Challenging the Norm: The Unrecognized Impact of Soluble Guanylyl Cyclase Subunits in Cancer.

Since the discovery of nitric oxide (NO), a long journey has led us to the present, during which much knowledge has been gained about its pathway members and their roles in physiological and various pathophysiological conditions. Soluble guanylyl cyclase (sGC), the main NO receptor composed of the sGCα1 and sGCß1 subunits, has been one of the central figures in this narrative. However, the sGCα1 and sGCß1 subunits remained obscured by the focus on sGC's enzymatic activity for many years. In this review, we restore the significance of the sGCα1 and sGCß1 subunits by compiling and analyzing available but previously overlooked information regarding their roles beyond enzymatic activity. We delve into the basics of sGC expression regulation, from its transcriptional regulation to its interaction with proteins, placing particular emphasis on evidence thus far demonstrating the actions of each sGC subunit in different tumor models. Exploring the roles of sGC subunits in cancer offers a valuable opportunity to enhance our understanding of tumor biology and discover new therapeutic avenues.

Soluble guanylyl cyclase beta1 subunit targets epithelial-to-mesenchymal transition and downregulates Akt pathway in human endometrial and cervical cancer cells.

Endometrial and cervical cancer are among the most frequently diagnosed malignancies globally. Nitric oxide receptor-soluble guanylyl cyclase (sGC) is a heterodimeric enzyme composed of two subunits, α1 and ß1. Previously we showed that sGCα1 subunit promotes cell survival, proliferation, and migration, but the role of sGCß1 subunit has not been addressed. The aim of the present work was to study the impact of sGCß1 restoration in proliferation, survival, migration, and cell signaling in endometrial and cervical cancer cells. We found that sGCß1 transcript levels are reduced in endometrial and cervical tumors vs normal tissues. We confirmed nuclear enrichment of sGCß1, unlike sGCα1. Overexpression of sGCß1 reduced cell viability and augmented apoptotic index. Cell migration and invasion were also negatively affected. All these sGCß1-driven effects were independent of sGC enzymatic activity. sGCß1 reduced the expression of epithelial-to-mesenchymal transition factors such as N-cadherin and ß-catenin and increased the expression of E-cadherin. sGCß1 impacted signaling in endometrial and cervical cancer cells through significant downregulation of Akt pathway affecting some of its main targets such as GSK-3ß and c-Raf. Our results show for the first time that sGCß1 exerts several antiproliferative actions in ECC-1 and HeLa cell lines by targeting key regulatory pathways.

From End-of-Life Care to Improved Quality of Life and Better Prognosis by Using Vericiguat: A Case Report From Costa Rica.

In this case report, we present the evolution of a heart failure with reduced ejection fraction (HFrEF) patient who was set to receive end-of-life care but demonstrated improvement following treatment with vericiguat in combination with foundational therapy. Vericiguat is a novel soluble guanylate cyclase stimulant that has been proven helpful for treating decompensated heart failure with HFrEF, decreasing hospitalization rates and mortality of cardiovascular causes. This medication is currently indicated in patients who require IV diuretics administration or hospitalization due to decompensated heart failure. This is a case study of a 62-year-old woman with dilated heart failure and reduced left ventricular ejection fraction (LVEF), who was a wheelchair user due to severe cardiovascular symptoms and various comorbidities, who was referred to our heart failure program for treatment. Despite previous treatment, the patient experienced persistent cardiovascular symptoms and required palliative care. After optimizing the foundational therapy, the patient's condition improved but continued to require hospitalization. Vericiguat was initiated as an add-on. After six months, the patient's LVEF improved by 9%, and she is now asymptomatic with a considerable decrease in pro-B-type natriuretic peptide levels and is wheelchair independent due to enhance exercise resistance. However, the echocardiogram revealed a progression in the dysfunction of both the mitral and aortic valves. The patient's renal function and quality of life scores also changed over time. Vericiguat therapy, as an adjunct to foundational therapy, improved exercise tolerance and symptom relief. However, further investigation is necessary to assess the effects of vericiguat on renal function and disease progression in individuals with HFrEF.

Oxygen sensing in crustaceans: functions and mechanisms.

