s).)

Asthma is characterized by airway remodeling and hyperreactivity. Our earlier studies determined that the nitric oxide (NO)-soluble guanylyl cyclase (sGC)-cGMP pathway plays a significant role in human lung bronchodilation. However, this bronchodilation is dysfunctional in asthma due to high NO levels, which cause sGC to become heme-free and desensitized to its natural activator, NO. In order to determine how asthma impacts the various lung segments/lobes, we mapped the inflammatory regions of lungs to determine whether such regions coincided with molecular signatures of sGC dysfunction. We demonstrate using murine models of asthma (OVA and CFA/HDM) that the inflamed segments of these murine lungs can be tracked by upregulated expression of HO1 and these regions in turn overlap with regions of heme-free sGC as evidenced by a decreased sGC-α1β1 heterodimer and an increased response to heme-independent sGC activator, BAY 60-2770, relative to naïve uninflamed regions. We also find that NO generated from iNOS upregulation in the inflamed segments has a higher impact on developing heme-free sGC as increasing iNOS activity correlates linearly with elevated heme-independent sGC activation. This excess NO works by affecting the epithelial lung hemoglobin (Hb) to become heme-free in asthma, thereby causing the Hb to lose its NO scavenging function and exposing the underlying smooth muscle sGC to excess NO, which in turn becomes heme-free. Recognition of these specific lung segments enhances our understanding of the inflamed lungs in asthma with the ultimate aim to evaluate potential therapies and suggest that regional and not global inflammation impacts lung function in asthma., (© 2024 Federation of American Societies for Experimental Biology.)

Chronic kidney disease (CKD) is one of the greatest health burdens with an increasing global prevalence. Renal fibrosis (RF) is the hallmark of all forms of CKD which shows a strong positive correlation with severity of the disease. However, there are no therapeutic options available for treatment of RF. In the present study, we used an animal model based on unilateral ureteral obstruction (UUO), for renal injury and fibrosis. The UUO animals were treated with soluble guanylyl cyclase (sGC) stimulator, riociguat (RIO) (1, 3 and 10 mg/kg) to investigate its possible renoprotective effects. Kidneys of animals treated with RIO were found to show less abnormalities as compared to UUO control. Further, the levels of proinflammatory cytokines were reduced in RIO treated group. Furthermore, administration of RIO reduced expression of collagen-1, TGF-β, CTGF, α-SMA, vimentin along with transcription factors including Snail and Slug. The results of the present study provided strong evidence to support the antifibrotic activity of RIO. • Riociguat, a soluble guanylyl cyclase stimulator ameliorates renal injury. • Riociguat shows potent antioxidant and anti-inflammatory activities. • The Riociguat inhibited master regulators of EMT, Snail and Slug. [ABSTRACT FROM AUTHOR]

For the treatment of systemic hypertension, pharmacological intervention in nitric oxide-cyclic guanosine monophosphate signaling is a well-explored but unexploited option. In this review, we present the identified drug targets, including oxidases, mitochondria, soluble guanylyl cyclase, phosphodiesterase 1 and 5, and protein kinase G, important compounds that modulate them, and the current status of (pre)clinical development. The mode of action of these compounds is discussed, and based upon this, the clinical opportunities. We conclude that drugs that directly target the enzymes of the nitric oxide-cyclic guanosine monophosphate cascade are currently the most promising compounds, but that none of these compounds is under investigation as a treatment option for systemic hypertension. [ABSTRACT FROM AUTHOR]

Epileptic seizure is phenomenon of abnormal synchronous neuronal discharge of a set of neurons in brain as a result of neuronal excitation. Evidence shows the nitric oxide (NO) involvement in neuronal excitability. Moreover, the role of cyclic guanosine monophosphate (cGMP) activation in seizure pathogenesis is well-established. Sumatriptan is a selective agonist of 5-Hydroxytryptamine1B/D auto-receptor, has been reassessed for its neuroprotection. This study was aimed to explore the anticonvulsant effect of sumatriptan through possible involvement of NO-cGMP pathway in mice. For this purpose, the protective effect of sumatriptan on PTZ-induced clonic seizure threshold (CST) was measured using NO-cGMP pathway inhibitors including N(G)-nitro-L-arginine (L-NNA, 1, 5, and 10 mg/kg), 7-nitroindazole (7-NI, 30, 45, and 60 mg/kg), aminoguanidine (AG, 30, 50, and 100 mg/kg), methylene blue (MB, 0.1, 0.5, and 1 mg/kg) and sildenafil (5, 10, and 20 mg/kg). The involvement of nitrergic system was further confirmed by measurement of nitrite levels by Griess reaction. The gene expression of neuronal nitric oxide synthase (nNOS) and subunits of soluble guanylyl cyclase (sGC) was studied using qRT-PCR analysis. Acute administration of sumatriptan (1.2 and 0.3 mg/kg) in combination with subeffective doses of NOS, sGC, and phosphodiesterase 5 inhibitors significantly reversed the PTZ-induced CST (P ≤ 0.001). The nitrite level in prefrontal cortex was significantly attenuated by sumatriptan (P ≤ 0.01). Furthermore, sumatriptan downregulated the PTZ-induced mRNA expression of nNOS (P ≤ 0.01), α1 (P ≤ 0.001), α2 (P ≤ 0.05), and β1 (P ≤ 0.05) genes in cerebral cortex of mice. In conclusion, the anticonvulsant activity of sumatriptan at least, in part, is mediated through inhibiting NO-cGMP pathway. [ABSTRACT FROM AUTHOR]

