Assumpta Peral

Involvement of melatonin MT3 receptors in the regulation of intraocular pressure in rabbits

Melatonin, a neurohormone secreted by the pineal gland, can stimulate three subtypes of receptors, namely: mt(1), MT(2) and MT(3). We examined the ability of melatonin and the selective MT(3) receptor agonist, 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT), to modify intraocular pressure in rabbits. Both compounds significantly reduced intraocular pressure, maximally by 24% and 43%, respectively, with IC(50) values of 363+/-23.0 and 423+/-30.0 ng/10 microl (1.6+/-0.1 and 1.8+/-0.1 nmol, respectively). The non-specific melatonin receptor antagonist, luzindole, abolished the depressant effect of both compounds, thus confirming the involvement of melatonin receptors. Our results show, for the first time, a functional response that may be regulated by melatonin MT(3) receptors, and provide evidence that supports a role of melatonin in the circadian changes of intraocular pressure.

Ocular hypotensive effects of melatonin receptor agonists in the rabbit: further evidence for an MT3 receptor

Melatonin is involved in the control of intraocular pressure during the night and day photoperiod. We have investigated the receptor that regulates intraocular pressure in New Zealand white rabbits by means of agonists and antagonists of melatonin receptors. Melatonin and its analogues: 2‐Phe‐melatonin, 6‐Cl‐melatonin, 2‐I‐melatonin, 5‐ methoxycarbonylamino‐N‐acetyltryptamine (5‐MCA‐NAT) and N‐acetyltryptamine all produced a reduction in intraocular pressure. Dose‐response analysis for these compounds gave pD2 values of 9.3±0.24 for melatonin; 9.0±0.09 for 6‐Cl‐melatonin; 9.0±0.84 for 2‐I‐melatonin; 8.9±0.07 for 5‐MCA‐NAT; 8.7±0.18 for 2‐Phe‐melatonin and 9.4±0.30 for N‐acetyltryptamine (all n=8). At a dose of 0.5 nmol (in 10 μl) melatonin and the selective melatonin MT3 agonist 5‐MCA‐NAT, induced greater reductions of intraocular pressure (22.8±2.3% and 32.5±1.4%, respectively) than the other compounds. The melatonin‐receptor antagonists, prazosin, DH‐97 and 4‐P‐PDOT, reversed the effect of 5‐MCA‐NAT in a dose‐dependent manner, with pA2 values of 13.5±0.17 for prazosin, 10.6±0.16 for DH‐97 and 9.4±0.20 for 4‐P‐PDOT (n=8). Cholinoceptor antagonists (hexamethonium and atropine) and α2‐ and β2‐adrenoceptor antagonists (yohimbine and ICI 118,551) partially reversed the effects produced by melatonin and 5‐MCA‐NAT, suggesting the possible involvement of cholinergic and noradrenergic systems in the hypotensive actions mediated by melatonin agonists. The α1‐adrenoceptor antagonist, corynanthine, had no significant effect. The strong hypotensive effect of the MT3 agonist, 5‐MCA‐NAT, suggests that this compound may be a useful agent for treating those pathologies where intraocular pressure is abnormally elevated.

UTP and diadenosine tetraphosphate accelerate wound healing in the rabbit cornea.

Nucleotides are naturally occurring substances present in tear film that can stimulate tear secretion in animals and humans. We investigated whether certain nucleotides can affect the rate of wound healing in the cornea of white rabbits. In the absence of any added compound, the rate of healing was 72.4 ± 2.2 μm h−1. Of all the tested nucleotides, UTP and Ap4A were the most active ones, maximally increasing the rate of healing to 121.6 ± 3.7 and 93.7 ± 3.2 μm h−1, respectively. Responses to UTP were dose dependent. UTP had a pD2 value of 8.9 ± 0.1 (EC50: 1.25 nM). P2 purinoceptor antagonists such as suramin and reactive blue‐2, inhibited the effect of UTP indicating the involvement of P2Y receptors. Mitogen‐activated protein kinase (MAPK) cascade inhibitors also abolished the effects of UTP, suggesting that P2Y receptors are coupled to the MAPK cascade, and that this is involved in controlling the rate of epithelial cell migration.

