Activation Of Soluble Guanylate Cyclase Research Articles

A Novel Selective Soluble Guanylate Cyclase Activator, MGV354, Lowers Intraocular Pressure in Preclinical Models, Following Topical Ocular Dosing.

The nitric oxide/soluble guanylate cyclase/protein kinase G (NO/sGC/PKG) is known to be involved in the regulation of intraocular pressure (IOP) and may be dysregulated in glaucoma. The purpose is to demonstrate that the sGC activator MGV354 lowers IOP in a monkey model of glaucoma and could be considered as a possible new clinical drug candidate. Changes to cGMP were assessed in primary human trabecular meshwork (hNTM) cells and binding studies were conducted using human sGC full-length protein. Ocular safety tolerability, exposure, and efficacy studies were conducted in rabbit and monkey models following topical ocular dosing of MGV354. sGC was highly expressed in the human and cynomolgus monkey outflow pathways. MGV354 had a 7-fold greater Bmax to oxidized sGC compared to that of reduced sGC and generated an 8- to 10-fold greater cGMP compared to that of a reduced condition in hTM cells. A single topical ocular dose with MGV354 caused a significant dose-dependent reduction of 20% to 40% (versus vehicle), lasting up to 6 hours in pigmented rabbits and 24 hours postdose in a cynomolgus monkey model of glaucoma. The MGV354-induced IOP lowering was sustained up to 7 days following once-daily dosing in a monkey model of glaucoma and was greater in magnitude compared to Travatan (travoprost)-induced IOP reduction. Mild to moderate ocular hyperemia was the main adverse effect noted. MGV354 represents a novel class of sGC activators that can lower IOP in preclinical models of glaucoma. The potential for sGC activators to be used as effective IOP-lowering drugs in glaucoma patients could be further determined in clinical studies.

Smooth Muscle Cullin‐3 Deficiency Causes Vascular Dysfunction, Arterial Stiffness and Severe Hypertension

Cullin‐3 (CUL3) mutations resulting in exon 9 skipping (CUL3D9) cause human hypertension. We recently showed that selective expression of CUL3D9 protein in smooth muscle caused mild hypertension. Herein, we hypothesized that complete genetic deletion of CUL3 in smooth muscle would cause severe hypertension. Mice carrying a conditional allele of CUL3 were bred with mice expressing a tamoxifen‐inducible CRE‐recombinase driven by a smooth muscle myosin heavy chain promoter. Mice were administered tamoxifen i.p. (75 mg/kg) for 5 consecutive days to generate smooth muscle CUL3 knockout mice (S‐CUL3KO). CUL3 protein was undetectable whereas Cullin‐1 and Cullin‐5 proteins were preserved in aorta. CUL3 protein was also preserved in heart and kidney. We assessed vascular function in the cerebral basilar artery and aorta using pressurized and wire myograph, respectively. Pulse wave velocity (PWV) as index of aortic stiffness was measured by Doppler ultrasound, and blood pressure (BP) was measured by radiotelemetry. Aorta from S‐CUL3KO mice exhibited extremely impaired vasorelaxation to acetylcholine (ACh) compared to controls (at 100 μM: 1±3% vs 77±5%, p<0.0001), and to the nitric oxide donor sodium nitroprusside (SNP) (at 100 μM: 15±4% vs 96±1%, p<0.001). Cerebral basilar artery from S‐CUL3KO mice also exhibited equivalent impairment to ACh‐ and SNP‐mediated vasorelaxation. Although RhoA/Rho‐kinase signaling was significantly elevated in aorta from S‐CUL3KO mice, pre‐incubation with a specific Rho‐kinase inhibitor, Y27632 did not restore vasorelaxation suggesting importance of an alternative pathway in response to CUL3 deletion. Conversely, S‐CUL3KO aorta exhibited only modestly impaired relaxation to a cGMP analogue (8‐pCPT‐cGMP, at 100 μM: 70±3% vs 98±2% controls) and to the heme‐independent soluble guanylate cyclase activator (BAY 58–2667, at 10 μM: 94±2% vs 101±1% controls) with a preservation of maximal relaxation. Aorta from S‐CUL3KO mice and CUL3‐deficient primary aortic smooth muscle cells (AoSMC) exhibited a significant two‐fold reduction in the expression of guanylate cyclase β1 and α1 subunits. As consequence, CUL3‐deficient primary AoSMC exhibited impaired cGMP production following stimulation with SNP (baseline cGMP pmol/mg protein: 0.9±0.1 vs 1.3±0.4 controls, SNP‐induced cGMP pmol/mg protein: 3.4±0.5 vs 8.7±1.6 controls). Interestingly, systolic BP (SBP) progressively increased in S‐CUL3KO mice upon tamoxifen‐induced CUL3 deletion (2 weeks SBP mmHg: 145±1 vs 115±2, p<0.001; 4 weeks SBP: 169±1 vs 115±3, p<0.001, n=6/genotype). Pulse wave velocity was also significantly increased in S‐CUL3KO mice (3.7±0.1 m/s vs 2.2±0.1, P<0.001), suggesting increased arterial stiffness. We conclude that smooth muscle CUL3 is a major BP determinant, and identifying novel CUL3 substrates in smooth muscle would be beneficial in the search for new anti‐hypertensive targets.Support or Funding InformationThis project is funded by R01 HL125603 and R01 HL131689 to Dr. Curt Sigmund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Renal Nitric Oxide‐Dependent Mechanisms in Early Sunitinib‐Induced Hypertension

