scholarly journals The G-protein regulator LGN modulates the activity of the NO receptor soluble guanylate cyclase

2012 ◽  
Vol 446 (3) ◽  
pp. 445-453 ◽  
Author(s):  
Swati Chauhan ◽  
Filip Jelen ◽  
Iraida Sharina ◽  
Emil Martin
Keyword(s):  
G Protein ◽  
Guanylate Cyclase ◽  
Soluble Guanylate Cyclase ◽  
Direct Interaction ◽  
Inhibitory Effect ◽  
Hill Coefficient ◽  
Activated State ◽  
Ic50 Values

sGC (soluble guanylate cyclase) is the main mediator of NO signalling. Biochemical and physiological studies suggest that, besides NO, in vivo regulation of sGC involves direct interaction with other proteins. Using yeast two-hybrid screening, we identified that the multidomain LGN (Leu-Gly-Asn repeat-enriched protein) interacts with both α1 and β1 sGC subunits. LGN and sGC co-localized in the cell cytoplasm, and the LGN–sGC complex was co-immunoprecipitated from cells expressing both proteins and from native tissues. Their interaction requires the N-terminal tetratricopeptide repeats of LGN, but does not require the N-terminal portions of α1 or β1 sGC subunits. Overexpression of LGN decreases the activity of cellular sGC, whereas knockdown of LGN mRNA and protein correlated with increased sGC activity. Although purified LGN interacts directly with purified sGC, the inhibitory effect in vitro is observed only after supplementation of cell lysate to the reaction. Although resting sGC and sGC activated by the stimulator BAY41-2272 have very similar LGN-IC50 values to the NO-stimulated sGC, they have a much higher Hill coefficient, suggesting co-operative binding with respect to LGN in the low-activated state of sGC. AGS3 (activator of G-protein signalling 3), the closest LGN homologue, also inhibits sGC. The interaction of sGC with these scaffolding proteins may expand the cross-talk between NO/cGMP signalling and other cellular pathways and tailor sGC function to specific tissues or signals.

α2-Adrenergic-mediated tubular NO production inhibits thick ascending limb chloride absorption

2001 ◽  
Vol 281 (4) ◽  
pp. F679-F686 ◽  
Author(s):  
Craig F. Plato ◽  
Jeffrey L. Garvin
Keyword(s):  
Guanylate Cyclase ◽  
Soluble Guanylate Cyclase ◽  
Chloride Transport ◽  
Receptor Activation ◽  
Inhibitory Effect ◽  
No Production ◽  
Thick Ascending Limb ◽  
Chloride Absorption ◽  
Ly 83583

Stimulation of α2-adrenergic receptors inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (THAL) expresses α2-receptors. We hypothesized that selective α2-receptor activation decreases NaCl absorption by cortical THALs through activation of NOS and increased production of NO. We found that the α2-receptor agonist clonidine (10 nM) decreased chloride flux ( J Cl) from 119.5 ± 15.9 to 67.4 ± 13.8 pmol · mm−1 · min−1 (43% reduction; P < 0.02), whereas removal of clonidine from the bath increased J Cl by 20%. When NOS activity was inhibited by pretreatment with 5 mM N G-nitro-l-arginine methyl ester, the inhibitory effects of clonidine on THAL J Clwere prevented (81.7 ± 10.8 vs. 71.6 ± 6.9 pmol · mm−1 · min−1). Similarly, when the NOS substrate l-arginine was deleted from the bath, addition of clonidine did not decrease THAL J Cl from control (106.9 ± 11.6 vs. 132.2 ± 21.3 pmol · mm−1 · min−1). When we blocked the α2-receptors with rauwolscine (1 μM), we found that the inhibitory effect of 10 nM clonidine on THAL J Cl was abolished, verifying that α2, rather than I1, receptors mediate the effects of clonidine in the THAL. We investigated the mechanism of NOS activation and found that intracellular calcium concentration did not increase in response to clonidine, whereas pretreatment with 150 nM wortmannin abolished the clonidine-mediated inhibition of THAL J Cl, indicating activation of phosphatidylinositol 3-kinase and the Akt pathway. We found that pretreatment of THALs with 10 μM LY-83583, an inhibitor of soluble guanylate cyclase, blocked clonidine-mediated inhibition of THAL J Cl. In conclusion, α2-receptor stimulation decreases THAL J Cl by increasing NO release and stimulating guanylate cyclase. These data suggest that α2-receptors act as physiological regulators of THAL NO synthesis, thus inhibiting chloride transport and participating in the natriuretic and diuretic effects of clonidine in vivo.

scholarly journals The soluble guanylate cyclase stimulator IWP‐550 inhibits neuroinflammation in vitro and in vivo