Nucleotides in ocular secretions: Their role in ocular physiology

The eye is the sense organ that permits the detection of light owing to the existence of a sophisticated neuronal array, called the retina, which is responsive to photons. The correct functioning of this complex system requires the coordination of several intraocular structures that ultimately permit the perfect focusing of images on the neural retina. Light has to pass through different media: the tear, the cornea, aqueous humour, lens, and vitreous humour before it reaches the retina. Moreover, the composition and structure of some of these media can change due to several physiological mechanisms. Nucleotides are active components of the humours bathing relevant ocular structures. The tear contains nucleotides and dinucleotides that control the process of tearing, wound healing and protects of superficial infections. In the inner eye, the aqueous humour also presents a collection of mono and dinucleotides that affect pupil contraction, aqueous humour production and accommodation. Behind the lens and between this structure and the retina the vitreous humour can modify the physiology of the retinal cells, mostly the ganglion cells. By investigating the actions of nucleotides and dinucleotide present in the ocular humours we will be able not only to understand the functioning of the ocular structures but also to develop new pharmacological therapies for pathologies such as dry eye, glaucoma or retinal detachment.

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.

Diadenosine polyphosphates in tears of Sjogren syndrome patients.

PURPOSE To analyze the levels of diadenosine tetraphosphate (Ap(4)A) and diadenosine pentaphosphate (Ap(5)A) in tears of subjects with Sjögren syndrome and to compare them with those in a control group. METHODS Twelve subjects with a diagnosis of Sjögren syndrome and 20 healthy control subjects were invited to participate in the present study. Schirmer strips were used to measure tear secretion (Schirmer I test) and to collect tears. Ap(4)A and Ap(5)A were measured by high-pressure liquid chromatography (HPLC), and a dry eye questionnaire (DEQ) was used to evaluate dry eye symptomatology. RESULTS The mean concentrations of Ap(4)A and Ap(5)A in the Sjögren syndrome group were 2.54 ± 1.02 and 26.13 ± 6.95 μM, respectively. This group of patients was divided in two subgroups: four patients with normal tear production and eight patients with low tear production. Concentrations of Ap(4)A, and Ap(5)A in patients with normal tear production (Schirmer test result, 12.3 ± 1.2 mm) were 0.47 ± 0.20 and 8.03 ± 3.27 μM, respectively. In the patients with low tear production (Schirmer test result, 1.0 ± 0.3 mm), the concentrations were 4.09 ± 1.36 and 39.51 ± 8.46 μM, respectively and in the control group, 0.13 ± 0.03 and 0.04 ± 0.02 μM, respectively. CONCLUSIONS Patients with Sjögren syndrome have abnormally elevated concentrations of diadenosine polyphosphates, indicating that these compounds could be used in the diagnosis of this disease.

Silencing of P2Y2 receptors reduces intraocular pressure in New Zealand rabbits

P2 receptors are involved in the regulation of ocular physiological processes like intraocular pressure (IOP). In the present study, the involvement of P2Y2 receptors in the hypertensive effect of nucleotides was investigated by use of antagonists and of a siRNA designed for the P2Y2 receptor.

