Generation and Characterization of a Stable Soluble Guanylate Cyclase-Overexpressing CHO Cell Line

Becker, Erika; Wunder, Frank; Kast, Raimund; Robyr, Chantal; Hoenicka, Markus; Gerzer, R.; Schröder, H.; Stasch, Johannes Peter

doi:10.1006/niox.1999.0207

Cited by 22 publications

(16 citation statements)

References 36 publications

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“…The cGMP readout cell transiently transfected with WT-sGC displayed an activation profile similar to that of the isolated enzyme responding to DEA/NO and/or BAY 41-2272 (13,25,33). Additionally, incubation with BAY 58-2667 led to an increase in sGC activity that was potentiated in the presence of ODQ as described for the purified enzyme (14,15).…”

Section: Discussionmentioning

confidence: 67%

Identification of Residues Crucially Involved in the Binding of the Heme Moiety of Soluble Guanylate Cyclase

Schmidt

Schramm

Schröder

et al. 2004

Journal of Biological Chemistry

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155

153

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Soluble guanylate cyclase (sGC), a heterodimeric hemeprotein, is the only receptor for the biological messenger nitric oxide (NO) identified to date and is intimately involved in various signal transduction pathways. By using the recently discovered NO-and hemeindependent sGC activator BAY 58-2667 and a novel cGMP reporter cell, we could distinguish between hemecontaining and heme-free sGC in an intact cellular system. Using these novel tools, we identified the invariant amino acids tyrosine 135 and arginine 139 of the ␤ 1 -subunit as crucially important for both the binding of the heme moiety and the activation of sGC by BAY 58-2667. The heme is displaced by BAY 58-2667 due to a competition between the carboxylic groups of this compound and the heme propionic acids for the identified residues tyrosine 135 and arginine 139. This displacement results in the release of the axial heme ligand histidine 105 and to the observed activation of sGC. Based on these findings we postulate a signal transmission triad composed of histidine 105, tyrosine 135, and arginine 139 responsible for the enzyme activation by this compound and probably also for transducing changes in heme status and porphyrin geometry upon NO binding into alterations of sGC catalytic activity.

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Section: Discussionmentioning

confidence: 67%

Identification of Residues Crucially Involved in the Binding of the Heme Moiety of Soluble Guanylate Cyclase

Schmidt

Schramm

Schröder

et al. 2004

Journal of Biological Chemistry

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155

153

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“…In purified preparations, ODQ is a potent inhibitor of sGC. 24,25 At the same concentrations used in these experiments, ODQ does not inhibit endothelial nitric oxide synthase or vascular responses to cGMP, adenosine, or papaverine. 9,11,26 -28 Consistent with these studies, we found that ODQ did not alter relaxation of primate cerebral arteries to papaverine.…”

Section: Didion Et Al No-mediated Relaxation Of Cerebral Arteriesmentioning

confidence: 88%

Mechanisms That Produce Nitric Oxide–Mediated Relaxation of Cerebral Arteries During Atherosclerosis