Almost fifty years ago, experiments on isolated veins showed that acute hypoxia augments venoconstrictor responses in vitro and that such facilitation relied on anaerobic glycolysis. Over the years, this phenomenon was extended to a number of arterial preparations of different species and revisited, from a mechanistic point of view, with the successive demonstration that it depends on calcium handling in the vascular smooth muscle cells, is endothelium‐dependent and requires the production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) and the activation of soluble guanylyl cyclase (sGC). However, rather than the vasodilator cyclic nucleotide 3′,5′‐cyclic guanosine monophosphate (cGMP), its canonical product, the latter enzyme produces 3′,5′‐cyclic inosine monophosphate (cIMP) instead during acute hypoxia; this non‐canonical cyclic nucleotide facilitates the contractile process in the vascular smooth muscle cells. This 'biased' activity of soluble guanylyl cyclase appears to involve stimulation of NAD(P)H:quinone oxidoreductase 1 (NQO‐1). The exact interactions between hypoxia, anaerobic metabolism and NQO‐1 leading to biased activity of soluble guanylyl cyclase remain to be established. [ABSTRACT FROM AUTHOR]

Neuronal nitric oxide synthase (nNOS) catalyses the production of the neurotransmitter nitric oxide. nNOS is expressed in the dorsal raphe nucleus (DRN), a source of ascending serotonergic projections. In this study, we examined the distribution nNOS and the function of nitric oxide in the DRN and adjacent median raphe nucleus (MRN) of the rat. We hypothesized that nNOS is differentially expressed across the raphe nuclei and that nitric oxide influences the firing activity of a subgroup of 5‐HT neurons. Immunohistochemistry revealed that, nNOS is present in around 40% of 5‐HT neurons, throughout the DRN and MRN, as well as in some non‐5‐HT neurons immediately adjacent to the DRN and MRN. The nitric oxide receptor, soluble guanylyl cyclase, was present in all 5‐HT neurons examined in the DRN and MRN. In vitro extracellular electrophysiology revealed that application of the nitric oxide donor, diethylamine NONOate (30–300 µM) inhibited 60%–70% of putative 5‐HT neurons, excited approximately 10% of putative 5‐HT neurons and had no effect on the rest. The inhibitory response to nitric oxide was blocked by [1H‐[1,2,4]oxadiazolo‐[4, 3‐a]quinoxalin‐1‐one (ODQ, 30 or 100 µM), indicating mediation by soluble guanylyl cyclase. Juxtacellular labelling revealed that nitric oxide inhibits firing in both putative 5‐HT neurons which express nNOS and those which do not express nNOS. Our data are consistent with the notion that nitric oxide acts as both a trans‐synaptic and autocrine signaller in 5‐HT neurons in the DRN and MRN and that its effects are widespread and primarily inhibitory. [ABSTRACT FROM AUTHOR]

Coronary microvessel endothelial dysfunction and nitric oxide (NO) depletion contribute to elevated passive tension of cardiomyocytes, diastolic dysfunction and predispose the heart to heart failure with preserved ejection fraction. We examined if diastolic dysfunction at the level of the cardiomyocytes precedes coronary endothelial dysfunction in prediabetes. Further, we determined if myofilaments other than titin contribute to impairment. Utilizing synchrotron microangiography we found young prediabetic male rats showed preserved dilator responses to acetylcholine in microvessels. Utilizing synchrotron X-ray diffraction we show that cardiac relaxation and cross-bridge dynamics are impaired by myosin head displacement from actin filaments particularly in the inner myocardium. We reveal that increased PKC activity and mitochondrial oxidative stress in cardiomyocytes contributes to rho-kinase mediated impairment of myosin head extension to actin filaments, depression of soluble guanylyl cyclase/PKG activity and consequently stiffening of titin in prediabetes ahead of coronary endothelial dysfunction. • Sarcomeric dysfunction precedes coronary microvascular dysfunction in prediabetes. • PKC/rho-kinase activation displaces myosin heads from actin filaments in prediabetes. • Oxidative stress and inflammation reduce sGC activity to increase titin stiffness. • Mild hyperglycemia induces cardiomyocyte diastolic dysfunction ahead of remodeling. [ABSTRACT FROM AUTHOR]

