1 Under voltage-clamp conditions, the activity of agonists and antagonists at a recombinant P2X 2 receptor expressed in Xenopus oocytes was examined at di erent levels of extracellular pH (pH e ). 2 In normal Ringer (Mg 2+ ions absent), the amplitude of submaximal inward currents to ATP was increased by progressively lowering pH e (8.0 ± 5.5). ATP-responses reached a maximum at pH 6.5 with a 5 fold increase in ATP-a nity; the apparent pK a was 7.05+0.05. 3 Receptor a nity for ATP was lowered when extracellular Ca 2+ ions were replaced with equimolar Mg 2+ ions. However, the amplitude of the ATP-responses was still enhanced under acidic conditions, reaching maximal activity at pH 6.5 with a 5 fold increase in ATP-a nity; the apparent pK a was 7.35+0.05. 4 ATP species present in the superfusate (for the above ionic conditions and pH levels) were calculated to determine the forms of ATP which activate P2X 2 receptors: possible candidates include HATP, CaHATP and MgHATP. However, levels of these protonated species increase below pH 6.5, suggesting that receptor protonation rather than agonist protonation is more important. 5 The potency order for agonists of P2X 2 receptors was: ATP42-MeS-ATP5ATPgS4 ATPaS44CTP5BzATP, while other nucleotides were inactive. EC 50 and n H values for full agonists were determined at pH 7.4 and re-examined at pH 6.5. Extracellular acidi®cation increased the a nity by approximately 5 fold for full agonists (ATP, 2-MeSATP, ATPgS and ATPaS), without altering the potency order. 6 The potency order for antagonists at P2X 2 receptors was: Reactive blue-24trinitrophenol-ATP5Palatine fast black5Coomassie brilliant blue5PPADS4suramin (at pH 7.4). IC 50 values and slopes of the inhibition curves were re-examined at di erent pH levels. Only blockade by suramin was a ected signi®cantly by extracellular acidi®cation (IC 50 values: 10.4+2 mM, at pH 7.4; 78+5 nM, at pH 6.5; 30+6 nM, at pH 5.5). 7 In summary, a lowered pH e enhanced the activity of all agonists at P2X 2 receptors but, with the exception of suramin, not antagonists. Since a lowered pH e is also known to enhance agonist activity at P 2X receptors on sensory neurones containing P2X 2 transcripts, the sensitization by metabolic acidosis of native P 2X receptors containing P2X 2 subunits may have a signi®cant e ect on purinergic cell-to-cell signalling.

A full pharmacological characterization was carried out on a recombinant ATP-gated ion channel (P2X, purinoceptor) expressed in Xenopus oocytes. This slowly-desensitizing neuronal P2X2 purinoceptor, activated by ATP (EC50=4.6+1 jM at pH 7.4; n=4), showed the agonist potency order: ATP ,2-MeSATP = ATPyS ATPoSS> > Bz-ATP. The receptor affinity for ATP was enhanced 5-10 fold by acidifying the bathing solution (to pH 6.5) but was diminished 4-5 fold in an alkaline solution (pH 8.0).The maximum activity of P2X2 purinoceptors and the activity order of a series of nucleotides were unaltered by changing extracellular pH. Interestingly, ATP sensitivity at a recombinant P2Y purinoceptor remained unaltered with changing extracellular pH. These results indicate that acidotic conditions in the synaptic cleft could strengthen purinergic transmission at neuronal P2X2 purinoceptors.

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This review highlights recent findings that describe how purines modulate the physiological and pathophysiological responses of ocular tissues. For example, in lacrimal glands the cross-talk between P2X7 receptors and both M3 muscarinic receptors and α1D-adrenergic receptors can influence tear secretion. In the cornea, purines lead to post-translational modification of EGFR and structural proteins that participate in wound repair in the epithelium and influence the expression of matrix proteins in the stroma. Purines act at receptors on both the trabecular meshwork and ciliary epithelium to modulate intraocular pressure (IOP); ATP-release pathways of inflow and outflow cells differ, possibly permitting differential modulation of adenosine delivery. Modulators of trabecular meshwork cell ATP release include cell volume, stretch, extracellular Ca2+ concentration, oxidation state, actin remodeling and possibly endogenous cardiotonic steroids. In the lens, osmotic stress leads to ATP release following TRPV4 activation upstream of hemichannel opening. In the anterior eye, diadenosine polyphosphates such as Ap4A act at P2 receptors to modulate the rate and composition of tear secretion, impact corneal wound healing and lower IOP. The Gq11-coupled P2Y1-receptor contributes to volume control in Müller cells and thus the retina. P2X receptors are expressed in neurons in the inner and outer retina and contribute to visual processing as well as the demise of retinal ganglion cells. In RPE cells, the balance between extracellular ATP and adenosine may modulate lysosomal pH and the rate of lipofuscin formation. In optic nerve head astrocytes, mechanosensitive ATP release via pannexin hemichannels, coupled with stretch-dependent upregulation of pannexins, provides a mechanism for ATP signaling in chronic glaucoma. With so many receptors linked to divergent functions throughout the eye, ensuring the transmitters remain local and stimulation is restricted to the intended target may be a key issue in understanding how physiological signaling becomes pathological in ocular disease.