2018 ◽  
Vol 32 (S1) ◽  
Author(s):  
Guang Liu ◽  
Susana S. Correia ◽  
Sylvie G. Bernier ◽  
Kim Tang ◽  
Sarah Jacobson ◽  
...  
Keyword(s):  
Guanylate Cyclase ◽  
Soluble Guanylate Cyclase ◽  
Soluble Guanylate Cyclase Stimulator

scholarly journals Cinaciguat, a soluble guanylate cyclase activator, augments cGMP after oxidative stress and causes pulmonary vasodilation in neonatal pulmonary hypertension

2011 ◽  
Vol 301 (5) ◽  
pp. L755-L764 ◽  
Author(s):  
Marc Chester ◽  
Gregory Seedorf ◽  
Pierre Tourneux ◽  
Jason Gien ◽  
Nancy Tseng ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Guanylate Cyclase ◽  
Soluble Guanylate Cyclase ◽  
Ductus Arteriosus ◽  
No Donor ◽  
Fetal Sheep ◽  
Cgmp Production ◽  
Pulmonary Vasodilation

Although inhaled NO (iNO) therapy is often effective in treating infants with persistent pulmonary hypertension of the newborn (PPHN), up to 40% of patients fail to respond, which may be partly due to abnormal expression and function of soluble guanylate cyclase (sGC). To determine whether altered sGC expression or activity due to oxidized sGC contributes to high pulmonary vascular resistance (PVR) and poor NO responsiveness, we studied the effects of cinaciguat (BAY 58-2667), an sGC activator, on pulmonary artery smooth muscle cells (PASMC) from normal fetal sheep and sheep exposed to chronic intrauterine pulmonary hypertension (i.e., PPHN). We found increased sGC α1- and β1-subunit protein expression but lower basal cGMP levels in PPHN PASMC compared with normal PASMC. To determine the effects of cinaciguat and NO after sGC oxidation in vitro, we measured cGMP production by normal and PPHN PASMC treated with cinaciguat and the NO donor, sodium nitroprusside (SNP), before and after exposure to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an sGC oxidizer), hyperoxia (fraction of inspired oxygen 0.50), or hydrogen peroxide (H2O2). After treatment with ODQ, SNP-induced cGMP generation was markedly reduced but the effects of cinaciguat were increased by 14- and 64-fold in PPHN fetal PASMC, respectively ( P < 0.01 vs. controls). Hyperoxia or H2O2enhanced cGMP production by cinaciguat but not SNP in PASMC. To determine the hemodynamic effects of cinaciguat in vivo, we compared serial responses to cinaciguat and ACh in fetal lambs after ductus arteriosus ligation. In contrast with the impaired vasodilator response to ACh, cinaciguat-induced pulmonary vasodilation was significantly increased. After birth, cinaciguat caused a significantly greater fall in PVR than either 100% oxygen, iNO, or ACh. We conclude that cinaciguat causes more potent pulmonary vasodilation than iNO in experimental PPHN. We speculate that increased NO-insensitive sGC may contribute to the pathogenesis of PPHN, and cinaciguat may provide a novel treatment of severe pulmonary hypertension.

scholarly journals Differential MMP-14 Targeting by Lumican-Derived Peptides Unraveled by In Silico Approach

Cancers ◽  
2021 ◽  
Vol 13 (19) ◽  
pp. 4930
Author(s):  
Jonathan Dauvé ◽  
Nicolas Belloy ◽  
Romain Rivet ◽  
Nicolas Etique ◽  
Pierre Nizet ◽  
...  
Keyword(s):  
Amino Acids ◽  
In Silico ◽  
Primary Melanoma ◽  
Direct Interaction ◽  
Inhibitory Effect ◽  
Melanoma Tumor ◽  
And Migration ◽  
Key Residues

Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), displays anti-tumor properties through its direct interaction with MMP-14. Lumican-derived peptides, such as lumcorin (17 amino acids) or L9M (10 amino acids), are able to inhibit the proteolytic activity of MMP-14 and melanoma progression. This work aimed to visualize the interactions of lumican-derived peptides and MMP-14. Molecular modeling was used to characterize the interactions between lumican-derived peptides, such as lumcorin, L9M, and cyclic L9M (L9Mc, 12 amino acids), and MMP-14. The interaction of L9Mc with MMP-14 was preferential with the MT-Loop domain while lumcorin interacted more with the catalytic site. Key residues in the MMP-14 amino acid sequence were highlighted for the interaction between the inhibitory SLRP-derived peptides and MMP-14. In order to validate the in silico data, MMP-14 activity and migration assays were performed using murine B16F1 and human HT-144 melanoma cells. In contrast to the HT-144 melanoma cell line, L9Mc significantly inhibited the migration of B16F1 cells and the activity of MMP-14 but with less efficacy than lumican and lumcorin. L9Mc significantly inhibited the proliferation of B16F1 but not of HT-144 cells in vitro and primary melanoma tumor growth in vivo. Thus, the site of interaction between the domains of MMP-14 and lumcorin or L9Mc were different, which might explain the differences in the inhibitory effect of MMP-14 activity. Altogether, the biological assays validated the prediction of the in silico study. Possible and feasible improvements include molecular dynamics results.

