Adenine nucleotide effect on intraocular pressure: Involvement of the parasympathetic nervous system

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Abstract

Nucleotides are present in the aqueous humor possibly exerting physiological effects on intraocular pressure (IOP). To determine the effect of nucleotides such as ATP and its related derivatives on IOP, New Zealand white rabbits were used. IOP was measured in rabbits treated topically either with saline (control) or with a single dose (10 μg/μL) of adenine nucleotides (ATP, 2-meS-ATP, ATP-γ-S, α,β-meADP, α,β-meATP and β,γ-meATP). Those nucleotides reducing IOP (α,β-meATP and β,γ-meATP) were then tested in concentrations ranging from 1 to 100 μg/μL to obtain the IC50 value. Several antagonists for the P2 and adenosine A1 receptors (all at 10 μg/μL) were assayed 30 min before the application of the hypotensive nucleotide β,γ-meATP. To see whether the nucleotide was acting directly on the structures involved in aqueous humor dynamics or on the autonomic nerves controlling IOP, animal denervation and sympathetic (yohimbine and ICI-118,551 at 10 μg/μL) and parasympathetic (atropine and hexametonium at 10 μg/μL) receptors' antagonists were used 30 min before the instillation of β,γ-meATP. α,β-meATP and β,γ-meATP decreased IOP to 60% of control value (basal IOP = 23.2 ± 1.3 mmHg), with IC50 of 1.59 ± 0.21 μg/μL and 0.56 ± 0.62 μg/μL, which corresponds to 3 mM and 1 mM respectively. Denervation completely abolished the effect of β,γ-meATP. Sympathetic antagonists did not modify the hypotensive effect of β,γ-meATP, but parasympathetic antagonists were able to abolish it. Among the series of adenine nucleotide tested, α,β-meATP and β,γ-meATP presented hypotensive actions on IOP. β,γ-meATP seems to stimulate cholinergic terminals being its final effect the IOP reduction. Therefore, these two nucleotides are interesting pharmacological tools for those pathologies related with high intraocular pressure.

Introduction

Many efforts have been done to fully understand the mechanisms that control and regulate intraocular pressure (IOP) in mammals. The study of the balance between aqueous humor production and its drainage and how this system is modulated by different neurotransmitters is highly complex (Davson, 1993). Nevertheless, some clear findings are actually accepted. Both, sympathetic and parasympathetic innervations are involved in the regulation of IOP as observed in many experimental models (Jumblatt, 2000). Sympathetic nerves release noradrenaline, which stimulate predominantly α-receptors present in the iris and ciliary body and therefore influencing IOP. The stimulation of α-receptors (in particular α2-receptors) in the ciliary processes produce a reduction in IOP, and this fact had invited the ocular pharmacologists to study the potential of lowering IOP by using adrenergic receptor agonists (Burke and Potter, 1986). Moreover, the effect mediated by α2-receptors can be enhanced by blocking β2 adrenergic receptors, this indicating that a combined mixture of agonists and antagonists of the adrenergic receptors could be a good approach for glaucoma therapy (Nathanson, 1980).

Parasympathetic nerves also play a role in regulating different processes in the eye such as pupil size, lens accommodation and IOP (Judge and Flitcroft, 2000, Jumblatt, 2000). The effect of acetylcholine (ACh) released from parasympathetic nerves on intraocular pressure is the result of various actions on the aqueous humor outflow, related to the stimulation of muscarinic receptors present in the ciliary muscle (for a review see Stone, 1996). The stimulation of these receptors by means of muscarinic agonists produces the contraction of the ciliary muscle. The way the muscle affects the humor outflow is related to the anatomic connection of the anterior tendons of the ciliary muscle with the scleral spur and the trabecular meshwork. The ciliary muscle contraction pulls the tendons, which produce an unfolding of the trabecular meshwork permitting a widening of the canal lumen this allowing the facilitation of the aqueous outflow from the anterior chamber (Rohen, 1964). Different cholinergic agonists such as pilocarpine or carbamylcholine are commonly used in glaucoma therapy, although many side effects have been described both in the eye (ciliary congestion, accommodative myopia and pupilary constriction among others) and systemically, such as nausea diarrhea or bradycardia (Leopold, 1994).