The natural compound thymoquinone, extracted from Nigella sativa (black cumin), is widely used in humans for its anti-oxidative properties. Thymoquinone is known for its acute endothelium-independent vasodilator effects in isolated rat aortae and pulmonary arteries, depending in part on activation of adenosine triphosphate-sensitive potassium channels and inhibition of voltage-dependent calcium channels. The compound also improves endothelial dysfunction in mesenteric arteries of ageing rodents and in aortae of rabbits treated with pyrogallol, by inhibiting oxidative stress. Serendipitously, thymoquinone was found to augment contractions in isolated arteries with endothelium of both rats and pigs. The endothelium-dependent augmentation it causes counterintuitively depends on biased activation of soluble guanylyl cyclase (sGC) producing inosine 3ʹ,5ʹ-cyclic monophosphate (cyclic IMP) rather than guanosine 3ʹ,5ʹ-cyclic monophosphate. This phenomenon shows a striking mechanistic similarity to the hypoxic augmentation previously observed in porcine coronary arteries. The cyclic IMP preferentially produced under thymoquinone exposure causes an increased contractility of arterial smooth muscle by interfering with calcium homeostasis. This brief review summarizes the vascular pharmacology of thymoquinone, focussing in particular on how the compound causes endothelium-dependent contractions by biasing the activity of sGC.

Abstract Background Volatile anesthetics increase levels of the neurotransmitter nitric oxide (NO) and the secondary messenger molecule cyclic guanosine monophosphate (cGMP) in the brain. NO activates the enzyme guanylyl cyclase (GC) to produce cGMP. We hypothesized that the NO-GC-cGMP pathway contributes to anesthesia-induced unconsciousness. Methods Sevoflurane-induced loss and return of righting reflex (LORR and RORR, respectively) were studied in wild-type mice (WT) and in mice congenitally deficient in the GC-1α subunit (GC-1−/− mice). Spatial distributions of GC-1α and the GC-2α subunit in the brain were visualized by in situ hybridization. Brain cGMP levels were measured in WT and GC-1−/− mice after inhaling oxygen with or without 1.2% sevoflurane for 20 min. Results Higher concentrations of sevoflurane were required to induce LORR in GC-1−/− mice than in WT mice (1.5 ± 0.1 vs. 1.1 ± 0.2%, respectively, n = 14 and 14, P

This study investigated the effect of fermented garlic extract (FGE) on pulmonary hypertension in monocrotaline (MCT)-treated rats. Sprague-Dawley rats were subcutaneously injected with either 50 mg/kg MCT or a vehicle 3 days after the FGE feeding (0.97 mg nitrite/ml/day) was initiated. MCT treatment increased the weight, systolic pressure, and atrial natriuretic peptide concentration in the right ventricle but not in the left ventricle. FGE feeding attenuated these effects as well as the endothelial damage and medial hypertrophy of the pulmonary arterioles and the pulmonary fibrosis induced by MCT. These FGE effects were blocked by a soluble guanylyl cyclase (sGC) inhibitor. Increases in VCAM-1 and MMP-9 protein expressions, and decreases in PKG and eNOS protein expressions in the lung of MCT rats were attenuated by FGE feeding. These findings suggest that FGE ameliorates MCT-induced pulmonary hypertension by decreasing the inflammatory reaction via the NO–sGC–PKG pathway.

Septic shock typically requires the administration of vasopressors. Adrenergic agents remain the first choice, namely norepinephrine. However, their use to counteract life-threatening hypotension comes with potential adverse effects, so that non-adrenergic vasopressors may also be considered. The use of agents that act through different mechanisms may also provide an advantage. Nitric oxide (NO) is the main driver of the vasodilation that leads to hypotension in septic shock, so several agents have been tested to counteract its effects. The use of non-selective NO synthase inhibitors has been of questionable benefit. Methylene blue, an inhibitor of soluble guanylate cyclase, an important enzyme involved in the NO signaling pathway in the vascular smooth muscle cell, has also been proposed. However, more than 25 years since the first clinical evaluation of MB administration in septic shock, the safety and benefits of its use are still not fully established, and it should not be used routinely in clinical practice until further evidence of its efficacy is available., (© 2024. The Author(s).)

Ion channels activity is regulated through soluble guanylate cyclase (sGC) and adenylate cyclase (AC) pathways, while phosphodiesterases (PDE) control the intracellular levels of cAMP and cGMP. Here we applied RNA transcriptome sequencing to study changes in the gene expression of the sGC, AC, and PDE isoforms in isolated rat ventricular cardiomyocytes under conditions of microgravity and hypergravity. Our results demonstrate that microgravity reduces the expression of sGC isoform genes, while hypergravity increases their expression. For a subset of AC isoforms, gene expression either increased or decreased under both microgravity and hypergravity conditions. The expression of genes encoding 10 PDE isoforms decreased under microgravity, but increased under hypergravity. However, under both microgravity and hypergravity, the gene expression increased for 7 PDE isoforms and decreased for 3 PDE isoforms. Overall, our findings indicate specific gravity-dependent changes in the expression of genes of isoforms associated with the studied enzymes., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