Nitrite-dependent vasodilation is facilitated by hypoxia and is independent of known NO-generating nitrite reductase activities

2007 ◽  
Vol 292 (6) ◽  
pp. H3072-H3078 ◽  
Author(s):  
Thomas Dalsgaard ◽  
Ulf Simonsen ◽  
Angela Fago
Keyword(s):  
Xanthine Oxidase ◽  
Guanylate Cyclase ◽  
Nitrite Reductase ◽  
Soluble Guanylate Cyclase ◽  
Nitrite Concentration ◽  
Inositol Hexaphosphate ◽  
Relative Contribution ◽  
Norepinephrine Concentration

The reduction of circulating nitrite to nitric oxide (NO) has emerged as an important physiological reaction aimed to increase vasodilation during tissue hypoxia. Although hemoglobin, xanthine oxidase, endothelial NO synthase, and the bc1 complex of the mitochondria are known to reduce nitrite anaerobically in vitro, their relative contribution to the hypoxic vasodilatory response has remained unsolved. Using a wire myograph, we have investigated how the nitrite-dependent vasodilation in rat aortic rings is controlled by oxygen tension, norepinephrine concentration, soluble guanylate cyclase (the target for vasoactive NO), and known nitrite reductase activities under hypoxia. Vasodilation followed overall first-order dependency on nitrite concentration and, at low oxygenation and norepinephrine levels, was induced by low-nitrite concentrations, comparable to those found in vivo. The vasoactive effect of nitrite during hypoxia was abolished on inhibition of soluble guanylate cyclase and was unaffected by removal of the endothelium or by inhibition of xanthine oxidase and of the mitochondrial bc1 complex. In the presence of hemoglobin and inositol hexaphosphate (which increases the fraction of deoxygenated heme), the effect of nitrite was not different from that observed with inositol hexaphosphate alone, indicating that under the conditions investigated here deoxygenated hemoglobin did not enhance nitrite vasoactivity. Together, our results indicate that the mechanism for nitrite vasorelaxation is largely intrinsic to the vessel and that under hypoxia physiological nitrite concentrations are sufficient to induce NO-mediated vasodilation independently of the nitrite reductase activities investigated here. Possible reaction mechanisms for nitrite vasoactivity, including formation of S-nitrosothiols within the arterial smooth muscle, are discussed.

scholarly journals Chemical Group Profiling, In Vitro and In Silico Evaluation of Aristolochia ringens on α-Amylase and α-Glucosidase Activity

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
J. B. Ahmad ◽  
E. O. Ajani ◽  
S. Sabiu
Keyword(s):  
In Silico ◽  
Inhibitory Effect ◽  
Root Extract ◽  
Asiatic Acid ◽  
Standard Drug ◽  
Synthetic Drugs ◽  
Ic50 Values ◽  
Hypoglycaemic Agent

Diabetes mellitus (DM) has become a global scourge, and there is a continuous search for novel compounds as viable alternatives to synthetic drugs which are often accompanied by severe adverse effects. Aristolochia ringens is among the scientifically implicated botanicals effective in the management of several degenerative diseases including DM. The current study evaluated the inhibitory mechanism(s) of root extract of A. ringens on α-amylase and α-glucosidase in vitro and in silico, while its constituents were characterized using liquid chromatography-mass spectrometric technique. The extract had concentration-dependent inhibitory effect on the study enzymes, and the inhibition compared well with that of standard drug (acarbose) with respective IC50 values of 0.67 mg/mL (α-amylase) and 0.57 mg/mL (α-glucosidase) compared with that of the extract (0.63 and 0.54 mg/mL). The extract competitively and uncompetitively inhibited α-amylase and α-glucosidase, respectively. Of the identified compounds, dianoside G (−12.4, −12.5 kcal/mol) and trilobine (−10.0, −10.0 kcal/mol) had significant interactions with α-amylase and α-glucosidase, respectively, while magnoflorine and asiatic acid also interacted keenly with both enzymes, with quercetin 3-O-glucuronide and strictosidine showing better affinity towards α-glucosidase. These observations are suggestive of involvement of these compounds as probable ligands contributing to antidiabetic potential of the extract. While studies are underway to demystify the yet to be identified compounds in the extract, the data presented have lent scientific credence to the acclaimed in vivo antidiabetic potential of the extract and suggested it as a viable source of oral hypoglycaemic agent.

