The effect of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and charybdotoxin (CTX) on relaxations of isolated cerebral arteries to nitric oxide

doi:10.1016/S0006-8993(97)01393-0

Brain Research

Volume 785, Issue 1, 23 February 1998, Pages 107-113

https://doi.org/10.1016/S0006-8993(97)01393-0 Get rights and content

Abstract

The mechanism underlying smooth muscle relaxations of cerebral arteries in response to nitric oxide is still not completely understood. The present study was designed to determine the role of soluble guanylate cyclase in the relaxations to a nitric oxide/nucleophile complex, diethylaminodiazen-1-ium-1,2-dioate (DEA-NONOate). Rings of canine middle cerebral arteries without endothelium were suspended in Krebs–Ringer bicarbonate solution for isometric tension recording. The levels of guanosine 3′,5′-cyclic monophosphate (cyclic GMP) were measured by radioimmunoassay technique. During contractions to uridine 5′-triphosphate (UTP), DEA-NONOate (10⁻¹⁰ to 10⁻⁵ M) caused concentration-dependent relaxations. Measurements of cyclic GMP levels in cerebral arterial wall demonstrated that DEA-NONOate is a potent stimulator of guanylate cyclase and subsequent formation of cyclic GMP. Increasing concentrations of a selective soluble guanylate cyclase inhibitor, 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), caused concentration-dependent reduction of both cyclic GMP production and relaxations to DEA-NONOate. Interestingly, in the presence of the highest concentration (3×10⁻⁶ M) of ODQ, production of cyclic GMP in response to 10⁻⁶ M of DEA-NONOate was abolished, whereas the same concentration of DEA-NONOate caused almost complete relaxation, suggesting that mechanisms independent of cyclic GMP production may mediate relaxing effect of high concentration of a nitric oxide donor. A selective Ca²⁺-activated potassium channel blocker charybdotoxin (CTX) significantly reduced relaxations to DEA-NONOate resistant to ODQ, supporting the idea that in cerebral arteries nitric oxide may activate potassium channels independently of cyclic GMP. The results of our study suggest that under physiological conditions, guanylate cyclase is a key mediator of cerebral arterial relaxations to nitric oxide. However, under pathological conditions associated with induction of nitric oxide synthase and increased biosynthesis of nitric oxide (e.g., cerebral ischemia, inflammation, sepsis), mechanisms other than formation of cyclic GMP may be activated.

Introduction

Nitric oxide is an important mediator of endothelium-dependent relaxations in various vascular beds 6, 27, and it plays an essential role in regulation of the cerebral circulation under both physiological and pathological conditions 3, 5, 21, 36. It is generally accepted that nitric oxide relaxes smooth muscle cells by activation of soluble guanylate cyclase, subsequent accumulation of guanosine 3′,5′-cyclic monophosphate (cyclic GMP), and cyclic GMP-dependent modification of several intracellular processes [12]. However, a previous study has documented that besides activation of guanylate cyclase, nitric oxide itself may directly (independently of cyclic GMP) interact with a variety of other proteins involved in cellular signaling mechanisms [31]. A recent study demonstrated that nitric oxide can directly activate potassium (K⁺) channels (Ca²⁺-activated K⁺ channels; K_Ca channels) and causes relaxations in rabbit aortic smooth muscle cells [2]. In cerebral arteries, it has not been determined whether cyclic GMP-independent mechanisms are involved in relaxations induced by nitric oxide.

Previous studies examined the role of soluble guanylate cyclase in mediation of nitric oxide-induced vascular relaxations by using methylene blue and LY83583 (6-anilino-5,8-quinolinedione), which reportedly generate superoxide anions and may not be specific guanylate cyclase inhibitors 8, 19, 35. Recently, a novel and highly selective inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) has become available 7, 23. Additionally, in order to obtain the controlled delivery of nitric oxide into biological systems, nitric oxide/nucleophile complexes including diethylamine NONOate (DEA-NONOate), have been used 18, 22. The availability of these novel compounds made it possible to more precisely analyze the role of guanylate cyclase in nitric oxide-mediated relaxations in cerebral arteries. Therefore, the primary goal of the present study was designed to determine the effect of ODQ on formation of cyclic GMP and relaxations in response to DEA-NONOate in isolated canine middle cerebral arteries.