Animals that live in changing environments need to adjust their metabolism to maintain body functions, and sensing these changing conditions is essential for mediating the short- and long-term physiological and behavioral responses that make these adjustments. Previous research on nematodes and insects facing changing oxygen levels has shown that these animals rapidly respond using atypical soluble guanylyl cyclases (sGCs) as oxygen sensors connected to downstream cGMP pathways, and they respond more slowly using hypoxia-inducible transcription factors (HIFs) that are further modulated by oxygen-sensing prolyl hydroxylases (PHs). Crustaceans are known to respond in different ways to hypoxia, but the mechanisms responsible for sensing oxygen levels are more poorly understood than in nematodes and insects. Our paper reviews the functions of and mechanisms underlying oxygen sensing in crustaceans. Furthermore, using the oxygen sensing abilities of nematodes and insects as guides in analyzing available crustacean transcriptomes, we identified orthologues of atypical sGCs, HIFs, and PHs in crustaceans, including in their chemosensory organs and neurons. These molecules include atypical sGCs activated by hypoxia (Gyc-88E/GCY-31 and Gyc-89D/GCY-33) but not those activated by hyperoxia (GCY-35, GCY-36), as well as orthologues of HIF-α, HIF-ß, and PH. We offer possible directions for future research on oxygen sensing by crustaceans.

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.

Soluble Guanylyl Cyclase Alpha1 Subunit: A New Marker for Estrogenicity of Endocrine Disruptor Compounds.

Endocrine-disrupting chemicals (EDCs) include widespread naturally occurring and synthetic substances in the environment that adversely affect humans and wildlife. Because of the increasing numbers of EDCs, screening methods and ideal biomarkers to determine EDC potencies at relevant environmental concentrations need to be drastically improved. Soluble guanylyl cyclase α1 subunit (sGCα1) is an abundant cytosolic protein ubiquitously expressed in most tissues. We previously showed that sGCα1 is specifically and highly up-regulated by estrogen (E2) in vivo and in vitro, even though it lacks estrogen-responsive elements. The aim of the present study was to evaluate sGCα1 protein expression as a potential marker for xenoestrogenic EDC exposure in the E2-responsive lactosomatotroph-derived pituitary cell line GH3. Cells were incubated with a wide variety of EDCs such as heavy metals and a metalloid, synthetic E2 derivatives, plastic byproducts, and pesticides at a range of doses including those with proven xenoestrogenic activity. We demonstrated that E2 increased sGCα1 expression in GH3 cells as well as in other E2-responsive tumor cell lines. Moreover, this effect was fully dependent on estrogen receptor (ER) activation. Importantly, sGCα1 protein levels were strongly up-regulated by all the EDCs tested, even by those exhibiting low or null ER binding capacity. We provide evidence that the in vitro sGCα1 protein assay may be a very sensitive and powerful tool to identify compounds with estrogenic activity, which could improve current mammalian-based screening methods. Environ Toxicol Chem 2019;38:2719-2728. © 2019 SETAC.

Repurposing an established drug: an emerging role for methylene blue in L-DOPA-induced dyskinesia.

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.

Balance between S-nitrosylation and denitrosylation modulates myoblast proliferation independently of soluble guanylyl cyclase activation.

Nitric oxide (NO) contributes to myogenesis by regulating the transition between myoblast proliferation and fusion through cGMP signaling. NO can form S-nitrosothiols (RSNO), which control signaling pathways in many different cell types. However, neither the role of RSNO content nor its regulation by the denitrosylase activity of S-nitrosoglutathione reductase (GSNOR) during myogenesis is understood. Here, we used primary cultures of chick embryonic skeletal muscle cells to investigate whether changes in intracellular RSNO alter proliferation and fusion of myoblasts in the presence and absence of cGMP. Cultures were grown to fuse most of the myoblasts into myotubes, with and without S-nitrosocysteine (CysNO), 8-Br-cGMP, DETA-NO, or inhibitors for NO synthase (NOS), GSNOR, soluble guanylyl cyclase (sGC), or a combination of these, followed by analysis of GSNOR activity, protein expression, RSNO, cGMP, and cell morphology. Although the activity of GSNOR increased progressively over 72 h, inhibiting GSNOR (by GSNOR inhibitor - GSNORi - or by knocking down GSNOR with siRNA) produced an increase in RSNO and in the number of myoblasts and fibroblasts, accompanied by a decrease in myoblast fusion index. This was also detected with CysNO supplementation. Enhanced myoblast number was proportional to GSNOR inhibition. Effects of the GSNORi and GSNOR knockdown were blunted by NOS inhibition, suggesting their dependence on NO synthesis. Interestingly, GSNORi and GSNOR knockdown reversed the attenuated proliferation obtained with sGC inhibition in myoblasts, but not in fibroblasts. Hence myoblast proliferation is enhanced by increasing RSNO, and regulated by GSNOR activity, independently of cGMP production and signaling.