Guanylate cyclase-G, expressed in the Grueneberg ganglion olfactory subsystem, is activated by bicarbonate

2010 ◽  
Vol 432 (2) ◽  
pp. 267-273 ◽  
Author(s):  
Ying-Chi Chao ◽  
Chien-Jui Cheng ◽  
Hsiu-Ting Hsieh ◽  
Chih-Ching Lin ◽  
Chien-Chang Chen ◽  
...  
Keyword(s):  
G Protein ◽  
Guanylate Cyclase ◽  
Activation Mechanism ◽  
Cgmp Level ◽  
Carbonic Anhydrases ◽  
Grueneberg Ganglion ◽  
Biochemical Analyses ◽  
Ganglion Neurons

GC (guanylate cyclase)-G is the most recently identified member of the receptor GC family. However, the regulation of its activity and protein expression in the mammalian olfactory system remains unclear. In the present study, we used a GC-G-specific antibody to validate that the GC-G protein is expressed in Grueneberg ganglion neurons, a newly recognized olfactory subsystem co-expressing other cGMP signalling components such as the cGMP-regulated PDE2A (phosphodiesterase 2A) and the cGMP-gated ion channel CNGA3 (cyclic nucleotide-gated cation channel α-3). Further molecular and biochemical analyses showed that heterologously expressed GC-G protein, specifically the C-terminal cyclase domain, was directly stimulated by bicarbonate in both in vivo cellular cGMP accumulation assays in human embryonic kidney-293T cells and in vitro GC assays with a purified recombinant protein containing the GC domain. In addition, overexpression of GC-G in NG108 neuronal cells resulted in a CO2-dependent increase in cellular cGMP level that could be blocked by treatment with acetazolamide, an inhibitor of carbonic anhydrases, which implies that the stimulatory effect of CO2 requires its conversion to bicarbonate. Together, our data demonstrate a novel CO2/bicarbonate-dependent activation mechanism for GC-G and suggest that GC-G may be involved in a wide variety of CO2/bicarbonate-regulated biological processes such as the chemosensory function in Grueneberg ganglion neurons.

Formation and Structure of Casein Micelles in Lactating Mammary Tissue

1970 ◽  
Vol 28 ◽  
pp. 150-151
Author(s):  
Robert J. Carroll ◽  
Marvin P. Thompson ◽  
Harold M. Farrell
Keyword(s):  
Size Distribution ◽  
G Protein ◽  
Milk Proteins ◽  
Mammary Tissue ◽  
Colloidal System ◽  
Casein Micelles ◽  
The Stability

Milk is an unusually stable colloidal system; the stability of this system is due primarily to the formation of micelles by the major milk proteins, the caseins. Numerous models for the structure of casein micelles have been proposed; these models have been formulated on the basis of in vitro studies. Synthetic casein micelles (i.e., those formed by mixing the purified αsl- and k-caseins with Ca2+ in appropriate ratios) are dissimilar to those from freshly-drawn milks in (i) size distribution, (ii) ratio of Ca/P, and (iii) solvation (g. water/g. protein). Evidently, in vivo organization of the caseins into the micellar form occurs in-a manner which is not identical to the in vitro mode of formation.

The Effect of Barbituric Acid Derivatives on Platelet Function in Vitro and in Vivo

1973 ◽  
Vol 30 (02) ◽  
pp. 315-326
Author(s):  
J. Heinz Joist ◽  
Jean-Pierre Cazenave ◽  
J. Fraser Mustard
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Barbituric Acid ◽  
Platelet Rich Plasma ◽  
Inhibitory Effect ◽  
Primary Response ◽  
Sodium Pentobarbital ◽  
Barbituric Acid Derivatives

SummarySodium pentobarbital (SPB) and three other barbituric acid derivatives were found to inhibit platelet function in vitro. SPB had no effect on the primary response to ADP of platelets in platelet-rich plasma (PRP) or washed platelets but inhibited secondary aggregation induced by ADP in human PRP. The drug inhibited both phases of aggregation induced by epinephrine. SPB suppressed aggregation and the release reaction induced by collagen or low concentrations of thrombin, and platelet adherence to collagen-coated glass tubes. The inhibition by SPB of platelet aggregation was readily reversible and isotopically labeled SPB did not become firmly bound to platelets. No inhibitory effect on platelet aggregation induced by ADP, collagen, or thrombin could be detected in PRP obtained from rabbits after induction of SPB-anesthesia.

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