The pathogenesis of complex diseases such as pulmonary arterial hypertension (PAH) is entirely rooted in changes in the expression of some vasoactive factors. These play a significant role in the onset and progression of the disease. Indeed, PAH has been associated with pathophysiologic alterations in vascular function. These are often dictated by increased oxidative stress and impaired modulation of the nitric oxide (NO) pathway. NO reduces the uncontrolled proliferation of vascular smooth muscle cells that leads to occlusion of vessels and an increase in pulmonary vascular resistances, which is the mainstay of PAH development. To date, two classes of NO-pathway modulating drugs are approved for the treatment of PAH: the phosphodiesterase-5 inhibitors (PD5i), sildenafil and tadalafil, and the soluble guanylate cyclase activator (sGC), riociguat. Both drugs provide considerable improvement in exercise capacity and pulmonary hemodynamics. PD5i are the recommended drugs for first-line PAH treatment, whereas sGCs are also the only drug approved for the treatment of resistant or inoperable chronic thromboembolic pulmonary hypertension. In this review, we will focus on the current information regarding the nitric oxide pathway and its modulation in PAH.

Keloids are characterized by excessive proliferation of fibroblasts and invasion of surrounding healthy skin. High levels of Nitric Oxide (NO) are thought to be the crucial factor within the micro-environment in promoting keloid formation. However, the effects and mechanisms of NO on the proliferation of Keloid Fibroblasts (KDFs) remain unclear. In this study, we investigated the effect of NO on KDFs proliferation by Sodium Nitroprusside (SNP), an NO donor. Our results show that SNP significantly enhanced KDFs proliferation. Moreover, with prolonged treatment with SNP after cell confluence, the growth of KDFs escape contact inhibition and experience significant pile up growth. Furthermore, PTIO, an NO scavenger, attenuated SNP-enhanced cell proliferation effectively. The mechanism involved in SNP-induced KDFs proliferation was soluble Guanylyl Cyclase (sGC) and cGMP independent. ODQ, a specific sGC inhibitor, failed to suppress SNP-enhanced KDFs proliferation. 8-Bromo-c GMP, a cell-permeable cGMP analogue, could not stimulate KDFs proliferation. Erk and Akt provide important signaling for cell growth. U0126 and LY294002, inhibitors of Erk and Akt respectively, block SNP-enhanced KDFs proliferation effectively. As expected, a Western blot showed that SNP up-regulated the phosphorylation levels of Erk and Akt. Moreover, it decreased the expression of p27, a cell cycle inhibitor. Our results reveal that SNP induced KDFs proliferation and loss contact inhibition led to pile up growth via activation of the Erk and Akt pathways, as well as a decreased expression of p27. Thus, we speculate that the pathological feature of continuous expansion in keloids is caused by NO-induced KDFs sustained growth. Image 1 [ABSTRACT FROM AUTHOR]

Migraine is a complex disorder that is characterized by an assortment of neurological and systemic effects. While headache is the most prominent feature of migraine, a host of symptoms affecting many physiological functions are also observed before, during, and after an attack. Furthermore, migraineurs are heterogeneous and have a wide range of responses to migraine therapies. The recent approval of calcitonin gene‐related‐peptide based therapies has opened up the treatment of migraine and generated a renewed interest in migraine research and discovery. Ongoing advances in migraine research have identified a number of other promising therapeutic targets for this disorder. In this review, we highlight emergent treatments within the following biological systems: pituitary adenylate cyclase activating peptdie, 2 non‐mu opioid receptors that have low abuse liability – the delta and kappa opioid receptors, orexin, and nitric oxide‐based therapies. Multiple mechanisms have been identified in the induction and maintenance of migraine symptoms; and this divergent set of targets have highly distinct biological effects. Increasing the mechanistic diversity of the migraine tool box will lead to more treatment options and better patient care. [ABSTRACT FROM AUTHOR]