Sunitinib is an antiangiogenic receptor tyrosine kinase inhibitor used in cancer treatment. A major adverse effect of sunitinib is a rapidly developing hypertension associated with reduced renal nitric oxide (NO) availability and increased renal fractional sodium reabsorption (FRNa). We tested if NO‐dependent regulation of renal hemodynamics and FRNa are compromised and if nitrate supplementation has antihypertensive effects in early sunitinib‐induced hypertension.Male Wistar rats were treated with sunitinib at 15 mg/(kg*d). Four days after initiation of sunitinib treatment, renal hemodynamics and renal sodium excretion were investigated. Radiotelemetric experiments were performed to study the effects of nitrate supplementation and soluble guanylate cyclase (sGC) activation on arterial pressure. Nitric oxide synthase (NOS) as well as sGC mRNA and protein abundances were studied in renal tissue.Within four days, sunitinib elicited a 20–25 mmHg arterial pressure rise. NOS inhibition elicited similar reductions in total and renal medullary blood flow in sunitinib‐ and vehicle‐treated rats. NO‐dependent and independent components of endothelium‐dependent renal vasodilation were not compromised in early sunitinib‐induced hypertension. Low dose intrarenal NOS inhibition did not differentially affect FRNa in sunitinib‐ and vehicle‐treated rats. In sunitinib‐treated rats, renal nitrite/nitrate excretion was 60 per cent less than in controls (p < 0.05). However, nitrate supplementation did not reduce arterial pressure and renal sodium balance in sunitinib‐treated rats. While sunitinib treatment had no effects on renal NOS mRNA abundances, it significantly reduced renal cortical sGC α‐ and β‐subunit mRNA abundances as well as renal cortical guanylate cyclase protein expression. Pharmacological sGC activation caused a rapid 15–20 mmHg arterial pressure fall in sunitinib‐treated rats (p < 0.05).We conclude that the NO‐dependent regulation of renal hemodynamics is largely unaltered in early sunitinib‐induced hypertension despite evidence for reduced NO formation. Reduced renal sGC expression and activity may contribute to the early arterial pressure rise in sunitinib‐induced hypertension and cause its nitrate resistance.Support or Funding InformationSupported by the German Research Foundation, Grant‐No. KO 4936/2‐1.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

GENIe: A bright new genetically‐encoded fluorescent biosensor for monitoring cGMP

