A. Mediero

Immunolocalisation of P2Y receptors in the rat eye

Nucleotides present an important role in ocular physiology which has been demonstrated by recent works that indicate their involvement in many ocular processes. P2Y are important among P2 receptors since they can control tear production, corneal wound healing, aqueous humour dynamics and retinal physiology. Commercial antibodies have allowed us to investigate the distribution of P2Y receptors in the cornea, anterior and posterior chamber of the eye and retina. The P2Y1 receptor was present mainly in cornea, ciliary processes, and trabecular meshwork. The P2Y2 receptors were present in cornea, ciliary processes and retinal pigmented epithelium. P2Y4 was present in cornea, ciliary processes, photoreceptors, outer plexiform layer and ganglion cell layer. The P2Y6 presented almost an identical distribution as the P2Y4 receptor. The P2Y11 was also detectable in the retinal pigmented epithelium. The detailed distribution of the receptors clearly supports the recent findings indicating the relevant role of nucleotides in the ocular function.

Corneal re-epithelialization stimulated by diadenosine polyphosphates recruits RhoA/ROCK and ERK1/2 pathways.

PURPOSE To investigate the role of ERK1/2 and RhoA/ROCK intracellular pathways in the modification of corneal re-epithelialization when stimulated by the diadenosine polyphosphates Ap(4)A and Ap(3)A. METHODS In wounded confluent SIRC (Statens Seruminstitut rabbit cornea) cell monolayers and in the presence or absence of Ap(4)A or Ap(3)A 100 microM, a battery of P2 receptor antagonists and inhibitors of tyrosin kinases, MAPK, and cytoskeleton pathways (AG1478 100 microM, U0126 100 microM, Y27632 100 nM, and (-)-blebbistatin 10 microM; n = 8 each) were assayed. Also, the activation of ERK1/2 and ROCK-I was examined by Western blot assay after treatment with Ap(4)A and Ap(3)A (100 microM), with or without suramin, RB-2, U0126, and Y27632. The intracellular distribution of pERK and ROCK-I was examined in the presence of Ap(4)A or Ap(3)A (100 microM) with U0126 and Y27632 (100 nM). RESULTS In the presence of Ap(4)A, U0126, Y27632, AG1478, and (-)-blebbistatin, reduced the migration rate compared to the effect of Ap(4)A alone (P < 0.0001, P < 0.001, P < 0.01, and P < 0.1 versus Ap(4)A, respectively). In the presence of Ap(3)A 100 microM, U0126 and Y27632 accelerated the migration rate when compared with the effect of Ap(3)A alone, whereas AG1478 and (-)-blebbistatin (P < 0.0001 versus Ap(3)A) slowed the migration rate. Western blot assays demonstrated that both dinucleotides activated the ERK1/2 pathway but only Ap(4)A activated the ROCK-I pathway. The intracellular distribution of pERK1/2 and ROCK-I reflected cross-talk between these two pathways. CONCLUSIONS The activation of the Ap(4)A/P2Y(2) receptor, accelerates corneal epithelial cell migration during wound healing with the activation of MAPK and cytoskeleton pathways, whereas activation of the Ap(3)A/P2Y(6) receptor signals only the MAPK pathway.

Sympathetic nervous system modulates the ocular hypotensive action of MT2‐melatonin receptors in normotensive rabbits

Abstract:  The aim of this study was to investigate the hypotensive effect of the melatonin analogue, N‐butanoyl‐2‐(2‐methoxy‐6H‐isoindolo[2,1‐a]indol‐11‐yl)ethanamine (IIK7), through MT2‐melatonin receptors and the involvement of the sympathetic nervous system in this action in New Zealand rabbit eyes. The topical application of melatonin or IIK7 produced a reduction in intraocular pressure of 20.2 ± 5.3% and 38.5 ± 3.2% respectively. This effect was concentration‐dependent; it was blocked by selective MT2 receptor antagonists and was severely diminished after chemical sympathectomy. Immunohistochemistry and western blot analysis showed the ciliary processes as the site of this action and no co‐localization of MT2‐melatonin receptor with the sympathetic nervous system was observed. The β‐adrenergic agonists, terbutaline and salbutamol, potentiated the hypotensive effect of IIK7 reducing intraocular pressure (IOP) 41.75 ± 4.26% and 44.7 ± 5.6% respectively. Also, IIK7 in presence of the nonspecific protein phosphatase inhibitor okadaic acid, lowered IOP 32.2 ± 4.5% and in presence of forskolin plus 3‐isobutyl‐1‐methylxanthine decreased IOP in 32.2 ± 5.47%. These data suggest that the melatonin agonist IIK7 reduces intraocular pressure by acting through MT2‐melatonin receptors presumably decreasing aqueous humour formation. Also, in the presence of β‐adrenoceptor agonists MT2‐melatonin receptors activity increase their ability to reduce IOP.