The nitric oxide (NO) system has been proven to be a valuable modulator of L‐DOPA‐induced dyskinesia in Parkinsonian rodents. NO activates the enzyme soluble guanylyl cyclase and elicits the synthesis of the second‐messenger cGMP. Although we have previously described the anti‐dyskinetic potential of NO synthase inhibitors on L‐DOPA‐induced dyskinesia, the effect of soluble guanylyl cyclase inhibitors remains to be evaluated. The aim of this study was to analyze whether the clinically available non‐selective inhibitor methylene blue, or the selective soluble guanylyl cyclase inhibitor ODQ (1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one), could mitigate L‐DOPA‐induced dyskinesia in 6‐hydroxydopamine‐lesioned rats. Here, we demonstrated that methylene blue was able to reduce L‐DOPA‐induced dyskinesia incidence when chronically co‐administered with L‐DOPA during 3 weeks. Methylene blue chronic (but not acute) administration (2 weeks) was effective in attenuating L‐DOPA‐induced dyskinesia in rats rendered dyskinetic by a previous course of L‐DOPA chronic treatment. Furthermore, discontinuous methylene blue treatment (e.g., co‐administration of methylene blue and L‐DOPA for 2 consecutive days followed by vehicle and L‐DOPA co‐administration for 5 days) was effective in attenuating dyskinesia. Finally, we demonstrated that microinjection of methylene blue or ODQ into the lateral ventricle effectively attenuated L‐DOPA‐induced dyskinesia. Taken together, these results demonstrate an important role of NO‐soluble guanylyl cyclase‐cGMP signaling on L‐DOPA‐induced dyskinesia. The clinical implications of this discovery are expected to advance the treatment options for patients with Parkinson's disease. In this work, we demonstrated an important role of striatal neuronal nitric oxide synthase (nNOS) – nitric oxide (NO) – soluble guanylyl cyclase (sGC) signaling pathway on L‐DOPA‐induced dyskinesia. Our data indicate that inhibition of nNOS/sGC with methylene blue and the specific inhibition of sGC with ODQ were able to attenuate L‐DOPA‐induced dyskinesia in the 6‐OHDA‐lesioned rat model of Parkinson's disease. [ABSTRACT FROM AUTHOR]

Aims Aged arteries are characterized by attenuated vasodilator and enhanced vasoconstrictor responses, which contribute to the development of diseases such as arterial hypertension, atherosclerosis, and heart failure. SIRT1 is a longevity regulator exerting protective functions against vascular ageing, although the underlying mechanisms remain largely unknown. This study was designed to elucidate the signalling pathways involved in endothelial SIRT1-mediated vasodilator responses in the arteries of young and old mice. In particular, the contributions of nitric oxide (NO), endothelial NO synthase (eNOS), cyclooxygenase (COX), and/or soluble guanylyl cyclase (sGC) were examined. Methods and results Wild type (WT) or eNOS knockout (eKO) mice were cross-bred with those overexpressing human SIRT1 selectively in the vascular endothelium (EC-SIRT1). Arteries were collected from the four groups of mice (WT, EC-SIRT1, eKO, and eKO-SIRT1) to measure isometric relaxations/contractions in response to various pharmacological agents. Reduction of NO bioavailability, hyper-activation of COX signalling, and down-regulation of sGC collectively contributed to the decreased vasodilator and increased vasoconstrictor responses in arteries of old WT mice. Overexpression of endothelial SIRT1 did not block the reduction in NO bioavailability but attenuated the hyper-activation of COX-2, thus protecting mice from age-induced vasoconstrictor responses in arteries of EC-SIRT1 mice. Deficiency of eNOS did not affect endothelial SIRT1-mediated anti-contractile activities in arteries of eKO-SIRT1 mice. Mechanistic studies revealed that overexpression of endothelial SIRT1 enhanced Notch signalling to up-regulate sGCβ1 in smooth muscle cells. Increased expression and activity of sGC prevented age-induced hyper-activation of COX-2 as well as the conversion of endothelium-dependent relaxations to contractions in arteries of EC-SIRT1 mice. Conclusion Age-induced down-regulation of sGC and up-regulation of COX-2 in arteries are at least partly attributable to the loss-of-endothelial SIRT1 function. Enhancing the endothelial expression and function of SIRT1 prevents early vascular ageing and maintains vasodilator responses, thus representing promising drug targets for cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Abstract Extensive alterations in splicing is one of the molecular indicator for human cancers. Soluble guanylyl cyclase (sGC), an obligatory heterodimer, is composed of α1 and β1 subunits. Each subunit is encoded by a separate gene, GUCY1a3 and GUCY1b3, correspondingly. sGC activity has been regulated by an alternative splicing and it has an important effect on the breast cancer. sGC alternative splicing has been evaluated in the 55 malignant, 25 benign and 30 normal breast tissues using qRT-PCR and RT-PCR. The differences between groups were analyzed by Mann-Whitney U. The expression of six different splice forms have been detected, three for α1 and three for β1 sGC. Expressions of Tr1, Tr2 β1 sGC and Tr7, Tr6 α1 sGC mRNA in the malignant breast tumors were significantly lower than those of benign and normal breast tissues. However, the expression of Tr3 α1 sGC mRNA was significantly higher than that of benign and normal tissues. Present data have provided some evidences for an alteration in the expression of α1 and β1 sGC alternative splicing forms which may contribute to the loss of sGC functions in the breast cancer. The observed information might be discussed by the cGMP status. Highlights • Soluble guanylate cyclase (sGC) activity has been regulated by an alternative splicing. • Low expressions of Tr1, Tr2 β1 sGC (GUCY1b3) and Tr7, Tr6 α1 sGC (GYCY1a3) mRNA were found in the malignant breast tumors. • High expression of Tr3 α1 sGC mRNA was detected in the malignant breast tumor. • No significant differences were observed for the benign breast tumors. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is a neurotransmitter synthesized in the brain by neuronal nitric oxide synthase (nNOS). Using immunohistochemistry and confocal imaging in the inferior colliculus (IC, auditory midbrain) of the guinea pig (Cavia porcellus, male and female), we show that nNOS occurs in two distinct cellular distributions. We confirm that, in the cortices of the IC, a subset of neurons show cytoplasmic labeling for nNOS, whereas in the central nucleus (ICc), such neurons are not present. However, we demonstrate that all neurons in the ICc do in fact express nNOS in the form of discrete puncta found at the cell membrane. Our multi-labeling studies reveal that nNOS puncta form multiprotein complexes with NMDA receptors, soluble guanylyl cyclase (sGC), and PSD95. These complexes are found apposed to glutamatergic terminals, which is indicative of synaptic function. Interestingly, these glutamatergic terminals express both vesicular glutamate transporters 1 and 2 denoting a specific source of brainstem inputs. With in vivo electrophysiological recordings of multiunit activity in the ICc, we found that local application of NMDA enhances sound-driven activity in a concentration-dependent and reversible fashion. This response is abolished by blockade of nNOS or sGC, indicating that the NMDA effect is mediated solely via the NO and cGMP signaling pathway. This discovery of a ubiquitous, but highly localized, expression of nNOS throughout the ICc and demonstration of the dramatic influence of the NMDA activated NO pathway on sound-driven neuronal activity imply a key role for NO signaling in auditory processing. [ABSTRACT FROM AUTHOR]

