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

Timely activation of the luteinizing hormone receptor (LHCGR) is critical for fertility. Activating mutations in LHCGR cause familial male-limited precocious puberty (FMPP) due to premature synthesis of testosterone. A mouse model of FMPP (KiLHRD582G), expressing a constitutively activating mutation in LHCGR, was previously developed in our laboratory. KiLHRD582G mice became progressively infertile due to sexual dysfunction and exhibited smooth muscle loss and chondrocyte accumulation in the penis. In this study, we tested the hypothesis that KiLHRD582G mice had erectile dysfunction due to impaired smooth muscle function. Apomorphine-induced erection studies determined that KiLHRD582G mice had erectile dysfunction. Penile smooth muscle and endothelial function were assessed using penile cavernosal strips. Penile endothelial cell content was not changed in KiLHRD582G mice. The maximal relaxation response to acetylcholine and the nitric oxide donor, sodium nitroprusside, was significantly reduced in KiLHRD582G mice indicating an impairment in the nitric oxide (NO)-mediated signaling. Cyclic GMP (cGMP) levels were significantly reduced in KiLHRD582G mice in response to acetylcholine, sodium nitroprusside and the soluble guanylate cyclase stimulator, BAY 41-2272. Expression of NOS1, NOS3 and PKRG1 were unchanged. The Rho-kinase signaling pathway for smooth muscle contraction was not altered. Together, these data indicate that KiLHRD582G mice have erectile dysfunction due to impaired NO-mediated activation of soluble guanylate cyclase resulting in decreased levels of cGMP and penile smooth muscle relaxation. These studies in the KiLHRD582G mice demonstrate that activating mutations in the mouse LHCGR cause erectile dysfunction due to impairment of the NO-mediated signaling pathway in the penile smooth muscle.

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Soluble Guanylate Cyclase Stimulators and Activators: Where are We and Where to Go?

Mini-Reviews in Medicinal Chemistry ◽

10.2174/1389557519666190730110600 ◽

2019 ◽

Vol 19 (18) ◽

pp. 1544-1557 ◽

Cited By ~ 6

Author(s):

Sijia Xiao ◽

Qianbin Li ◽

Liqing Hu ◽

Zutao Yu ◽

Jie Yang ◽

...

Keyword(s):

Guanylate Cyclase ◽

Soluble Guanylate Cyclase ◽

Muscle Relaxation ◽

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Smooth Muscle Relaxation ◽

Secondary Messenger ◽

Physiological Processes ◽

Cgmp Pathway ◽

Preclinical And Clinical Studies

Soluble Guanylate Cyclase (sGC) is the intracellular receptor of Nitric Oxide (NO). The activation of sGC results in the conversion of Guanosine Triphosphate (GTP) to the secondary messenger cyclic Guanosine Monophosphate (cGMP). cGMP modulates a series of downstream cascades through activating a variety of effectors, such as Phosphodiesterase (PDE), Protein Kinase G (PKG) and Cyclic Nucleotide-Gated Ion Channels (CNG). NO-sGC-cGMP pathway plays significant roles in various physiological processes, including platelet aggregation, smooth muscle relaxation and neurotransmitter delivery. With the approval of an sGC stimulator Riociguat for the treatment of Pulmonary Arterial Hypertension (PAH), the enthusiasm in the discovery of sGC modulators continues for broad clinical applications. Notably, through activating the NO-sGC-cGMP pathway, sGC stimulator and activator potentiate for the treatment of various diseases, such as PAH, Heart Failure (HF), Diabetic Nephropathy (DN), Systemic Sclerosis (SS), fibrosis as well as other diseases including Sickle Cell Disease (SCD) and Central Nervous System (CNS) disease. Here, we review the preclinical and clinical studies of sGC stimulator and activator in recent years and prospect for the development of sGC modulators in the near future.