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RNA interference (RNAi) can be used to inhibit the expression of specific genes in vitro and in vivo, thereby providing an extremely useful tool for investigating gene function. Progress in the understanding of RNAi-based mechanisms has opened up new perspectives in therapeutics for the treatment of several diseases including ocular disorders. The eye is currently considered a good target for RNAi therapy mainly because it is a confined compartment and, therefore, enables local delivery of small-interfering RNAs (siRNAs) by topical instillation or direct injection. However, delivery strategies that protect the siRNAs from degradation and are suitable for long-term treatment would be help to improve the efficacy of RNAi-based therapies for ocular pathologies. siRNAs targeting critical molecules involved in the pathogenesis of glaucoma, retinitis pigmentosa and neovascular eye diseases (age-related macular degeneration, diabetic retinopathy and corneal neovascularization) have been tested in experimental animal models, and clinical trials have been conducted with some of them. This review provides an update on the progress of RNAi in ocular therapeutics, discussing the advantages and drawbacks of RNAi-based therapeutics compared to previous treatments. AbbreviationsAMD, age-related macular degeneration; Apaf-1, apoptotic protease-activating factor-1; CNV, choroidal neovascularization; CTGF, connective tissue growth factor; DR, diabetic retinopathy; dsRNA, double-stranded RNA; ECM, extracellular matrix; HIF-1α, hypoxia-inducible factor-1α; IOP, intraocular pressure; ONC, optic nerve crush; POAG, open-angle glaucoma; RGC, retinal ganglion cell; RISC, RNA-induced silencing complex; RNAi, RNA interference; RP, retinitis pigmentosa; Smad, signalling mathers against decapentaplegic; SPARC, secreted protein acidic and rich in cysteine; TLR3, toll-like receptor 3; TM, trabecular meshwork; TXNIP, thioredoxin interacting protein IntroductionRNA interference (RNAi) technology has been used to elucidate gene function, to generate model systems and to identify new molecular targets. In addition, RNAi is currently progressing from basic research to potential therapeutic applications. Because any gene that causes or contributes to a disease is susceptible to suppression by RNAi, RNAi therapy represents a promising biomedical strategy for treating a diverse range of diseases including cancer, cardiovascular diseases, neurodegenerative diseases, inflammatory conditions, viral infections and ocular diseases (Guo et al., 2010). In particular, the accessibility of the eye facilitates small-interfering RNA (siRNA) delivery, and naked siRNA has been efficiently applied by topical administration to the anterior segment or by intravitreal injection to the posterior segment (de Fougerolles, 2008). As the localized delivery of siRNA to the eye is less challenging than for other tissues, there has been significant progress made towards its use as a therapeutic procedure for eye diseases. In fact, several siRNA-based therapeutic agents for o...

In the ocular pharmacology market, there is a noteworthy unmet demand for more efficacious delivery of ocular therapeutics. Contact lenses are emerging as an alternative ophthalmic drug delivery system to resolve the drawbacks of the conventional topical application methods. Thus, contact lenses drug delivery systems have been developed to provide an increased residence time of the drug at the surface of the eye leading to enhanced bioavailability and more convenient and efficacious therapy. Several research groups have already explored the feasibility and potential of contact lenses loading conventional drugs used to treat anterior eye disorders. Drug incorporation to the lens body is achieved with techniques, like simple soaking, inclusion of drug-loaded colloidal nanoparticles, or molecular imprinting. Regardless of the technique used, key properties of the contact lens, such as transparency and oxygen permeability, should be preserved. In this article, we reviewed the different techniques used for drug delivery through contact lenses, analyzing their advantages and disadvantages, and focused on articles describing contact lens-based ophthalmic drug delivery systems with significant potential to use in ocular therapeutics.