GENIe is a Genetically Encoded Nucleotide Indicator for monitoring cGMP, a second messenger in cell signaling that mediates many important physiological processes. Phosphodiesterase (PDE) inhibitors and soluble guanylate cyclase (sGC) activators modulate cGMP levels to produce beneficial effects on pulmonary and heart disease, erectile dysfunction, and other conditions. The timing, location and strength of cGMP signaling is tightly regulated by nearly 100 isoforms of cyclic nucleotide PDEs (Bender and Bravo 2006). Some isoforms are specific for cGMP, while others are specific for cAMP. Some are nonspecific and act on both cAMP and cGMP.We have developed a new, very bright mNeonGreen fluorescent biosensor to monitor cGMP levels in any living cell type. The sensor produces robust changes in fluorescence intensity that are readily detected on fluorescence imaging systems or plate readers. Since different PDE isoforms have different substrate specificities, we did parallel assays of cGMP and cAMP in HEK293 cells expressing either the cGMP biosensor (GENIe) or the cAMP biosensor (cADDis). These sensors produced markedly different fluorescent profiles of cGMP and cAMP responses when treated with different PDE inhibitors. Our results indicate that the combination of these assays provide useful insights into the specific action of a particular PDE inhibitor in a living cell. Since these sensors can be genetically targeted, and since many biologically relevant cell types express more than one PDE, these tools provide a new approach to understanding the specific action of PDE inhibitors in health and disease.We tested GENIe in HEK293T cells on a BioTek Synergy MX automated fluorescence plate reader. cGMP production was initiated by adding sodium nitroprusside (SNP), a nitric oxide donor, in both the presence and absence of the PDE5 selective inhibitor, Sildenafil. Fluorescence intensity changed dramatically in the presence of Sildenafil, as degradation of cGMP is inhibited by the drug. To optimize the response in HEK293T cells, we over‐expressed sGC, treated with SNP, and observed a significant improvement in the amplitude of the signal accompanied by faster kinetics and an increased sensitivity to the nitric oxide donor SNP. We expect that endogenous levels of sGC will vary in different cell types and these parameters should be varied accordingly.To examine PDE inhibitor specificity, we monitored responses from both cADDis and GENIe in the presence of different PDE inhibitors. IBMX, a non‐specific inhibitor, produced similar responses from both sensors. Sildenafil and Rolipram indicated cGMP vs cAMP specificity. Sildenafil exhibited selective inhibition of the cGMP‐specific PDE5, while Rolipram exhibited selective inhibition of the cAMP specific PDE4.Support or Funding InformationFunding from NSF SBIR Phase IIB 1430878This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In Endothelial Cells, the Activation or Stimulation of Soluble Guanylyl Cyclase Induces the Nitric Oxide Production by a Mechanism Dependent of Nitric Oxide Synthase Activation.

In endothelial cells, investigate if the soluble guanylate cyclase (sGC) activation or stimulation is able to potentiate the relaxation in vessels. Aortic and coronary rings with and without endothelium were placed in a myograph and cumulative concentration-effect curves for DETA-NO or ataciguat were performed. Nitric oxide (NO) were measured by fluorescence or by selective electrode in human umbilical endothelial cells (HUVECs) in response to some treatments, including ataciguat, 8-Br-cGMP and A23187. The presence of the endothelium potentiated the relaxation induced by DETA-NO in aortic and coronary rings. In addition, in aortic rings the endothelium potentiated the relaxation induced by ataciguat. In the presence of nitric oxide synthase (NOS) inhibitor, the endothelium effect was abolished to DETA-NO or ataciguat, in both vessels. Ataciguat, 8-Br-cGMP and A23187 were able to induce NO production in HUVECs cells. In the presence of NOS inhibitor, the NO production induced by ataciguat and 8-Br-cGMP was abolished. Our results suggest that in aortic and coronary rings the endothelium potentiates the relaxation induced by activation or stimulation of sGC through a mechanism dependent of NOS activation.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Modulation of BAY 41-2272-induced-relaxation in normotensive and hypertensive rat aorta by perivascular adipose tissue

In spontaneously hypertensive rat (SHR), perivascular adipose tissue (PVAT) has been recently identified as the effector of vascular dysfunction. In order to restore this alteration, therapeutic strategies target the cGMP/PKG pathway which is involved in both vascular and adipose tissue functions. However, direct activation of soluble guanylate cyclase, a key enzyme of this signalling pathway has not been studied yet in arterial hypertension. This study was designed to determine whether relaxation induced by BAY 41-2272, a soluble guanylate cyclase stimulator agent, was modified in SHR aorta and whether PVAT modulates this effect. Aortic rings isolated from 12-week-old Wistar Kyoto (WKY) rats and SHR were mounted in organ baths and pre-constricted with phenylephrine. Cumulative concentration-response curves to BAY 41-2272 (0.1 nM to 30 μM) were constructed in different experimental conditions in the absence or presence of endothelium and PVAT. In the absence of PVAT, BAY 41-2272 produced concentration-dependent relaxation, which was inhibited by L-NAME (100 μM), a nitric oxide (NO) synthase inhibitor only in WKY rat aorta. Endothelium denudation enhanced the BAY 41-2272-induced-relaxation only in SHR aorta. In both rat strains, relaxant effect to BAY 41-2272 was attenuated by PVAT and this was associated with a decrease in tissue cGMP content. NO and cyclo-oxygenases (COX)-derived products do not appear to participate to this impairment since 100 μM L-NAME and 10 μM indomethacin, a non-specific COX inhibitor, do not modify the BAY 41-2272-induced-relaxation. We showed for the first time that BAY 41-2272 partially relaxes WKY rat aorta by NO-dependent-mechanism and that the endothelium negatively modulates the BAY 41-2272-induced-relaxation only in hypertensive rats. The decrease of BAY 41-2272-induced-relaxation in SHR aorta by PVAT suggests the release of adipose contracting factors that need further elucidation.

