Elsevier

Biochemical Pharmacology

Volume 92, Issue 4, 15 December 2014, Pages 661-668
Biochemical Pharmacology

Regulation of vascular tone in rabbit ophthalmic artery: Cross talk of endogenous and exogenous gas mediators

https://doi.org/10.1016/j.bcp.2014.10.011Get rights and content

Abstract

Nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H2S) modulate vascular tone. In view of their therapeutic potential for ocular diseases, we examined the effect of exogenous CO and H2S on tone of isolated rabbit ophthalmic artery and their interaction with endogenous and exogenous NO. Ophthalmic artery segments mounted on a wire myograph were challenged with cumulative concentrations of phenylephrine (PE) in the presence or absence of NG-nitro-l-arginine (LNNA) to inhibit production of NO, the CO-releasing molecules CORMs or the H2S-donor GYY4137. The maximal vasoconstriction elicited by PE reached 20–30% of that induced by KCl but was dramatically increased by incubation with LNNA. GYY4137 significantly raised PE-mediated vasoconstriction, but it did not change the response to PE in the presence of LNNA or the relaxation to sodium nitroprusside (SNP). CORMs concentration-dependently inhibited PE-induced constriction, an effect that was synergistic with endogenous NO (reduced by LNNA), but insensitive to blockade of guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3,-α]quinoxalin-1-one (ODQ). In vascular tissues cyclic GMP (cGMP) levels seemed reduced by GYY4137 (not significantly), but were not changed by CORM. These data indicate that CO is able per se to relax isolated ophthalmic artery and to synergize with NO, while H2S counteracts the effect of endogenous NO. CO does not stimulate cGMP production in our system, while H2S may reduce cGMP production stimulated by endogenous NO. These findings provide new insights into the complexities of gas interactions in the control of ophthalmic vascular tone, highlighting potential pharmacological targets for ocular diseases.

Introduction

Nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H2S) are gaseous molecules that have long been known as environmental pollutants and highly toxic gases [1]. Since the seminal discovery of the endothelium-derived relaxing factor (EDRF) by Furchgott [2], later identified as NO [3], CO [4] and H2S [5] have also been found to be endogenously produced and to act as signalling molecules in vertebrates. Nowadays these molecules are studied not only as endogenous regulators, but also as potentially exploitable therapeutics. In fact, although NO was initially characterized as the main mediator of endothelium-dependent vasodilatation, CO and H2S have also been shown to affect vascular tone among other important biological actions. Collectively, these three signalling gases have become known as gasotransmitters [6]. While a large body of studies during the last three decades has focused on the molecular and cellular mechanisms of NO and its functions in different biological systems, including the eye, much less is known about CO and H2S, for which the mechanisms underlying their pharmacologic effects are still mostly unclear.

Interestingly, CO, NO and H2S are endogenously produced in vertebrate retinas and have been studied in several non-clinical and clinical ocular paradigms [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19].

A number of chemicals able to deliver CO and H2S to tissues in a controlled manner have been developed and could potentially be used in human therapy alongside NO-donors, which are approved drugs used to relieve acute cardiac ischemia and hypertension [20].

A potential cross talk between the three gas mediators has been hypothesized but poorly examined. Thus, the aim of the present study was to investigate the effects of exogenous CO and H2S on tone of isolated rabbit ophthalmic artery and their interaction with endogenous and exogenous NO. Furthermore, in order to gain insight on the mechanism/s whereby these gases exert the regulation of vascular tone, we measured cyclic GMP (cGMP) levels in rabbit ophthalmic arteries.

Section snippets

Preparation of ophthalmic arteries

Animal use was approved by the subcommittee for research and animal care at the University of Catania according to guidelines from Italian Ministry of Health. Male New Zealand rabbits (Charles River, Calco, Italy; 200–250 g) were euthanatized with a single injection of zoletil® 100 in the peripheral vein of the hear. Ophthalmic arteries from both eyes were dissected, immersed in physiological salt solution (PSS, composition in mM: NaCl, 118; KCl, 4.6; NaHCO3, 25; MgSO4, 1.2; KH2PO4, 1.2; CaCl2,

Characterization of NO-dependent responses of isolated ophthalmic artery

Because basal, unstimulated, NO release from endothelium is well known to reduce responses to vasoconstrictor agonists [26], [27], [28], we attempted to functionally assess endothelial NO-production in isolated ophthalmic artery by: (i) comparing vasoconstriction to PE before and after blockade of basal NO synthesis by LNNA; (ii) by measuring vasodilatation of PE-preconstricted preparations following stimulation of NO release with acetylcholine (ACh, Fig. 2, Fig. 3).

In control conditions, PE

Discussion

NO, CO and H2S have been evaluated for potential clinical applications for ocular diseases, particularly in conditions like glaucoma, where the ocular blood flow regulation is crucial [30], [31], [32], [33].

In this study we used isolated rabbit ophthalmic artery to explore the impact of these gases on ocular circulation. However, because ophthalmic artery supplies also many extra-ocular structures, the significance of our data on retinal or choroidal blood flow regulation may be limited. We

Conflict of interest statement

The authors do not have competing interests to disclose.

Acknowledgements

This work was supported in part by a National grant PON01-00110, the Aremcar Foundation and the International Ph.D. Program in Neuropharmacology, University of Catania, Italy. We thank Prof. Brian Mann for the synthesis of CORM 371.

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