Pilar Alarma-Estrany

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.

5‐MCA‐NAT does not act through NQO2 to reduce intraocular pressure in New‐Zealand white rabbit

Abstract:  Solid data support the idea that the MT3 melatonin binding site is an enzyme, quinone reductase 2 (NQO2), rather than a membrane melatonin receptor. However, the melatonin analogue, 5‐methoxycarbonylamino‐N‐acetyltryptamine (5‐MCA‐NAT), reduces intraocular pressure (IOP) via MT3 melatonin receptors. Therefore, the aim of this work was to test whether the melatonin binding site, MT3, is indeed the enzyme NQO2 in New Zealand rabbit eyes. To investigate this, the action of several substrates and inhibitors for NQO2 was compared to 5‐MCA‐NAT in their ability to modify IOP. Also, the effect of 5‐MCA‐NAT on IOP produced after NQO2 silencing by means of a siRNA was determinated. Altogether, the results led us to conclude that the in vivo effect of the MT3 ligand 5‐MCA‐NAT on IOP is not mediated by the enzyme NQO2, suggesting the existence of another melatonin 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.

Requirement of intact sympathetic transmission for the ocular hypotensive effects of melatonin and 5-MCA-NAT

Melatonin and its analogue, 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT), potently reduce intraocular pressure, and may be good candidates for the treatment of ocular hypertension and glaucoma. After chemical sympathectomy by reserpine or 6-hydroxydopamine, the hypotensive effects of melatonin and 5-MCA-NAT are severely inhibited. This indicates that the sympathetic nervous system is involved in the production and drainage of aqueous humour by the ciliary body and trabecular meshwork, and that it mediates the effects of melatonin and its analogue, 5-MCA-NAT.

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.

Electrogenic Ion Transport in Mammalian Colon Involves an Ammonia-Sensitive Apical Membrane K+ Conductance

It is remarkable that high ammonia concentrations can be present within the colonic lumen without compromising normal epithelial function. We investigated the impact of luminal ammonia on Cl− secretion in native tissue. Stripped human colonic mucosa and unstripped rat distal colon were used. Paired samples were mounted in modified Ussing chambers for electrophysiological studies. In rat distal colon, apical ammonia dose-dependently blocked forskolin-activated short-circuit current with an IC50 ≈ 5 mM. Basolateral NH4Cl was less effective. Luminal methylamine (50 mM), chromanol 293 B (10–50 μM), and Ba2+ (5 mM) blocked cAMP-activated short-circuit current but apical clotrimazole (100 μM) was without effect. In stripped human colonic mucosa, luminal but not basolateral NH4Cl (10 mM) and luminal Ba2+ (5 mM) suppressed forskolin-activated short-circuit current. Ammonia may be an endogenous regulator of colonic water and salt secretion. Apical K+ channels may be involved in the regulation of cAMP-stimulated Cl− secretion in mammalian colon.

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.

Regulation of ocular adrenoceptor genes expression by 5-MCA-NAT: implications for glaucoma treatment.

We have demonstrated that 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT), reduces intraocular pressure (IOP) in rabbits. In addition, we have reported a link between hypotensive effect of 5-MCA-NAT and sympathetic nervous system. Moreover, it is known that aqueous humour production is controlled by the activation of adrenoceptors (ADRs) present in the ocular ciliary epithelium. Thus, the aim of this study is to investigate if the hypotensive effect of 5-MCA-NAT is due to a regulation of ciliary ADR genes expression. To confirm this we followed the effect of 5-MCA-NAT on rabbit IOP for 144 consecutive hours. A sustained IOP reduction for up to 72 h (P<0.01) was seen. In addition, changes in ADRB2 and ADRA2A mRNA were measured in cultured rabbit nonpigmented ciliary epithelial cells. After 5-MCA-NAT treatment, a significant downregulation of ADRB2 and upregulation of ADRA2A was observed. These results provide the regulation of ADRs mRNA by 5-MCA-NAT.

Progesterone inhibits chloride transport in human intestinal epithelial cells.