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A theoretical model of nitric oxide transport in arterioles: frequency- vs. amplitude-dependent control of cGMP formation

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00525.2003 ◽

2004 ◽

Vol 286 (3) ◽

pp. H1043-H1056 ◽

Cited By ~ 51

Author(s):

Nikolaos M. Tsoukias ◽

Mahendra Kavdia ◽

Aleksander S. Popel

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Smooth Muscle ◽

Soluble Guanylate Cyclase ◽

Muscle Tone ◽

Muscle Relaxation ◽

Smooth Muscle Relaxation ◽

No Production ◽

Cgmp Formation

Nitric oxide (NO) plays many important physiological roles, including the regulation of vascular smooth muscle tone. In response to hemodynamic or agonist stimuli, endothelial cells produce NO, which can diffuse to smooth muscle where it activates soluble guanylate cyclase (sGC), leading to cGMP formation and smooth muscle relaxation. The close proximity of red blood cells suggests, however, that a significant amount of NO released will be scavenged by blood, and thus the issue of bioavailability of endothelium-derived NO to smooth muscle has been investigated experimentally and theoretically. We formulated a mathematical model for NO transport in an arteriole to test the hypothesis that transient, burst-like NO production can facilitate efficient NO delivery to smooth muscle and reduce NO scavenging by blood. The model simulations predict that 1) the endothelium can maintain a physiologically significant amount of NO in smooth muscle despite the presence of NO scavengers such as hemoglobin and myoglobin; 2) under certain conditions, transient NO release presents a more efficient way for activating sGC and it can increase cGMP formation severalfold; and 3) frequency-rather than amplitude-dependent control of cGMP formation is possible. This suggests that it is the frequency of NO bursts and perhaps the frequency of Ca2+ oscillations in endothelial cells that may limit cGMP formation and regulate vascular tone. The proposed hypothesis suggests a new functional role for Ca2+ oscillations in endothelial cells. Further experimentation is needed to test whether and under what conditions in silico predictions occur in vivo.

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Roles of CaM kinase II and phospholamban in SNP-induced relaxation of murine gastric fundus smooth muscles

AJP Cell Physiology ◽

10.1152/ajpcell.00397.2005 ◽

2006 ◽

Vol 291 (2) ◽

pp. C337-C347 ◽

Cited By ~ 27

Author(s):

Minkyung Kim ◽

In Soo Han ◽

Sang Don Koh ◽

Brian A. Perrino

Keyword(s):

Smooth Muscle ◽

Soluble Guanylate Cyclase ◽

Muscle Relaxation ◽

Smooth Muscles ◽

Cyclopiazonic Acid ◽

Gastric Fundus ◽

Smooth Muscle Relaxation ◽

Cam Kinase Ii ◽

Cam Kinase ◽

No Donor

The mechanisms by which nitric oxide (NO) relaxes smooth muscles are unclear. The NO donor sodium nitroprusside (SNP) has been reported to increase the Ca2+ release frequency (Ca2+ sparks) through ryanodine receptors (RyRs) and activate spontaneous transient outward currents (STOCs), resulting in smooth muscle relaxation. Our findings that caffeine relaxes and hyperpolarizes murine gastric fundus smooth muscles and increases phospholamban (PLB) phosphorylation by Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM kinase II) suggest that PLB phosphorylation by CaM kinase II participates in smooth muscle relaxation by increasing sarcoplasmic reticulum (SR) Ca2+ uptake and the frequencies of SR Ca2+ release events and STOCs. Thus, in the present study, we investigated the roles of CaM kinase II and PLB in SNP-induced relaxation of murine gastric fundus smooth muscles. SNP hyperpolarized and relaxed gastric fundus circular smooth muscles and activated CaM kinase II. SNP-induced CaM kinase II activation was prevented by KN-93. Ryanodine, tetracaine, 2-aminoethoxydiphenylborate, and cyclopiazonic acid inhibited SNP-induced fundus smooth muscle relaxation and CaM kinase II activation. The Ca2+-activated K+ channel blockers iberiotoxin and apamin inhibited SNP-induced hyperpolarization and relaxation. The soluble guanylate cyclase inhibitor 1 H-[1,2,4]oxadiazolo-[4,3-α]quinoxalin-1-one inhibited SNP-induced relaxation and CaM kinase II activation. The membrane-permeable cGMP analog 8-bromo-cGMP relaxed gastric fundus smooth muscles and activated CaM kinase II. SNP increased phosphorylation of PLB at Ser16 and Thr17. Thr17 phosphorylation of PLB was inhibited by cyclopiazonic acid and KN-93. Ser16 and Thr17 phosphorylation of PLB was sensitive to 1 H-[1,2,4]oxadiazolo-[4,3-α]quinoxalin-1-one. These results demonstrate a novel pathway linking the NO-soluble guanylyl cyclase-cGMP pathway, SR Ca2+ release, PLB, and CaM kinase II to relaxation in gastric fundus smooth muscles.