Effects of soluble guanylate cyclase activation on cardiovascular reactivity in spontaneously hypertensive rats with metabolic syndrome

There is an increasing evidence regarding the relationship between the metabolic syndrome and risks of cardiovascular diseases. Several studies have shown a link between metabolic syndrome components and endothelial dysfunction. To further develop this scientific point and as little is known about the impact of metabolic syndrome on cardiovascular function in the spontaneously hypertensive rat (SHR), effects of long-term administration of BAY 41–2272, a soluble guanylate cyclase (GC) activator, on the cardiovascular reactivity of SHR rats with metabolic syndrome were investigated. Twenty-four 9-week-old rats were randomly divided into 3 groups: control group, cafeteria diet fed group and cafeteria diet fed group receiving a daily oral dose of BAY 41–2272 (5 mg/kg). Measurements of body weight, abdominal circumference, blood pressure and glucose tolerance test were performed. Cumulative concentration-response curves to isoprenaline (0.1 nM–1 μM), acetylcholine (1 nM–10 μM), phenylephrine (1 nM–10 μM) and insulin (1 nM–3 μM) as well as cGMP assays were determined on isolated perfused heart and thoracic aorta. We showed that 12 weeks of cafeteria diet induced several components of the metabolic syndrome in cafeteria diet fed group. These metabolic disorders were associated with a decrease in ß-adrenoceptor-induced cardiac inotropy and coronary perfusion and an alteration of α 1 -adrenergic response as well as insulin-induced vasorelaxation. However, no difference was observed in acetylcholine-mediated vasorelaxation between groups which is consistent with the cGMP assays. BAY 41-2272 improved markedly the in vivo measured parameters, the α 1 -adrenergic-induced vasoconstriction and significantly restored the cardiac inotropy. These results suggest that long-term activation of guanylate cyclase improved cardiovascular reactivity in SHR rats with an induced metabolic syndrome by modulating the GC-cGMP pathway.

The Discovery of (S)-1-(6-(3-((4-(1-(Cyclopropanecarbonyl)piperidin-4-yl)-2-methylphenyl)amino)-2,3-dihydro-1H-inden-4-yl)pyridin-2-yl)-5-methyl-1H-pyrazole-4-carboxylic Acid, a Soluble Guanylate Cyclase Activator Specifically Designed for Topical Ocular Delivery as a Therapy for Glaucoma

Soluble guanylate cyclase (sGC), the endogenous receptor for nitric oxide (NO), has been implicated in several diseases associated with oxidative stress. In a pathological oxidative environment, the heme group of sGC can be oxidized becoming unresponsive to NO leading to a loss in the ability to catalyze the production of cGMP. Recently a dysfunctional sGC/NO/cGMP pathway has been implicated in contributing to elevated intraocular pressure associated with glaucoma. Herein we describe the discovery of molecules specifically designed for topical ocular administration, which can activate oxidized sGC restoring the ability to catalyze the production of cGMP. These efforts culminated in the identification of compound (+)-23, which robustly lowers intraocular pressure in a cynomolgus model of elevated intraocular pressure over 24 h after a single topical ocular drop and has been selected for clinical evaluation.

Mechanistic Insights into the Activation of Soluble Guanylate Cyclase by Carbon Monoxide: A Multistep Mechanism Proposed for the BAY 41-2272 Induced Formation of 5-Coordinate CO-Heme.

Soluble guanylate cyclase (sGC) is a heme-containing enzyme that catalyzes cGMP production upon sensing NO. While the CO adduct, sGC-CO, is much less active, the allosteric regulator BAY 41-2272 stimulates the cGMP productivity to the same extent as that of sGC-NO. The stimulatory effect has been thought to be likely associated with Fe-His bond cleavage leading to 5-coordinate CO-heme, but the detailed mechanism remains unresolved. In this study, we examined the mechanism under the condition including BAY 41-2272, 2'-deoxy-3'-GMP and foscarnet. The addition of these effectors caused the original 6-coordinate CO-heme to convert to an end product that was an equimolar mixture of a 5- and a new 6-coordinate CO-heme, as assessed by IR spectral measurements. The two types of CO-hemes in the end product were further confirmed by CO dissociation kinetics. Stopped-flow measurements under the condition indicated that the ferrous sGC bound CO as two reversible steps, where the primary step was assigned to the full conversion of the ferrous enzyme to the 6-coordinate CO-heme, and subsequently followed by the slower second step leading a partial conversion of the 6-coordinate CO-heme to the 5-coordinate CO-heme. The observed rates for both steps linearly depended on CO concentrations. The unexpected CO dependence of the rates in the second step supports a multistep mechanism, in which the 5-coordinate CO-heme is led by CO release from a putative bis-carbonyl intermediate that is likely provided by the binding of a second CO to the 6-coordinate CO-heme. This mechanism provides a new aspect on the activation of sGC by CO.