Abstract Defects in neuronal nitric oxide synthase (nNOS) splice variant localization and signaling in skeletal muscle are a firmly established pathogenic characteristic of many neuromuscular diseases, including Duchenne and Becker muscular dystrophy (DMD and BMD, respectively). Therefore, substantial efforts have been made to understand and therapeutically target skeletal muscle nNOS isoform signaling. The purpose of this review is to summarize recent salient advances in understanding of the regulation, targeting, and function of nNOSμ and nNOSβ splice variants in normal and dystrophic skeletal muscle, primarily using findings from mouse models. The first focus of this review is how the differential targeting of nNOS splice variants creates spatially and functionally distinct nitric oxide (NO) signaling compartments at the sarcolemma, Golgi complex, and cytoplasm. Particular attention is given to the functions of sarcolemmal nNOSμ and limitations of current nNOS knockout models. The second major focus is to review current understanding of cGMP-mediated nNOS signaling in skeletal muscle and its emergence as a therapeutic target in DMD and BMD. Accordingly, we address the preclinical and clinical successes and setbacks with the testing of phosphodiesterase 5 inhibitors to redress nNOS signaling defects in DMD and BMD. In summary, this review of nNOS function in normal and dystrophic muscle aims to advance understanding how the messenger NO is harnessed for cellular signaling from a skeletal muscle perspective. Highlights • nNOS splice variant diversity, targeting and function in skeletal muscle. • Limitations of current mouse models for deciphering nNOS function. • The role of nNOS in the pathogenesis of Duchenne muscular dystrophy. • cGMP-dependent nNOS signaling as a therapeutic target in Duchenne muscular dystrophy. • Limitations of phosphodiesterase 5 inhibitor therapy for Duchenne muscular dystrophy. [ABSTRACT FROM AUTHOR]

Garlic (Allium sativum L.) has long been used as an anti-hypertensive. The study investigated the effect of fermented garlic extract (Fgarlic) on systolic blood pressure (SBP) and its molecular basis using spontaneously hypertensive rats (SHRs). Fgarlic contained high content of stable nitrite (0.975 mg nitrite/ml). Acute feeding of different amounts (0.3, 0.6, and 0.9 ml) of concentrated Fgarlic (9.75 mg nitrite/ml) reduced SBP dose-dependently. Chronic feeding of Fgarlic (containing 27 mg nitrite/day) for 12 days reduced SBP with increased expressions of eNOS and PKG proteins in aortic tissues, which were attenuated by a soluble guanylyl cyclase (sGC) inhibitor. The relaxation responses of thoracic aorta to acetylcholine and sodium nitroprussside were improved in SHRs fed Fgarlic. These results suggest that nitrite in Fgarlic, which converts to NO in the body, functions as an anti-hypertensive molecule and its effect is mediated through sGC-cGMP-PKG pathway.