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Guanylate cyclase and not ATP-dependent K+ channels seems temperature-dependent in smooth muscle relaxation of human umbilical arteries

European Journal of Pharmacology ◽

10.1016/s0014-2999(00)00529-x ◽

2000 ◽

Vol 406 (1) ◽

pp. 79-84 ◽

Cited By ~ 4

Author(s):

Alberto Tiritilli

Keyword(s):

Smooth Muscle ◽

Guanylate Cyclase ◽

Muscle Relaxation ◽

Smooth Muscle Relaxation ◽

Temperature Dependent ◽

K Channels ◽

Umbilical Arteries

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Effects of sulfhydryl reagents on nitroglycerin-induced relaxation of bovine coronary artery

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y86-236 ◽

1986 ◽

Vol 64 (11) ◽

pp. 1395-1401 ◽

Cited By ~ 30

Author(s):

Carl A. Gruetter ◽

Sally M. Lemke

Keyword(s):

Smooth Muscle ◽

Coronary Artery ◽

Vascular Smooth Muscle ◽

Guanylate Cyclase ◽

Ethacrynic Acid ◽

Muscle Relaxation ◽

Alkylating Agents ◽

Sulfhydryl Groups ◽

Smooth Muscle Relaxation

The mechanism whereby nitroglycerin relaxes vascular smooth muscle remains uncertain. A current hypothesis suggests that nitroglycerin reacts with critical cellular sulfhydryl groups to form an intermediate, which activates guanylate cyclase, resulting in cGMP accumulation and relaxation. This study investigated further the potential involvement of sulfhydryls in nitroglycerin-induced vascular smooth muscle relaxation by evaluating effects of a variety of sulfhydryl alkylating and reducing agents on responses to nitroglycerin and other relaxants in bovine coronary arterial strips submaximally contracted using 30 mM K. Whereas 10−4 M 5,5′-dithiobis-(2-nitrobenzoicacid), 10−5M N-ethylmaleimide, and 10−4M N-naphthylmaleimide did not affect nitroglycerin-induced relaxation, 10−4 M N-ethylmaleimide and 10−4 M ethacrynic acid significantly inhibited relaxation induced by nitroglycerin. Both ethacrynic acid and N-ethylmaleimide at 10−4 M also inhibited relaxation induced by sodium nitroprus-side. N-ethylmaleimide, but not ethacrynic acid, inhibited relaxation induced by isoproterenol and forskolin. Ethacrynic acid significantly reduced both relaxation and cGMP elevation induced by both 10−7 M nitroglycerin and 10−7 M sodium nitroprusside. Ethacrynic acid, but not N-ethylmaleimide, significantly reduced relaxation induced by 8-Br-cGMP. Pretreatment with the sulfhydryl-containing agents N-acetylcysteine, 2-mercaptoethanol, or dithiothreitol, at 10−3 M did not affect nitroglycerin-induced relaxation in nontolerant arteries. Similarly, N-acetylcysteine and dithiothreitol did not alter the depressed responses to nitroglycerin in arteries in which tolerance to nitroglycerin was induced in vitro. A slight but statistically significant reversal of nitroglycerin-tolerance occurred after treatment of tolerant arteries with 2-mercaptoethanol. As none of the sulfhydryl alkylating agents tested inhibited selectively nitroglycerin-induced relaxation, and none of the sulfhydryl-containing agents markedly enhanced nitroglycerin-induced relaxation in either nontolerant or tolerant arteries, the results provide little evidence to support the hypothesis that critical sulfhydryl groups are involved specifically in nitroglycerin-induced relaxation of arterial smooth muscle. The results do suggest that ethacrynic acid may selectively inhibit relaxation induced in bovine coronary artery by relaxants that act by activating guanylate cyclase, and that the inhibition by ethacrynic acid may involve two mechanisms, an inhibition of guanylate cyclase activation and an inhibition of the relaxant effect of cGMP.