Section snippets

In vitro studies

The experiments were performed on rings (4 mm long) of middle cerebral arteries taken from dogs (15 to 20 kg) anesthetized with 30 mg kg⁻¹ i.v. pentobarbital sodium. All procedures were conducted in accordance with institutional guidelines. The arterial rings were placed in modified Krebs–Ringer bicarbonate solution (control solution) of the following composition (mM): NaCl: 118.3; KCl: 4.7; CaCl₂: 2.5; MgSO₄: 1.2; KH₂PO₄: 1.2; NaHCO₃: 25.0; calcium–ethylenediamine-tetraacetic acid: 0.026;

Effect of ODQ on relaxations to DEA-NONOate and forskolin

During contractions induced by UTP (10⁻⁵ M), DEA-NONOate (10⁻¹⁰ to 10⁻⁵ M) induced concentration-dependent relaxations in canine middle cerebral arteries without endothelium. ODQ (3×10⁻⁷ to 3×10⁻⁶ M) caused concentration-dependent reduction of the relaxations to DEA-NONOate (Fig. 1A), whereas ODQ in the highest concentration (3×10⁻⁶ M) did not cause any effect on relaxations to forskolin (Fig. 1B).

Time-course of DEA-NONOate effect on cyclic GMP levels

In canine middle cerebral artery rings without endothelium, DEA-NONOate (10⁻⁸ M) produced

Discussion

It is generally accepted that nitric oxide as well as exogenous nitrovasodilators relax vascular smooth muscle cells by an activation of soluble guanylate cyclase [12]. However, recent studies indicated that besides guanylate cyclase, a variety of other proteins are targets for nitric oxide [31], indicating that nitric oxide may directly (independently of cyclic GMP production) interact with proteins involved in cellular signaling mechanisms responsible for vascular smooth muscle relaxations.

Acknowledgements

This work was supported in part by National Heart, Lung, and Blood Institute grant HL-53524, and the Mayo Foundation. Dr. Onoue was supported by a scholarship from Uehara Memorial Foundation (Tokyo, Japan). The authors would like to thank Janet Beckman for preparing the manuscript.

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Research reportThe effect of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and charybdotoxin (CTX) on relaxations of isolated cerebral arteries to nitric oxide

Abstract

Introduction

Section snippets

In vitro studies

Effect of ODQ on relaxations to DEA-NONOate and forskolin

Time-course of DEA-NONOate effect on cyclic GMP levels

Discussion

Acknowledgements

Eur. J. Pharmacol.

Trends Neurosci.

Cell

Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase

Proc. Natl. Acad. Sci. U.S.A.

Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle

Nature

Nitric oxide: a physiologic messenger molecule

Annu. Rev. Biochem.

Nitric oxide and the cerebral circulation

Stroke

The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine

Nature

Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one

Mol. Pharmacol.

Inhibition by LY83583 of endothelium-dependent relaxation of rabbit aorta

FASEB J.

Purification, sequence, and model structure of charybdotoxin, a potent selective inhibitor of calcium-activated potassium channels

Proc. Natl. Acad. Sci. U.S.A.

Phosphodiesterase isozyme inhibition and the potentiation by zaprinast of endothelium-derived relaxing factor and guanylate cyclase stimulating agents in vascular smooth muscle

J. Pharmacol. Exp. Ther.

The pharmacological and physiological role of cyclic GMP in vascular smooth muscle relaxation

Annu. Rev. Pharmacol. Toxicol.

Vasopressin causes endothelium-dependent relaxations of the canine basilar artery

Circ. Res.

Oxytocin causes endothelium-dependent relaxations of canine basilar arteries by activating V1-vasopressinergic receptors

J. Pharmacol. Exp. Ther.

Endothelium-dependent contraction to stretch in canine basilar arteries

Am. J. Physiol.

Experimental pneumococcal meningitis: cerebrovascular alterations, brain edema, and meningeal inflammation are linked to the production of nitric oxide

Ann. Neurol.

Nitric oxide measured by a porphyrinic microsensor in rat brain after transient middle cerebral artery occlusion

J. Cereb. Blood Flow Metab.

Research report
The effect of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and charybdotoxin (CTX) on relaxations of isolated cerebral arteries to nitric oxide

Oxytocin causes endothelium-dependent relaxations of canine basilar arteries by activating V₁-vasopressinergic receptors