New treatments for ocular hypertension

Glaucoma is a neurodegenerative pathology that affects the optic nerve producing blindness. This disease is often a consequence of an abnormal increase of intraocular pressure (IOP) due to a reduction in the ability of the eye to drain a transparent fluid termed aqueous humour. The dynamics of the aqueous humour is highly controlled by the autonomic nervous system, mainly the sympathetic, regulating its production and parasympathetic controlling the evacuation of aqueous humour. This has led pharmaceutical companies to develop chemicals which, by acting via different targets can substantially reduce IOP. Parasympathomimetics, adrenergic antagonists, plus eventually adrenergic agonists, are commonly used for the reduction of IOP and therefore for treatment of glaucoma. New substances linked to the nervous system that innervates the eye are emerging as interesting candidates. Nucleotides, commonly costored with catecholamines or acetylcholine or the indole melatonin, present interesting properties reducing IOP. Moreover new technological ideas such as the use of siRNA (small interference RNA) to silence protein expression demonstrate the relevance of this method to approach ocular hypertension and glaucoma from a different point of view. These three main groups of molecules: nucleotides, melatonins and siRNAs, are reviewed since they appear as firm candidates for the treatment of glaucoma in the near future.

Phospholipase C/Protein Kinase C Pathway is Essential for Corneal Re-epithelialization Induced by Ap4A

Purpose: We have previously demonstrated the importance of P2Y2 receptors in the corneal re-epithelialization effect triggered by diadenosine tetraphosphate (Ap4A). In addition, we have also shown the ERK1/2 and ROCK-I activation in Ap4A-wound repair response. Phospholipase C/Protein Kinase C (PLC/PKC) pathway activation has been suggested as a molecular mechanism of growth factors-modulated corneal cell migration and P2Y2 agonists. Hence, the aim of this study is to investigate the role of PLC/PKC cascade in the modification of re-epithelialization rate triggered by Ap4A in an established corneal epithelial cell line (Statens Seruminstitut rabbit cornea [SIRC] cells). Methods: In wounded confluent SIRC cell monolayers and in the presence or absence of Ap4A 100 µM, a group of PLC/PKC inhibitors (U73122 3 µM, Staurosporine 1 nM and Bisindolylmaleimide-I 10 µM) and activator (PDBU 1 µM) were assayed and the migration rate was evaluated. Also, the activation of ERK1/2 and ROCK-I was examined by Western blot assay after treatment with or without Ap4A, U73122, Staurosporine, Bisindolylmaleimide-I and PDBU. Results: Pre-treatment of wounded SIRC cells with PLC/PKC inhibitors significantly diminished the Ap4A-stimulated cell migration rate. Furthermore, PLC/PKC inhibitors also reduced ERK1/2 phosphorylation and ROCK-I activation triggered by Ap4A. Conclusions: The present study shows the involvement of PLC/PKC pathway in the activation of ERK1/2 and ROCK-I downstream signal transduction pathways stimulated by Ap4A/P2Y2 receptor during corneal epithelial wound repair.

Effect of Melatonin and Analogues on Corneal Wound Healing: Involvement of Mt2 Melatonin Receptor

Abstract Purpose: We have investigated the effect of melatonin and its analogues on rabbit corneal epithelial wound healing. Methods: New Zealand rabbits were anaesthetised and wounds were made by placing Whatman paper discs soaked in n-heptanol on the cornea. Melatonin and analogues (all 10 nmol) were instilled. Wound diameter was measured every 2 hours by means of fluorescein application with a Topcon SL-8Z slit lamp. Melatonin antagonists (all 10 nmol) were applied 2 hours before the application of the n-heptanol-soaked disc and then every 6 hours together with melatonin. To confirm the presence of MT2 receptors in corneal epithelial cells immunohistochemistry, Western blot and RT-PCR assays in native tissue and in rabbit corneal epithelial cells were performed. The tear components were extracted then processed by HPLC to quantify melatonin in tears. Results: Migration assays revealed that melatonin and particularly the treatment with the MT2 agonist IIK7, accelerated the rate of healing (p < 0.001). The application of the non-selective melatonin receptor antagonist luzindole and the MT2 antagonist DH97 (but not prazosin), prevented the effect of melatonin on wound healing (both p < 0.001). Immunohistochemistry, Western blot and RT-PCR assays showed the presence of MT2 melatonin receptor in corneal epithelial cells. In addition, we have identified melatonin in tears and determined its daily variations. Conclusions: These data suggest that MT2 receptors are implicated in the effect of melatonin on corneal wound healing regulating migration rate. This suggests the potential use of melatonin and its analogues to enhance epithelial wound healing in ocular surface disease.