Vascular cognitive impairment (VCI) describes neurodegenerative disorders characterized by a vascular component. Pathologically, it involves decreased cerebral blood flow (CBF), white matter lesions, endothelial dysfunction, and blood-brain barrier (BBB) impairments. Molecularly, oxidative stress and inflammation are two of the major underlying mechanisms. Nitric oxide (NO) physiologically stimulates soluble guanylate cyclase (sGC) to induce cGMP production. However, under pathological conditions, NO seems to be at the basis of oxidative stress and inflammation, leading to a decrease in sGC activity and expression. The native form of sGC needs a ferrous heme group bound in order to be sensitive to NO (Fe(II)sGC). Oxidation of sGC leads to the conversion of ferrous to ferric heme (Fe(III)sGC) and even heme-loss (apo-sGC). Both Fe(III)sGC and apo-sGC are insensitive to NO, and the enzyme is therefore inactive. sGC activity can be enhanced either by targeting the NO-sensitive native sGC (Fe(II)sGC), or the inactive, oxidized sGC (Fe(III)sGC) and the heme-free apo-sGC. For this purpose, sGC stimulators acting on Fe(II)sGC and sGC activators acting on Fe(III)sGC/apo-sGC have been developed. These sGC agonists have shown their efficacy in cardiovascular diseases by restoring the physiological and protective functions of the NO-sGC-cGMP pathway, including the reduction of oxidative stress and inflammation, and improvement of vascular functioning. Yet, only very little research has been performed within the cerebrovascular system and VCI pathology when focusing on sGC modulation and its potential protective mechanisms on vascular and neural function. Therefore, within this review, the potential of sGC as a target for treating VCI is highlighted., Competing Interests: Declaration of Competing Interest JP has a proprietary interest in sGC agonists in the treatment of cognitive impairments. PS is a full time employee of Bayer AG, Pharmaceuticals. All other authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Portal hypertension is a severe complication of cirrhosis. This Phase Ib study (NCT03842761) assessed the safety, tolerability, and pharmacokinetics of soluble guanylyl cyclase activator BI 685509 in patients with mild or moderate hepatic impairment (Child-Pugh [CP] A or B cirrhosis) and healthy volunteers (HVs)., Methods: In this single-center, randomized, placebo-controlled study, patients received BI 685509 (maximum doses: 1, 2, or 3 mg, twice daily [BID]) or placebo for 28 days. HVs received one 0.5 mg dose of BI 685509 or placebo., Results: In total, 64 participants (CP-A, n=24; CP-B, n=25; HVs, n=15) were included; most commonly with NAFLD (36.7%), alcohol-associated (30.6%), or chronic viral hepatitis-related cirrhosis (28.6%). In patients with CP-A cirrhosis, drug-related adverse events (AEs) occurred in 5.6% of BI 685509-treated patients and 16.7% of placebo recipients. In patients with CP-B cirrhosis, drug-related AEs occurred in 26.3% of BI 685509-treated patients only. No serious AEs occurred in patients with CP-A cirrhosis; in patients with CP-B cirrhosis, serious AEs (not drug-related) occurred in 10.5% of BI 685509-treated patients and 16.7% of patients receiving placebo. BI 685509 was rapidly absorbed; exposure increased with dosage and was similar between etiologies and between patients with CP-A cirrhosis and patients with CP-A cirrhosis but lower in HVs. The mean percentage portal-systemic shunt fraction was measured in patients with CP-A cirrhosis and decreased at the end of treatment in the 2 mg BID (-11.2 ± 11.9%) and 3 mg BID (-14.0 ± 8.4%) BI 685509 dose groups, but not in the placebo group (+1.0 ± 27.3%)., Conclusion: BI 685509 was generally well tolerated, with 3 serious, not drug-related AEs reported in patients with CP-B cirrhosis. In patients with CP-A cirrhosis, portal-systemic shunt fraction in the exploratory efficacy analysis was reduced by 2 mg BID and 3 mg BID BI 685509., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