Thiocarbonyl-bound metallonitrosyl complexes with visible-light induced DNA cleavage and promising vasodilation activity

Nitric oxide has been involved in many key biological processes such as vasodilation, platelet aggregation, apoptosis, memory function, and this has drawn attention to the development of exogenous NO donors. Metallonitrosyl complexes are an important class of these compounds. Here, two new ruthenium nitrosyl complexes containing a thiocarbonyl ligand, with the formula cis-[Ru(phen)2(L)(NO)](PF6)3 (phen = phenantroline, L = thiourea or thiobenzamide), were synthesized and characterized by electronic spectroscopy, FTIR, NMR, mass spectrometry and voltammetric techniques. Theoretical calculations using Density Functional Theory (DFT) and Time-dependent Density Functional Theory (TD-DFT) were also used and further supported the characterizations of these complexes. An efficient release of nitric oxide by blue light was validated using a NO/HNO probe: 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, known as cPTIO. Interestingly, the complex containing thiourea cleaved DNA even in the dark, while both complexes showed great DNA photocleavage activity in blue light. This process might work mainly through NO and hydroxyl radical production. Additionally, these complexes showed promising vasodilator activity, whose mechanism of action was investigated using N-Nitro-l-arginine methyl ester hydrochloride (L-NAME) and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and compared to sodium nitroprusside. Both compounds were indeed NO-mediated heme-dependent activators of soluble guanylate cyclase. Additionally, they did not show any significant cytotoxicity against cancer cell lines U87 and GBM02. Altogether, these results supported both complexes having potential pharmacological applications that deserve further studies.

Nitrite potentiates the vasodilatory signaling of S-nitrosothiols

Nitrite and S-nitrosothiols (SNOs) are both byproducts of nitric oxide (NO) metabolism and are proposed to cause vasodilation via activation of soluble guanylate cyclase (sGC). We have previously reported that while SNOs are potent vasodilators at physiological concentrations, nitrite itself only produces vasodilation at supraphysiological concentrations. Here, we tested the hypothesis that sub-vasoactive concentrations of nitrite potentiate the vasodilatory effects of SNOs. Multiple exposures of isolated sheep arteries to S-nitroso-glutathione (GSNO) resulted in a tachyphylactic decreased vasodilatory response to GSNO but not to NO, suggesting attenuation of signaling steps upstream from sGC. Exposure of arteries to 1 μM nitrite potentiated the vasodilatory effects of GSNO in naive arteries and abrogated the tachyphylactic response to GSNO in pre-exposed arteries, suggesting that nitrite facilitates GSNO-mediated activation of sGC. In intact anesthetized sheep and rats, inhibition of NO synthases to decrease plasma nitrite levels attenuated vasodilatory responses to exogenous infusions of GSNO, an effect that was reversed by exogenous infusion of nitrite at sub-vasodilating levels. This study suggests nitrite potentiates SNO-mediated vasodilation via a mechanism that lies upstream from activation of sGC.

A diseasome cluster-based drug repurposing of soluble guanylate cyclase activators from smooth muscle relaxation to direct neuroprotection

Network medicine utilizes common genetic origins, markers and co-morbidities to uncover mechanistic links between diseases. These links can be summarized in the diseasome, a comprehensive network of disease–disease relationships and clusters. The diseasome has been influential during the past decade, although most of its links are not followed up experimentally. Here, we investigate a high prevalence unmet medical need cluster of disease phenotypes linked to cyclic GMP. Hitherto, the central cGMP-forming enzyme, soluble guanylate cyclase (sGC), has been targeted pharmacologically exclusively for smooth muscle modulation in cardiology and pulmonology. Here, we examine the disease associations of sGC in a non-hypothesis based manner in order to identify possibly previously unrecognized clinical indications. Surprisingly, we find that sGC, is closest linked to neurological disorders, an application that has so far not been explored clinically. Indeed, when investigating the neurological indication of this cluster with the highest unmet medical need, ischemic stroke, pre-clinically we find that sGC activity is virtually absent post-stroke. Conversely, a heme-free form of sGC, apo-sGC, was now the predominant isoform suggesting it may be a mechanism-based target in stroke. Indeed, this repurposing hypothesis could be validated experimentally in vivo as specific activators of apo-sGC were directly neuroprotective, reduced infarct size and increased survival. Thus, common mechanism clusters of the diseasome allow direct drug repurposing across previously unrelated disease phenotypes redefining them in a mechanism-based manner. Specifically, our example of repurposing apo-sGC activators for ischemic stroke should be urgently validated clinically as a possible first-in-class neuroprotective therapy.

