Involvement of carbonic anhydrases in the ocular hypotensive effect of melatonin analogue 5-MCA-NAT Introduction In the search for new drugs suitable for reducing intraocular pressure (IOP), which has been recognized as the most important risk factor for the glaucoma, melatonin and its analogues arise as promising substances for the treatment of this pathology [1–3]. Melatonin has been reported as a regulator of circadian cycles including the day/night regu- lation of IOP [4]. Among melatonin and its analogues, the most potent and with greater efficacy reducing IOP is 5-methoxycarbonylamino-N-acetyltryptamine abbreviated 5-MCA-NAT. Pharmacological and in vivo RNAi assays have confirmed that the ocular hypotensive effect of 5-MCA- NAT is mediated by an unknown melatonin receptor present in the ciliary processes in clear contrast to the opinion of some authors who claim that 5-MCA-NAT acts through quinone reductase 2 [5]. However, the underlying molecular mechanisms of this receptor are still unknown. Melatonin has the ability to modify several physiological processes including the regulation of the expression of several genes [6, 7] and also, the activity of different enzymes such as the carbonic anhydrase (CA) [8, 9]. We have reported recently a genomic action of 5-MCA-NAT [10]. Among other possible target genes of 5-MCA-NAT, CA enzymes are interesting because these enzymes are direct regulators of aqueous humour formation [11]. Hence, administration of CA inhibitors is a common treatment for ocular hypertension and glaucoma because they produce a potent reduction in IOP by suppressing aqueous humour secretion [12]. In particular, it has been suggested by many authors that CAII and CAIV are the main enzymes that function in aqueous humour production, but, interestingly, the absence of CAIV enzyme in the human ciliary epithe- lium has been reported by other authors [13, 14]. Con- versely, an over-expression of CA12 mRNA has been described in patients with glaucoma [13]. As 5-MCA-NAT exhibits a long-term action on IOP that could be due to the regulation of gene expression and because CA are important regulators of IOP, the present experimental work investigated the effect of 5-MCA-NAT on CA2 (CAII) and CA12 (CAXII) expression of rabbit ciliary epithelium. Methods Animals and cultured cells A total of 48 New Zealand white rabbits, weighing 3–4 kg, were used for all IOP studies. The animals were kept in individual cages with free access to food and water. They Abstract: We have previously demonstrated that melatonin and its analogue, 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT), reduce intraocular pressure (IOP) in New Zealand rabbits. More recently, we have shown that 5-MCA-NAT can also regulate ciliary adrenoceptor gene expression. Like adrenoceptors, carbonic anhydrase (CA) enzymes are involved in aqueous humour secretion by the ocular ciliary epithelium. Moreover, CA enzymes have been reported to be regulated by melatonin. Hence, the aim of this study was to investigate whether the hypotensive effect of 5-MCA-NAT is also because of a regulation of CA genes and enzymes. Time course of 5-MCA-NAT effect on rabbit IOP was followed for 7 hr every day for up to 144 hr (6 days). 5-MCA-NAT reduced IOP, maximally by 51.30 ± 2.41% (at 3 hr), and the hypotensive effect was maintained for up to 96 hr with a single application. IOP studies with 5-MCA-NAT plus Trusopt Ò and immunohistochemical analysis confirmed that CA are molecular targets of 5-MCA-NAT. In addition, real-time quantitative PCR (qPCR) and immunocytochemical assays were performed to determine changes in CA2 (CAII) and CA12 (CAXII) expression in cultured rabbit nonpigmented ciliary epithelial cells (NPE) treated with 5-MCA-NAT. NPE cells showed a prominent decrease in both CA, at the mRNA and protein levels. These data confirm that the long-term hypotensive effect of 5-MCA- NAT is also due, to a down-regulation of CA2 (CAII) and CA12 (CAXII) expression. Almudena Crooke*, Fernando Huete-Toral*, Alejandro Martı ´nez- A ´ guila, Alba Martı ´n-Gil and Jesu ´s Pintor Departamento de Bioquı ´mica, E.U. O ´ ptica, Universidad Complutense de Madrid, Madrid, Spain Key words: 5-methoxycarbonylamino-N- acetyltryptamine, carbonic anhydrases, glaucoma, intraocular pressure, melatonin Address reprint requests to Jesu ´ s Pintor, Departamento de Bioquı ´mica y Biologı ´a Molecular IV, E.U. O ´ ptica, Universidad Complutense de Madrid, C/Arcos de Jalo ´n 118, 28037 Madrid, Spain. E-mail: jpintor@vet.ucm.es *Both authors have contributed equally to the present work. Received September 22, 2011; Accepted October 17, 2011. J. Pineal Res. 2011 Doi:10.1111/j.1600-079X.2011.00938.x Ó 2011 John Wiley & Sons A/S Journal of Pineal Research 1 Molecular, Biological, Physiological and Clinical Aspects of Melatonin