Several pieces of evidence suggest that female sex hormones may play a role in the regulation of electrolyte transport. We therefore hypothesized that female sex hormones might impair regulated transcellular chloride transport in human intestinal epithelial cells. The T84 cell line was used for electrophysiological studies. Changes in transepithelial resistance and short-circuit current (Isc) were measured via a dual voltage/current clamp in epithelial monolayers. Short-circuit current is equivalent to chloride secretion in T84 cells. Forskolin and 8-Br-cyclic adenosine monophosphate (cAMP) were used to activate cAMP-dependent Cl− transport. Ca2+-dependent secretion was stimulated by the receptor-mediated Ca2+ agonist carbachol. Acute exposure (30 minutes) to either progesterone or estradiol did not affect monolayer viability as reflected by transepithelial resistance. Moreover, the secretory response to both cAMP and Ca2+ agonists remained unaffected. In contrast, long-term exposure (24 hours) to physiological concentrations of progesterone (100 nM), but not estradiol, dose-dependently reduced the peak Isc induced by the cAMP-agonist forskolin from 125±2.7 µA · cm−2 in the control group to 96±2.5 µA · cm−2 in monolayers exposed to progesterone (n=6 for each group; p<0.001). When the cAMP-analogue 8-Br-cAMP was used, the same behavior was observed (peak Isc=112±1.6 µA · cm−2 vs 88±1.7 µA · cm−2 for control vs. progesterone-treated monolayers; n=6 for each group; p<0.001). Taken together, our results suggest that progesterone but not estradiol inhibits cAMP-stimulated Cl− secretion in intestinal epithelial cells at a site distal to cyclic nucleotide generation.RésuméIl existe plusieurs preuves suggérant que les hormones sexuelles féminines jouent un rôle dans la régulation du transport des electrolytes. Nous avons émis l’hypothèse que les hormones sexuelles féminines pourraient entraver le transport transcellulaire de chlorure dans les cellules épithéliales intestinales d’origine humaine. La lignée cellulaire T84 a été utilisée pour les études éiectrophysioôgiques. Les changements dans la résistance transepitheliale et le courrant court-circuité ont été mesurés par un câmp à voltage/courant dans les monocouches épithéliales. Le courant court-circuité est équivalent à la sécrétion de chlorure par les cellules T84. Le forskoline et le 8-Br-cAMP ont été utilisés pour activer le transport de chlorure cAMP-dépendant. La sécrétion Ca2+-dépendante a été stimulée par l’agoniste carbachole, médiateur des récepteurs Ca2+. L’exposition aiguë (30 minutes) à la progestérone ou à l’œstradiol n’influencent pas la viabilité monocouches fait attesté par la résistance transépithéliale. Cependant, la réponse sécrétoire aux agonistes cAMP et Ca2+ n’est pas affectée. En revanche, l’exposition longue (24 h) aux concentrations physiologiques de progestérone (100 nM), mais pas d’oestradiol, réduisait Fisc maximal induit par l’agoniste cAMP, le forskoline, de 125±2.7 µA · cm−2 des contrôles à 96±2.5 µA · cm−2 dans les monocouches exposées à la progestérone (n=6 pour chaque groupe; p<0.001). Le même comportement a été observé lorsqu’on a utilisé l’analogue cAMP, le 8-Br-cAMP (Isc max=112±1.6 µA · cm−2 vs. 88±1.7 µA · cm−2 pour les contrôles vs. monocouches traitées par la progestérone; n=6 pour chaque groupe; p<0.001). Dans l’ensemble, ces résultats suggèrent que la progestérone, mais pas l’œstradiol, inhibe la sécrétion de Cl− stimulée par le cAMP dans les cellules épithéliales intestinales à un site en amont de la génération des nucleotides cycliques.ResumenDiversos trabajos han sugerido que las hormonas femeninas podrían desempeñar algún papel en la regulación del transporte de electrolitos. Nuestra hipótesis es que las hormonas femeninas podrían alterar la regulación del transporte transcelular del cloro en las células epiteliales del intestino humano. Para nuestro estudio electrofisiológico utilizamos la cepa de células T84. En monocapas epiteliales registramos, mediante un clamp dual voltage/corriente, los cambios de la resistencia transepitelial y los potenciales eléctricos de membrana. Éste equivale a la secreción de cloro en las células T84. Para activar el transporte del Cl− dependiente del AMP cíclico utilizamos forskolin y 8-Br-cAMP. La secreción Ca2+ dependiente se estimuló mediante el carbachol, un agonista de los receptores mediadores del Ca2+. La exposición breve (30 minutos) a la progesterona y al estradiol no afectó la viabilidad de la monocapa, como quedó demostrado por la resistencia transepitelial; tampoco se modificó la respuesta secretora tanto al cAMP como a los agonistas del Ca2+. Por el contrario, la exposición larga (24 horas) a concentraciones fisiológicas de progesterona (100nM) pero no del estradiol reduce, dependiendo de la dosis, la concentración máxima del Isc inducida por el forskolin cAMP agonista, desde 125±2.7 µ A · cm−2 en el grupo control hasta 96±2.5 µ A cm−2 en las monocapas expuestas a la progesterona (n=6 en cada grupo; p<0.001). Cuando se empleó el cAMP-análogo 8-Br-cAMP se constató una conducta similar (concentración máxima Isc=112±1.6 µ A · cm−2 vs 88±1.7 µA · cm−2 en el grupo control vs monocapa tratada con progesterona (n=6 en cada grupo; p<0.001). Estos hallazgos sugieren que la progesterona (no el estradiol) inhibe la secreción del Cl− estimulado por el cAMP en las células epiteliales intestinales en un punto distal a la generación del nucleotido cíclico.