Binding of cholera toxin B subunit to intestinal epithelial cells

We have prepared 125I-labeled cholera toxin B subunit (125I-labeled CT-B, a specific activity of 98Ci/mmol) and found that it binds to rat IEC-6 and human Caco-2 intestinal epithelial cells with high affinity (Kd 3.6 and 3.7nM, respectively). The binding of labeled protein was completely inhibited by unlabeled thymosin-α1 (TM-α1), interferon-α2 (IFN-α2), and the synthetic peptide LKEKK that corresponds to residues 16–20 in TM-α1 and 131–135 in IFN-α2, but was not inhibited by the synthetic peptide KKEKL with inverted amino acid sequence (Ki>10μM). Thus, TM-α1, IFN-α2, and the peptide: LKEKK bind with high affinity and specificity to the cholera toxin receptor on IEC-6 and Caco-2 cells. It was found that CT-B and the peptide: LKEKK at concentrations of 10–1000nM increased in a dose-dependent manner the nitric oxide production and the soluble guanylate cyclase activity in IEC-6 and Caco-2 cells.

Stimulators and activators of soluble guanylate cyclase for urogenital disorders.

Lower urinary tract symptoms (LUTS), comprising storage (such as urinary incontinence and urinary frequency), voiding, and postmicturition symptoms, are highly prevalent conditions that affect millions of people worldwide. LUTS have a profound effect on quality of life and are a considerable cost to health care systems. In men specifically, BPH commonly leads to LUTS. Clinical studies also show an association of LUTS with erectile dysfunction (ED). Nitric oxide (NO) has long been recognized as an important nonadrenergic, noncholinergic (NANC) transmitter in bladder, urethra, prostate, and corpus cavernosum smooth muscle. Data from clinical and basic research show that oxidation and degradation of soluble guanylate cyclase (sGC; also known as GCS) and reduced cyclic GMP (cGMP) levels are involved in the physiopathology of genitourinary diseases. The NO-sGC-cGMP signalling pathway has a role in disease pathophysiology of the bladder, urethra, prostate, and corpus cavernosum in animal models and humans. Advances in targeting sGC directly to enhance cGMP production independently of endogenous NO have been made using NO-independent stimulators and activators of sGC. These molecules are potential therapeutics in the treatment of LUTS and ED.

Irreversible Activation and Stabilization of Soluble Guanylate Cyclase by the Protoporphyrin IX Mimetic Cinaciguat.

Belonging to the class of so-called soluble guanylate cyclase (sGC) activators, cinaciguat and BAY 60-2770 are interesting therapeutic tools for the treatment of various cardiovascular pathologies. The drugs are supposed to preferentially stimulate oxidized or heme-depleted, but not native sGC. Since this concept has been challenged by studies demonstrating complete relaxation of nondiseased vessels, this study was designed to reinvestigate the mode of action in greater detail. To this purpose, the effect of cinaciguat was studied on vessel tone of porcine coronary arteries and rat thoracic aortas. Organ bath studies showed that the compound caused time- and concentration-dependent relaxation of precontracted vessels with a maximal effect observed at 90 minutes. The dilatory response was not affected by extensive washout of the drug. Cinaciguat-induced vasodilation was associated with a time- and concentration-dependent increase of cGMP levels. Experiments with purified sGC in the presence of Tween 20 showed that cinaciguat activates the heme-free enzyme in a concentration-dependent manner with an EC50 value of ~0.2 μM and maximal cGMP formation at 10 μM. By contrast, the effect of cinaciguat on 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one–oxidized (ferric) sGC was moderate, reaching ~10%–15% of maximal activity. Dilution experiments of cinaciguat/Tween 20–preincubated sGC revealed the irreversible character of the drug. Assuming a sensitive balance between heme-free, ferric, and nitric oxide–sensitive ferrous sGC in cells and tissues, we propose that cinaciguat by virtue of its irreversible mode of action is capable of shifting this equilibrium toward the heme-free apo-sGC species.