Cross-talking between lymphocytes and platelets and its regulation by nitric oxide and peroxynitrite in physiological condition and endotoxemia.

AIMS: Cross-talk between platelets and lymphocytes may play a role in different pathological conditions like sepsis. This study aimed to investigate the effect of lymphocytes on platelet aggregation in lipopolysaccharide (LPS)-stimulated and non-stimulated cells. MAIN METHODS: Lymphocytes and platelet-rich plasma (PRP) were obtained from rat arterial blood. Platelets (1.2×108platelets/ml) were incubated with lymphocytes (0.8×106cells/ml) in the presence or not of LPS (100µg/ml), after which ADP (5µM)-induced platelet aggregation was carried out. KEY FINDINGS: Lymphocytes inhibited by 51% the platelet aggregation, which was significantly prevented by the non-selective NO inhibitor l-NAME (300µM) or the selective iNOS inhibitor 1400W (100µM), as well as by the soluble guanylyl cyclase (sGC) inhibitor ODQ (10µM). The platelet inhibition by lymphocytes was accompanied by 2-fold increase of intraplatelet cGMP levels. Next, lymphocytes and platelets were co-incubated with LPS for 6h. In LPS-treated cells, lymphocytes produced a larger inhibition of platelet aggregation (62%), despite the same elevation of cGMP levels (2.2-fold increase). This inhibitory effect was prevented by l-NAME and 1400W, but rather unaffected by ODQ. The peroxynitrite (ONOO-) scavenger -(-)epigallocatechin gallate (ECG, 100µM) abolished the inhibition by lymphocytes on platelet aggregation in LPS-treated cells, but not in non-treated cells. SIGNIFICANCE: Our results show that lymphocytes act to inhibit platelet aggregation via iNOS-derived NO release and cGMP generation. In presence of LPS, ONOO- production accounts for the platelet inhibition.

From Escherichia coli heat-stable enterotoxin to mammalian endogenous guanylin hormones

The isolation of heat-stable enterotoxin (STa) from Escherichia coli and cholera toxin from Vibrio cholerae has increased our knowledge of specific mechanisms of action that could be used as pharmacological tools to understand the guanylyl cyclase-C and the adenylyl cyclase enzymatic systems. These discoveries have also been instrumental in increasing our understanding of the basic mechanisms that control the electrolyte and water balance in the gut, kidney, and urinary tracts under normal conditions and in disease. Herein, we review the evolution of genes of the guanylin family and STa genes from bacteria to fish and mammals. We also describe new developments and perspectives regarding these novel bacterial compounds and peptide hormones that act in electrolyte and water balance. The available data point toward new therapeutic perspectives for pathological features such as functional gastrointestinal disorders associated with constipation, colorectal cancer, cystic fibrosis, asthma, hypertension, gastrointestinal barrier function damage associated with enteropathy, enteric infection, malnutrition, satiety, food preferences, obesity, metabolic syndrome, and effects on behavior and brain disorders such as attention deficit, hyperactivity disorder, and schizophrenia.

The soluble guanylyl cyclase activator BAY 60-2770 ameliorates overactive bladder in obese mice.

PURPOSE: Activators of soluble guanylyl cyclase are of potential interest as treatment for cardiovascular diseases but to our knowledge they have never been proposed to treat overactive bladder. We evaluated the effects of the soluble guanylyl cyclase activator BAY 60-2270 on voiding dysfunction and detrusor overactivity in a mouse model of obesity associated overactive bladder. MATERIALS AND METHODS: C57BL/6 male mice fed for 10 weeks with standard chow or a high fat diet were treated with 1 mg/kg BAY 60-2770 per day for 2 weeks via gavage. Cystometric evaluations were done and responses to contractile agents in isolated bladders were determined. RESULTS: Obese mice showed an irregular micturition pattern characterized by significant increases in voiding and nonvoiding contractions, which were normalized by BAY 60-2770. Carbachol, KCl and CaCl2 produced concentration dependent contractions in isolated bladder strips, which were markedly greater in obese than in lean mice. BAY 60-2770 normalized bladder contractions in the obese group. A 78% increase in reactive oxygen species generation in the bladder tissue of obese mice was observed, which was unaffected by BAY 60-2770. Treatment with BAY 60-2770 generated a tenfold increase in cyclic guanosine monophosphate in the bladders of obese mice without affecting the nucleotide level in the lean group. Protein expression of the soluble guanylyl cyclase α1 and ß1 subunits was decreased 40% in the bladder tissue of obese mice but restored by BAY 60-2770. CONCLUSIONS: Two-week BAY 60-2770 therapy increased cyclic guanosine monophosphate and rescued expression of the soluble guanylyl cyclase α1 and ß1 subunits in bladder tissue, resulting in great amelioration of bladder dysfunction.