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cGMP-independent mechanism of airway smooth muscle relaxation induced byS-nitrosoglutathione

AJP Cell Physiology ◽

10.1152/ajpcell.1998.275.2.c468 ◽

1998 ◽

Vol 275 (2) ◽

pp. C468-C474 ◽

Cited By ~ 38

Author(s):

William J. Perkins ◽

Christina Pabelick ◽

David O. Warner ◽

Keith A. Jones

Keyword(s):

Smooth Muscle ◽

Soluble Guanylate Cyclase ◽

Isometric Force ◽

Muscle Relaxation ◽

Smooth Muscle Relaxation ◽

Intracellular Protein ◽

No Donor ◽

Protein Thiols ◽

Independent Mechanism ◽

Independent Process

This study tested the hypothesis that the NO donor S-nitrosoglutathione (GSNO) relaxes canine tracheal smooth muscle (CTSM) in part by a cGMP-independent process that involves reversible oxidation of intracellular thiols. GSNO caused a concentration-dependent relaxation in ACh-contracted strips (EC50 ∼1.2 μM) accompanied by a concentration-dependent increase in cytosolic cGMP concentration ([cGMP]i). The soluble guanylate cyclase inhibitor methylene blue prevented the increase in [cGMP]iinduced by 1 and 10 μM GSNO, but isometric force decreased by 10 ± 4 and 55 ± 3%, respectively. After recovery of [cGMP]i to baseline, GSNO-induced relaxation persisted during continuous ACh stimulation. Dithiothreitol caused a rapid recovery of isometric force to values similar to those obtained with ACh alone in these strips. We conclude that GSNO relaxes CTSM contracted by ACh in part by oxidation of intracellular protein thiols.

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Characterization of nitroglycerine-induced relaxation in human corpus cavernosum smooth muscle: implications to erectile physiology and dysfunction

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y95-735 ◽

1995 ◽

Vol 73 (12) ◽

pp. 1714-1726 ◽

Cited By ~ 21

Author(s):

George J. Christ ◽

Daniel C. Kim ◽

Harvey C. Taub ◽

C. Marjorie Gondré ◽

Arnold Melman

Keyword(s):

Smooth Muscle ◽

Erectile Dysfunction ◽

Steady State ◽

Statistical Analysis ◽

Guanylate Cyclase ◽

Patient Population ◽

Muscle Relaxation ◽

Smooth Muscle Relaxation ◽

Induced Response ◽

Significant Heterogeneity

The importance of the nitric oxide – guanylate cyclase – cGMP system in modulating corporal smooth muscle tone and penile erection has been amply demonstrated. The goal of these studies was to evaluate the possibility that age- or disease-related alterations in human corporal smooth muscle responsivity to activation of this pathway might play a role in the etiology of erectile dysfunction. Thus, we utilized a previously described heuristic model to assess the kinetic and steady-state characteristics of relaxation of precontracted isolated corporal tissue strips elicited by nitroglycerine (NTG). Studies were conducted on corporal tissue strips excised from 26 patients with organic erectile dysfunction, and 7 patients with documented erections. For the purposes of statistical analysis the impotent patient population was stratified into two age groups (A, ≤59 years; B, ≥60 years) and further subdivided into two diagnostic categories, diabetic and nondiabetic patients, respectively. In ≈75% of precontracted corporal tissue strips derived from impotent patients (contracted to ≈75% of maximum with phenylephrine), the NTG-induced response was biphasic, consisting of a rapid relaxation response that reached steady state before onset of a more slowly developing regaining of tension, termed the desensitization response. In contrast, a biphasic response was observed much less frequently (≈30%) in corporal tissue strips derived from a potent patient population (p < 0.0001). Statistical analysis revealed significant heterogeneity among corporal tissue strips derived from patients with organic erectile dysfunction, with respect to both the kinetic and steady-state characteristics of the NTG-induced relaxation and desensitization responses. In particular, the maximal rate constant for both NTG-induced relaxation (krelmax; p < 0.01) and desensitization (kdes; p < 0.03) responses was significantly greater in corporal tissue strips excised from diabetic than nondiabetic patients. Furthermore, the EC50 for NTG-induced relaxation of precontracted corporal smooth muscle strips from potent patients (≈25 nM) was 0.90 log unit less than that for equivalently contracted corporal smooth muscle strips derived from impotent patients (≈180 nM; p < 0.03). Such observations suggest that alterations in corporal smooth muscle responsivity to activation of the guanylate cyclase – cGMP pathway, per se, may be a characteristic of organic erectile dysfunction. In the absence of compensatory changes in other vasodilatory mechanisms, this may contribute to incomplete corporal smooth muscle relaxation and the etiology of erectile dysfunction in some patients.Key words: nitroglycerine, corporal smooth muscle, relaxation, desensitization, guanylate cyclase.

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