Design of Novel Melatonin Analogs for the Reduction of Intraocular Pressure in Normotensive Rabbits

Melatonin, the MT2 melatonin receptor agonist IIK7 [N-butanoyl-2-(2-methoxy-6H-isoindolo[2,1-a]indol-11-yl)ethanamine], and the putative MT3 melatonin receptor agonist 5-MCA-NAT [5-methoxycarbonylamino-N-acetyltryptamine] have previously been shown to reduce intraocular pressure (IOP) in ocular normotensive rabbits. To gain a better understanding of the structure-activity relationship of compounds that activate MT2 and MT3 receptors mediating reductions in IOP, novel melatonin analogs with rationally varied substitutions were synthesized and tested for their effects on IOP in ocular normotensive rabbits (n = 160). All synthesized melatonin analogs reduced IOP. The best-effect lowering IOP was obtained with the analogs INS48848 [methyl-1-methylene-2,3,4,9-tetrahydro-1H-carbazol-6-ylcarbamate], INS48862 [methyl-2-bromo-3-(2-ethanamidoethyl)-1H-indol-5-ylcarbamate], and INS48852 [(E)-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-3-phenylprop-2-enamide]. These compounds produced dose-dependent decreases in IOP that were maximal at 0.1 mM (total dose of 0.259 μg for INS48848, 0.354 μg for INS48862, and 0.320 μg for INS48852) and 1 mM (total dose of 2.59 μg for INS48848, 3.54 μg for INS48862, and 3.20 μg for INS48852), with maximal reductions of 36.0 ± 4.0, 24.0 ± 1.5, and 30.0 ± 1.5% for INS48848, INS48862, and INS48852, respectively. Studies using melatonin receptor antagonists (luzindole, prazosin, and DH97 [N-pentanoyl-2-benzyltryptamine]) indicated that INS48862 and INS48852 activate preferentially a MT2 melatonin receptor and suggest that INS48848 may act mainly via a MT3 receptor. The most effective compounds were also well tolerated in a battery of standard ocular surface irritation studies. The implication of these findings to the design of novel drugs to treat ocular hypertension is discussed.

Ophthalmic formulations of the intraocular hypotensive melatonin agent 5-MCA-NAT

Melatonin is a hormone responsible for the regulation of circadian and seasonal rhythms. This hormone is synthesised in many tissues in the body including the eye, where it regulates important processes. During the recent years, the role of melatonin in the control of IOP has been investigated and it has been demonstrated that melatonin receptors are present and involved in the dynamics of the aqueous humour. 5-Methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) is a selective MT3 melatonin receptor agonist. Topical application of this product produces a clear reduction in intraocular pressure (IOP) in New Zealand white rabbits and in glaucomatous monkeys. In this work, the potent ocular hypotensive 5-MCA-NAT has been dissolved in excipients used in currently marketed drug formulations. Until now, this melatonin analogue had been dissolved in either DMSO or ethanol neither of which is suitable for ocular topical application in humans. Solubility assays in the different solvents were performed by the observation of the presence of drug crystals under optical microscopy. 5-MCA-NAT was completely dissolved in propylene glycol (PG) and polyethylene glycol 300 (PEG 300) within 24h. Ophthalmic formulations were prepared from different ratios of PG:PBS and the commercialized Systane product. Quantification of 5-MCA-NAT in the vehicles was assessed by HPLC. In vitro cytotoxicity of the formulations was evaluated by the MTT method and in vivo tolerance of 5-MCA-NAT in the solvents was analyzed by biomicroscopy and specular microscopy. Systane and proportions of PG:PBS up to 10% of PG did not show cytotoxicity in human corneal limbal epithelial cells (HCLE). In vivo experiments showed that the higher the ocular tolerance, the less amount of PG present. The ocular hypotensive effect of 5-MCA-NAT dissolved in the new formulations was checked measuring IOP for 8h after instillation of the substance. The best effect lowering IOP was obtained with 5-MCA-NAT dissolved in PG and diluted with PBS (PG 1.43%) in which 5-MCA-NAT produced a reduction of 28.11+/-2.0% and the effect lasted about 7h. In conclusion, new formulations accepted for ocular topical treatments different from DMSO or ethanol were capable of dissolving the melatonin analogue 5-MCA-NAT, preserving its ocular hypotensive ability. Therefore, the use of 5-MCA-NAT may be possible in the treatment of ocular hypertension and glaucoma.