Pharmacological preconditioning with gemfibrozil preserves cardiac function after heart transplantation

While heart transplantation (HTX) is the definitive therapy of heart failure, donor shortage is emerging. Pharmacological activation of soluble guanylate cyclase (sGC) and increased cGMP-signalling have been reported to have cardioprotective properties. Gemfibrozil has recently been shown to exert sGC activating effects in vitro. We aimed to investigate whether pharmacological preconditioning of donor hearts with gemfibrozil could protect against ischemia/reperfusion injury and preserve myocardial function in a heterotopic rat HTX model. Donor Lewis rats received p.o. gemfibrozil (150 mg/kg body weight) or vehicle for 2 days. The hearts were explanted, stored for 1 h in cold preservation solution, and heterotopically transplanted. 1 h after starting reperfusion, left ventricular (LV) pressure-volume relations and coronary blood flow (CBF) were assessed to evaluate early post-transplant graft function. After 1 h reperfusion, LV contractility, active relaxation and CBF were significantly (p < 0.05) improved in the gemfibrozil pretreated hearts compared to that of controls. Additionally, gemfibrozil treatment reduced nitro-oxidative stress and apoptosis, and improved cGMP-signalling in HTX. Pharmacological preconditioning with gemfibrozil reduces ischemia/reperfusion injury and preserves graft function in a rat HTX model, which could be the consequence of enhanced myocardial cGMP-signalling. Gemfibrozil might represent a useful tool for cardioprotection in the clinical setting of HTX surgery soon.

Cinaciguat ameliorates glomerular damage by reducing ERK1/2 activity and TGF-\xdf expression in type-1 diabetic rats

Decreased soluble guanylate cyclase activity and cGMP levels in diabetic kidneys were shown to influence the progression of nephropathy. The regulatory effects of soluble guanylate cyclase activators on renal signaling pathways are still unknown, we therefore investigated the renal molecular effects of the soluble guanylate cyclase activator cinaciguat in type-1 diabetic (T1DM) rats. Male adult Sprague-Dawley rats were divided into 2 groups after induction of T1DM with 60 mg/kg streptozotocin: DM, untreated (DM, n = 8) and 2) DM + cinaciguat (10 mg/kg per os daily, DM-Cin, n = 8). Non-diabetic untreated and cinaciguat treated rats served as controls (Co (n = 10) and Co-Cin (n = 10), respectively). Rats were treated for eight weeks, when renal functional and molecular analyses were performed. Cinaciguat attenuated the diabetes induced proteinuria, glomerulosclerosis and renal collagen-IV expression accompanied by 50% reduction of TIMP-1 expression. Cinaciguat treatment restored the glomerular cGMP content and soluble guanylate cyclase expression, and ameliorated the glomerular apoptosis (TUNEL positive cell number) and podocyte injury. These effects were accompanied by significantly reduced TGF-ß overexpression and ERK1/2 phosphorylation in cinaciguat treated diabetic kidneys. We conclude that the soluble guanylate cyclase activator cinaciguat ameliorated diabetes induced glomerular damage, apoptosis, podocyte injury and TIMP-1 overexpression by suppressing TGF-ß and ERK1/2 signaling.

Identification of a soluble guanylate cyclase in RBCs: preserved activity in patients with coronary artery disease

Endothelial dysfunction is associated with decreased NO bioavailability and impaired activation of the NO receptor soluble guanylate cyclase (sGC) in the vasculature and in platelets. Red blood cells (RBCs) are known to produce NO under hypoxic and normoxic conditions; however evidence of expression and/or activity of sGC and downstream signaling pathway including phopshodiesterase (PDE)-5 and protein kinase G (PKG) in RBCs is still controversial. In the present study, we aimed to investigate whether RBCs carry a functional sGC signaling pathway and to address whether this pathway is compromised in coronary artery disease (CAD). Using two independent chromatographic procedures, we here demonstrate that human and murine RBCs carry a catalytically active α1β1-sGC (isoform 1), which converts 32P-GTP into 32P-cGMP, as well as PDE5 and PKG. Specific sGC stimulation by NO+BAY 41-2272 increases intracellular cGMP-levels up to 1000-fold with concomitant activation of the canonical PKG/VASP-signaling pathway. This response to NO is blunted in α1-sGC knockout (KO) RBCs, but fully preserved in α2-sGC KO. In patients with stable CAD and endothelial dysfunction red cell eNOS expression is decreased as compared to aged-matched controls; by contrast, red cell sGC expression/activity and responsiveness to NO are fully preserved, although sGC oxidation is increased in both groups. Collectively, our data demonstrate that an intact sGC/PDE5/PKG-dependent signaling pathway exists in RBCs, which remains fully responsive to NO and sGC stimulators/activators in patients with endothelial dysfunction. Targeting this pathway may be helpful in diseases with NO deficiency in the microcirculation like sickle cell anemia, pulmonary hypertension, and heart failure.