Didion

Heistad

Faraci

2001

Stroke

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Background and Purpose-The first goal of the present study was to examine the hypothesis that relaxation of cerebral arteries to nitric oxide in primates is dependent on activation of soluble guanylate cyclase (sGC). The second goal was to determine whether the role of sGC in mediating responses to nitric oxide is altered in atherosclerosis. Methods-Basilar arteries from normal and atherosclerotic monkeys were studied in vitro. After precontraction with prostaglandin F 2␣ (0.1 to 1 mol/L), concentration-response curves to authentic nitric oxide (1 nmol/L to 1 mol/L), sodium nitroprusside (10 nmol/L to 10 mol/L; a nitric oxide donor), and papaverine (10 nmol/L to 10 mol/L; a non-nitric oxide, non-sGC-dependent stimulus) were generated in the presence and absence of 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 and 10 mol/L; an inhibitor of sGC). The effect of ODQ on basal tone of basilar arteries from normal and atherosclerotic monkeys was also examined. Results-Nitric oxide, sodium nitroprusside, and papaverine produced relaxation that was similar (PϾ0.05) in normal and atherosclerotic monkeys. ODQ produced marked inhibition (PϽ0.05) of vasorelaxation in response to nitric oxide and nitroprusside but not papaverine. For example, relaxation of the basilar artery in response to nitric oxide (0.1 mol/L) was inhibited by approximately 85% and 73% by ODQ (1 mol/L) in normal and atherosclerotic monkeys, respectively. ODQ produced contraction of the basilar arteries, and the increase in tension to ODQ was greater in normal (2.7Ϯ0.3 g; meanϮSE) than in atherosclerotic monkeys (1.4Ϯ0.4 g; PϽ0.05). In contrast, contraction to prostaglandin F 2␣ was similar in the basilar artery from normal and atherosclerotic monkeys. Conclusions-These findings suggest that (1) relaxation of cerebral arteries in primates in response to nitric oxide is normally dependent, in large part, on activation of sGC and (2) the influence of sGC (via reduced production and/or activity of basal nitric oxide) on cerebral vascular tone is reduced in atherosclerosis.

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“…sGC stimulation in platelets correlates with inhibition of aggregation, platelet cyclic GMP increase, prolongation of bleeding time, and antithrombotic eects in vitro (Hobbs, 2000;Stasch et al, 2002a, b;Becker et al, 1999;Teng et al, 1997). BAY 58-2667 potently inhibited platelet aggregation induced by the thromboxane mimic U46619 and collagen, whereas TRAP-6-and thrombin-mediated aggregation was not aected.…”

mentioning

confidence: 99%

NO‐ and haem‐independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle

Stasch

Schmidt

Alonso‐Alija

et al. 2002

British J Pharmacology

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287

280

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1 Soluble guanylyl cyclase (sGC) is the only proven receptor for the ubiquitous biological messenger nitric oxide (NO) and is intimately involved in many signal transduction pathways, most notably in regulating vascular tone and platelet function. sGC is a heterodimeric (a/û) protein that converts GTP to cyclic GMP; NO binds to its prosthetic haem group. Here, we report the discovery of a novel sGC activating compound, its interaction with a previously unrecognized regulatory site and its therapeutic implications. 2 Through a high-throughput screen we identi®ed BAY 58-2667, an amino dicarboxylic acid which potently activates sGC in an NO-independent manner. In contrast to NO, YC-1 and BAY 41-2272, the sGC stimulators described recently, BAY 58-2667 activates the enzyme even after it has been oxidized by the sGC inhibitor ODQ or rendered haem de®cient. 3 Binding studies with radiolabelled BAY 58-2667 show a high anity site on the enzyme. 4 Using photoanity labelling studies we identi®ed the amino acids 371 (a-subunit) and 231 ± 310 (û-subunit) as target regions for BAY 58-2667. 5 sGC activation by BAY 58-2667 results in an antiplatelet activity both in vitro and in vivo and a potent vasorelaxation which is not in¯uenced by nitrate tolerance. 6 BAY 58-2667 shows a potent antihypertensive eect in conscious spontaneously hypertensive rats. In anaesthetized dogs the hemodynamic eects of BAY 58-2667 and GTN are very similar on the arterial and venous system. 7 This novel type of sGC activator is a valuable research tool and may oer a new approach for treating cardiovascular diseases.

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Generation and Characterization of a Stable Soluble Guanylate Cyclase-Overexpressing CHO Cell Line

Cited by 22 publications

References 36 publications

Identification of Residues Crucially Involved in the Binding of the Heme Moiety of Soluble Guanylate Cyclase

Identification of Residues Crucially Involved in the Binding of the Heme Moiety of Soluble Guanylate Cyclase

Mechanisms That Produce Nitric Oxide–Mediated Relaxation of Cerebral Arteries During Atherosclerosis

NO‐ and haem‐independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle

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