Many other substances have been tested in their ability to reduce IOP: prostaglandins, corticosteroids, vasopressin and adenosine have been used as potential tools in the glaucoma treatment (Bito, 1987, McGhee, 1992, Crosson, 1995).

Adenosine together with ATP and adenine dinucleotides forms a group of neuroactive substances with physiological actions in neural and non-neural tissues (Abbracchio and Burnstock, 1994, Pintor et al., 2000). These purinergic compounds do not share the same membrane receptors. Adenosine can stimulate four different receptors' subtypes termed A1, A2A, A2B and A3, coupled to adenylate cyclase (Fredholm et al., 1994). ATP acts on two main groups of receptors termed P2X and P2Y. P2X receptors are Na+/Ca2+ ion channels (ATP ionotropic receptors) and it has been possible to describe up till 7 cloned subtypes (P2X1, …, P2X7) (von Kugelgen, 2006). On the other hand P2Y receptors are mainly coupled to phospholipase C (PLC), adenylate cyclase and thus they are nucleotide metabotropic receptors. There has been cloned and expressed 8 subtypes of these receptors which significantly differ in their responses to ATP, UTP and synthetic analogues (Ralevic and Burnstock, 1998, Jacobson et al., 2006).

Nucleotides and nucleosides, like adenosine, can exert physiological effects starting from on the ocular surface where they can modulate aspects such as tear secretion, lysozyme production, mucin release or corneal wound healing (Guzmán-Aranguez et al., 2007, Crooke et al., 2008), and they can also modify intraocular physiology. In this sense, Crosson and co-workers have described the effect of adenosine and specific adenosine receptor agonists on IOP in New Zealand rabbits (Crosson, 1995, Crosson and Gray, 1996). Various adenosine receptor subtypes are involved in its regulation, either raising or decreasing IOP (Avila et al., 2001), being adenosine receptors identified in the Schlemm canal responsible of at least part of these actions (Karl et al., 2005). Nevertheless, little is known regarding the effects of adenine nucleotides on IOP in rabbits and precisely where the actions of these nucleotides occur. In the present experimental work the differential effects of ATP and its synthetic analogues on IOP in New Zealand rabbits were studied, focusing in those compounds reducing intraocular pressure.

Section snippets

Animals

New Zealand white rabbits were used. The animals were submitted to controlled day–night cycles to avoid circadian effects in IOP measurements and kept in individual cages with free access to food and water. This study was adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and also the experiments were carried out in accordance with the European Communities Council Directive (86/609/EEC).

Intraocular pressure measurements

IOP was measured with a TONOPEN contact tonometer supplied by MENTOR

Effect of purinergic compounds on IOP

Prior to any nucleotide instillation, IOP was measured in all the rabbits to obtain the control value (23.2 ± 1.3 mmHg, n = 50). When the naturally occurring nucleotide ATP was tested at a concentration of 10 μg/μL (20 mM) a biphasic behavior was observed in IOP (Fig. 1A). An initial, fast IOP reduction was observed within 30 min after the nucleotide instillation, followed by an ongoing increase in the pressure, which remained above the control value for more than 6 h. The minimal IOP value obtained

Discussion

In the present experimental work the activity of a series of adenine mononucleotide derivatives in the modulation of intraocular pressure in New Zealand rabbits was investigated. Two main groups were observed among the different nucleotides assayed: those producing an increase in IOP and those which decrease IOP values.

The pharmacology of the compounds with a hypertensive effect fits appropriately with the classical pharmacology of the P2Y receptors (Pintor, 2000). This receptor has been

Acknowledgements

This work has been supported by a project from Comunidad de Madrid, NEUROTRANS-CM ref: P-SAL-0253-2006 and in part by SAF2005-0277, SAF2008-00529 and CSD2007-00023 from Ministerio de Educación y Ciencia and RETICS RD07/0062/0004.

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