Selective mastoporan inhibition of muscarinic activation of bovine tracheal smooth muscle

Muscarinic activation of bovine tracheal smooth muscle (BTSM) leading to smooth muscle contraction involves the generation of two cGMP signals (20 and 60 s), being 20s peak associated with soluble (sGC) and the second (60s) to membrane-bound Natriuretic Peptide- receptor-Guanylylcy clases (NPR-GC). In this study, we showed that pre-incubation of isolated BTSM strips with mastoparan and superactive mastoparan (mastoparan 7) decreased significantly the muscarinic dependent contractile smooth muscle responses in dose-dependent and non-competitive manner. Moreover, mastoparan (50 nM) inhibited completely the BTSM-muscarinic contractile responses and affected dramatically the carbachol-dependent cGMP signals being the first cGMP signal inhibited in a 63 ± 5%, whereas the second signal disappeared. Mastoparan inhibition of muscarinic activation is specific since other spasmogens as serotonin and histamine fully contracted these BTSM strips under mastoparan treatment. Cyclic GMP levels were evaluated by exposing BTSM strips to activators of NO-sensitive sGC as Sodium Nitroprussiate (SNP) and Natriuretic Peptides as CNP-53 for membrane-bound NPR-GC. Thus, SNP and CNP increased in a binary way, in more than 20 fold cGMP levels at 30-40 s being both increments inhibited by mastoparan. Furthermore, the Gi/o-protein involvement on mastoparan inhibition of cGMP elevations induced by CNP and SNP is suggested by Pertussis toxin pre-treatment, which reversed mastoparan effects. These results indicate that muscarinic signal transduction cascades leading to airway smooth muscle contractions involved two different guanylyl cyclases being both regulated by mastoparan-sensitive G-proteins. ANP, Natriuretic Peptide type A; ASM, Airway Smooth Muscle; BTSM, Bovine Tracheal Smooth Muscle; CNP-53, Natriuretic Peptide type C-53; GPCR, G-Protein Coupled Receptor; Gq16, Heterotrimeric G protein subtype 16; Gi/o, Heterotrimeric G protein subtype...

La activación muscarínica del músculo liso de las vías aéreasrelacionada a la contracción de dicho músculo liso esta asociada a la generación de dos señales de GMPc (20 y 60 s), siendo la señal de los 20s relacionado a la activación de la guanililciclasa soluble mientras que el pico de los 60s a la guanililciclasa unida membranas y sensible a péptidos natriuréticos (NPR-GC). En este trabajo, nosotros mostramos que la pre-incubación de fragmentos del músculo liso traqueal de bovino (BTSM) con mastoparan y su análogo superactivo (mastoparan 7), en una forma dosis dependiente, son capaces de disminuir de manera significativa la actividad contráctil dependiente de agentes muscarinicos. Adicionalmente, mastoparan (50 nM) inhibió completamente la respuesta contráctil muscarinica del BTSM y afectó dramáticamente los picos de GMPc asociados a la activación muscarinica siendola primera señal inhibida en un 63 ± 5%, mientras que la segunda señal desapareció completamente. Esta inhibición del mastoparan de la activación muscarínica es especifica ya que otros espamogenos como la serotonina y la histamina fueron capaces de inducir respuestas máximas en presencia del mastoparan y su análogos. Este efecto del mastoparan sobre los niveles del GMPc fue evaluado en presencia de otros agentes generadores de este segundo mensajero como son el nitroprusiato de sodio (SNP) que activa la guanililciclasa soluble sensible a NO y los péptidos natriureticos como el CNP-53 (CNP) activador de la NPR-GC asociada a membranas plasmáticas. Tanto, el SNP como el CNP aumentaronen mas de 50 veces los niveles de GMPc a los 30-40 s en forma bifasica, siendo estos incrementos inhibidos de manera significativa por el mastoparan. Ademas, se sugiere la participación de proteínas Gi/o en los efectos inhibitoriosdel mastoparan, porque la Toxina pertussis revertió los efectos inhibitorios. Estos resultados indican que la cascada de activación muscarinica que conduce...