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Asuncion Peral, Almudena Crooke, Fernando Huete-Toral, Alejandro Martinez-Aguilar, Pilar Alarma-Estrany, Jesus Pintor; 5-MCA-NAT Reduces Carbonic Anydrases Expression By Means Of MT3 Melatonin Receptors. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1517.
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In previous works we have demonstrated that the melatonin analogue, 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT), reduces intraocular pressure (IOP). This hypotensive effect keeps IOP lower than control for a few days. The aim of this study is to investigate if the long-term hypotensive effect of 5-MCA-NAT is due to the regulation of carbonic anhydrases expression.
IOP was measured by means of a Tonovet® contact tonometer. 100 µM 5-MCA-NAT (10 µl) and/or Trusopt® (40 µl) were instilled in New Zealand rabbit eyes. Another group of control animals received the same volume of saline (vehicle). IOP was measured twice before the application of the 5-MCA-NAT/Trusopt and after that once every hour for at least 6 hours. IOP was measured during the following days in the absence of any added compound. Cryostat sections of rabbit ciliary body were obtained for immunohistochemical experiments. Sections were incubated with AC12 antibody and subsequently incubated with a secondary IgG mouse labelled with TRITC and observed under confocal microscopy. Relative changes in CA2 and CA12 expression of ciliary non-pigmented epithelial cells (NPEsv) treated with 5-MCA-NAT, were determined by Real-time quantitative PCR (RT-qPCR) assays. Validation of the internal control gene and RT-qPCR data analysis were performed by 2-ΔCt method.
When 5-MCA-NAT was topically applied the maximal reduction of IOP was 40.8 ±5.0% (n=6). This hypotensive effect lasted up to 5 days until IOP returned to control (initial) values. Application of the carbonic anhydrase inhibitor Trusopt after 5-MCA-NAT treatment demonstrated a strong reduction in the hypotensive effect of this carbonic anhydrase inhibitor, suggesting a drop in the activity of these enzymes.Immunohystochemical studies showed that 5-MCA-NAT-treated animals had a marked reduction in CA12 expression along the ciliary processes, in comparison with control rabbits. On the other hand, a prominent decrease of CA2 and CA12 mRNA levels was detected in NPEsv cells treated with 5-MCA-NAT, by RT-qPCR assays. This reduction lasted up to 4 days in the case of CA12 and 24 h in the case of CA2.
The long-term hypotensive effect of 5-MCA-NAT is due, at least in part, to a down-regulation of Carbonic Anhydrases expression.
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