Expression of aquaporins in the rat eye

Purpose. The aquaporins (AQPs) are a family of intrinsic membrane proteins that function as water-selective channels in many water permeable tissues. Since the first aquaporin (AQP1) was cloned in 1992 five different mammalian isoforms were identified by homology cloning. AQP1, AQP3, AQP4 and AQP5 have been localized in the eye using in situ hybridization and northern analysis. Since disturbances in the water movement characterize several ocular diseases such as glaucoma, we sought to determine the semiquantitative expression of AQPs in rat eye tissues. Methods. The sense and antisense primers were designed corresponding to the nucleotide sequence of AQPs. RT-PCR was performed in the presence of [α-32P] dCTP. After PCR amplification, reaction mixture was analyzed on a agarose gel and the DNA band was cut and the radioactivity incorporated into DNA was measured. PCR product of G3PDH and β-actin were used as an internal controls. Serial dilution of reverse transcribed cDNAs of internal controls were subjected to amplifications with specific primers to generate calibration curves. Relative concentration of β-actin and G3PDH cDNAs in various ocular tissues were determined using the calibration curve. The cDNA concentrations were normalized to yield equivalent amounts of β-actin and G3PDH PCR products in each tissue before amplifying with AQP specific primers. The relative concentration of the AQP cDNAs in various ocular tissues were expressed as percentage of that in cornea. Results. A single DNA band of expected size corresponding to AQP1 was amplified from cornea, lens, iris, ciliary body, and retina. Similarly, AQP3 was amplified only from cornea; AQP4 was amplified from lens, iris, ciliary body, and retina; and AQP5 was amplified from cornea, and lens. Further, the restriction map for these fragments were identical to the known restriction map of the AQPs. Conclusion. Expression level of AQP1 is highest in lens followed by that in cornea, retina, iris and ciliary body. AQP2 is not detected in the eye. AQP3 is expressed only in cornea. Expression level of AQP4 is highest in retina followed by that in ciliary body, iris and lens. AQP5 is abundant in cornea but only weakly detectable in lens.