Interaction of Cholera Toxin B-subunit with Human T-lymphocytes.

In this work, 125I-labeled cholera toxin B-subunit (CT-B) (specific activity 98 Ci/mmol) was prepared, and its high-affinity binding to human blood T-lymphocytes (Kd= 3.3nM) was determined. The binding of the 125I-labeled CT-B was inhibited by unlabeled interferon-α2 (IFN-α2), thymosin-α1 (TM-α1), and by the synthetic peptide LKEKK, which corresponds to sequences 16-20 of human TM-α1 and 131-135 of IFN-α2 (Ki 0.8, 1.2, and 1.6nM, respectively), but was not inhibited by the unlabeled synthetic peptide KKEKL with inverted sequence (Ki > 1µM). In the concentration range of 10-1000nM, both CT-B and peptide LKEKK dose-dependently increased the activity of soluble guanylate cyclase (sGC) but did not affect the activity of membrane-bound guanylate cyclase. The KKEKL peptide tested in parallel did not affect sGC activity. Thus, the CT-B and peptide LKEKK binding to a common receptor on the surface of T-lymphocytes leads to an increase in sGC activity.

Abstract P243: Smooth Muscle Cullin-3 Deficiency Causes Severe Early Onset Hypertension and Nitric Oxide Resistance

Mutations in Cullin-3 ( Cul3 ) resulting in exon 9 skipping (CUL3α9) cause human hypertension. We demonstrated that selective expression of CUL3α9 in smooth muscle causes arterial stiffness and hypertension. We hypothesized that deletion of CUL3 in smooth muscle causes severe hypertension. Mice carrying a conditional allele of CUL3 were bred with mice expressing a tamoxifen-inducible CRE-recombinase driven by a smooth muscle promoter. Mice were administered tamoxifen i.p. (75 mg/kg) for 5 consecutive days to generate smooth muscle CUL3 knockout (S-CUL3KO). CUL3 protein was undetectable whereas Cullin-1 protein was preserved in aorta from S-CUL3KO mice. We assessed vascular function in the cerebral basilar artery and aorta using pressurized and wire myograph, respectively. Blood pressure (BP) was measured by radiotelemetry. S-CUL3KO mice exhibited significantly increased systolic BP (SBP) at 2 weeks and 4 weeks post tamoxifen compared to corn oil controls (2 wks SBP mmHg: 145±1 vs 115±2, p&lt;0.001; 4 weeks SBP: 169±1 vs 115±3, p&lt;0.001). Pulse wave velocity was also increased in S-CUL3KO mice (3.7±0.1 m/s vs 2.2±0.1, P&lt;0.001), suggesting increased arterial stiffness. Aorta from S-CUL3KO mice exhibited severely impaired vasorelaxation to acetylcholine (ACh) compared to controls (at 100 μM: 1.0±3% vs 77±5%, p&lt;0.0001), and to the nitric oxide donor sodium nitroprusside (SNP) (at 100 μM: 15±4% vs 96±1%, p&lt;0.001). In agreement with data from aorta, cerebral basilar artery from S-CUL3KO mice also exhibited significant impairment to ACh- and SNP-mediated vasorelaxation. Conversely, S-CUL3KO aorta retained an ability to relax in response to a cGMP analogue (8-pCPT-cGMP, at 100 μμ: 8-pCPT-cGMP 70±3%) and to the heme-independent soluble guanylate cyclase activator (BAY 58-2667, at 10 μμ: BAY 58-2667 94±2%), indicating that downstream mechanisms controlled by cGMP remain intact in the absence of CUL3. Captopril (120 mg/kg/day) was sufficient to normalized BP in S-CUL3KO mice to pre-tamoxifen levels (change in SBP mmHg: -50.2±1.9 S-CUL3KO vs -25.0±2 control, P&lt;0.0001). We conclude that smooth muscle CUL3 is a major BP determinant, and identifying novel CUL3 substrates in smooth muscle would be beneficial as therapeutic targets in the treatment of hypertension.