We studied the action of a new indolinone derivative GRS, acetylsalicylic acid (ASA), and their combination on platelet aggregation, vasodilatory endothelial function, neurological status, and cerebral infarction area in experimental focal cerebral ischemia/reperfusion in rats. GRS compound (10 mg/kg), ASA (10 mg/kg), and their combination in the same doses were administered orally once a day as a suspension in 1% starch solution over 5 days after pathology modeling. Sham-operated and control animals were administered 1% starch solution. On day 5 after pathology modeling, platelet aggregation and brain damage area were studied in a half of rats in each group, and the vasodilatory function of the endothelium was studied in the other half. Neurological deficit was assessed 4 h and 1, 3, and 5 days after pathology modeling. GRS compound and ASA equally effectively prevent platelet aggregation and the development of neurological deficit in rats. GRS compound restores the vasodilatory effects of the endothelium, but only ASA contributes to reduction of the cerebral infarction area. In case of combined administration, GRS and ASA do not exhibit synergy in their antiaggregant effect., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Background and Purpose: First-generation soluble guanylate cyclase (sGC) stimulators have shown clinical benefit in pulmonary hypertension (riociguat) and chronic heart failure (vericiguat). However, given the broad therapeutic opportunities for sGC stimulators, tailored molecules for distinct indications are required., Experimental Approach: We report the high-throughput screening (HTS)-based discovery of a second generation of sGC stimulators from a novel imidazo[1,2-a]pyridine lead series. An intense medicinal chemistry programme resulted in the discovery of the sGC stimulator BAY 1165747 (BAY-747). The pharmacokinetic profile of BAY-747 was determined in different species, and it was broadly characterized in pharmacological model systems relevant for vasodilatation and hypertension., Key Results: BAY-747 is a highly potent sGC stimulator in vitro. In addition, BAY-747 showed an excellent pharmacokinetic profile with long half-life and low peak-to-trough ratio. BAY-747 was investigated in experimental in vivo models of malignant and resistant hypertension (rHT). In spontaneously hypertensive (SH) rats, BAY-747 caused a dose-related and long-lasting decrease in mean arterial blood pressure (MAP). Oral treatment over 12 days resulted in a persistent decrease. BAY-747 provided additional benefit when dosed on top of losartan, amlodipine or spironolactone and even on top of triple combinations of frequently used antihypertensive drugs. In a new canine model of rHT, BAY-747 caused a dose-related and long-lasting (>6 h) MAP decrease., Conclusion and Implications: BAY-747 is a potent, orally available sGC stimulator. BAY-747 shows long-acting pharmacodynamic effects with a very low peak-to-trough ratio. BAY-747 could be a treatment alternative for patients with hypertension, especially those not responding to standard-of-care therapy., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Nitric oxide (NO) is an endogenously produced signaling molecule that regulates blood flow and platelet activation. However, intracellular and intravascular diffusion of NO are limited by scavenging reactions with several hemoproteins, raising questions as to how free NO can signal in hemoprotein-rich environments. We explore the hypothesis that NO can be stabilized as a labile ferrous heme-nitrosyl complex (Fe 2+ -NO, NO-ferroheme). We observe a reaction between NO, labile ferric heme (Fe 3+ ) and reduced thiols to yield NO-ferroheme and a thiyl radical. This thiol-catalyzed reductive nitrosylation occurs when heme is solubilized in lipophilic environments such as red blood cell membranes or bound to serum albumin. The resulting NO-ferroheme resists oxidative inactivation, is soluble in cell membranes and is transported intravascularly by albumin to promote potent vasodilation. We therefore provide an alternative route for NO delivery from erythrocytes and blood via transfer of NO-ferroheme and activation of apo-soluble guanylyl cyclase., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Chronic smoking causes dysfunction of vascular endothelial cells, evident as a reduction of flow-mediated dilation in smokers, but the role of nicotine is still controversial. Given the increasing use of e-cigarettes and other nicotine products, it appears essential to clarify this issue. We studied extracts from cigarette smoke (CSE) and vapor from e-cigarettes (EVE) and heated tobacco (HTE) for their effects on vascular relaxation, endothelial nitric oxide signaling, and the activity of soluble guanylyl cyclase. The average nicotine concentrations of CSE, EVE, and HTE were 164, 800, and 85 µM, respectively. At a dilution of 1:3, CSE almost entirely inhibited the relaxation of rat aortas and porcine coronary arteries to acetylcholine and bradykinin, respectively, while undiluted EVE, with a 15-fold higher nicotine concentration, had no significant effect. With about 50% inhibition at 1:2 dilution, the effect of HTE was between CSE and EVE. Neither extract affected endothelium-independent relaxation to an NO donor. At the dilutions tested, CSE was not toxic to cultured endothelial cells but, in contrast to EVE, impaired NO signaling and inhibited NO stimulation of soluble guanylyl cyclase. Our results demonstrate that nicotine does not mediate the impaired endothelium-dependent vascular relaxation caused by smoking., (© 2023. Springer Nature Limited.)

Red blood cells (RBCs) mediate cardioprotection via nitric oxide-like bioactivity, but the signaling and the identity of any mediator released by the RBCs remains unknown. We investigated whether RBCs exposed to hypoxia release a cardioprotective mediator and explored the nature of this mediator. Perfusion of isolated hearts subjected to ischemia-reperfusion with extracellular supernatant from mouse RBCs exposed to hypoxia resulted in improved postischemic cardiac function and reduced infarct size. Hypoxia increased extracellular export of cyclic guanosine monophosphate (cGMP) from mouse RBCs, and exogenous cGMP mimicked the cardioprotection induced by the supernatant. The protection induced by hypoxic RBCs was dependent on RBC-soluble guanylate cyclase and cGMP transport and was sensitive to phosphodiesterase 5 and activated cardiomyocyte protein kinase G. Oral administration of nitrate to mice to increase nitric oxide bioactivity further enhanced the cardioprotective effect of hypoxic RBCs. In a placebo-controlled clinical trial, a clear cardioprotective, soluble guanylate cyclase-dependent effect was induced by RBCs collected from patients randomized to 5 weeks nitrate-rich diet. It is concluded that RBCs generate and export cGMP as a response to hypoxia, mediating cardioprotection via a paracrine effect. This effect can be further augmented by a simple dietary intervention, suggesting preventive and therapeutic opportunities in ischemic heart disease.