Characterization of alpha(2)-adrenergic receptors and aquaporin-1 water channels in the human eye

Abstract

In most cells, water moves across the plasma membrane by simple diffusion. However, there are specialized epithelia, such as the kidney proximal tubules, that secrete or absorb water faster than simple diffusion predicts. While the identification of a proteinacious pore that transports water was elusive for many years, the recent discovery of aquaporin-1 (AQP-1) provides a molecular mechanism for the extraordinarily high permeability to water of epithelial cells in these tissues. Like the kidney, the human eye has several epithelial-lined structures that have a high permeability to water. One of these structures, the ciliary process, secretes aqueous humor (comprised mostly of water) into the eye and is regulated therapeutically by activating α₂-adrenergic receptors (α₂-AR) on its plasma membrane. The studies presented in this dissertation are structured to address four specific aims that were designed to test the hypothesis that the AQP-1 water channels are present in the human eye and are functionally regulated by α₂-ARs. Specific aim 1 characterized the distribution of AQP-1 in the human eye by immunofluorescence microscopy using affinity purified antibodies against purified AQP-1 protein. Using standard techniques in molecular biology, specific aim 2 generated antibodies to three fusion proteins; each containing a specific region of AQP-1. After screening several cell lines from the eye and the kidney with anti-AQP-1 and anti-α₂-AR IgY, specific aim 3 identified a cell line, human trabecular meshwork (HTM) cells, that contains both the AQP-1 water channels and α₂-ARs. Lastly, specific aim 4 investigated the functional relationship between the α₂-ARs and AQP-1 water channels. Using HTM cells as a model, the activation of α₂-ARs did not measurably affect AQP-1 messenger RNA or AQP-1 protein levels as compared to control. However, since the α₂-ARs primarily couple to cyclic AMP (cAMP) and several other aquaporins are regulated by cAMP, the effect of cAMP on AQP-1 was investigated. Using Xenopus oocytes expressing AQP-1 as a model, stimulation of oocytes with forskolin, a drug which increases intracellular cAMP, increased the permeability of AQP-1 to water. This observation provided evidence that is consistent with the general hypothesis that AQP-1 water channels and α₂-ARs are functionally coupled.