Stress-induced changes in neuronal Aquaporin-9 (AQP9) in a retinal ganglion cell-line

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Abstract

The water channel, Aquaporin-9 (AQP9) is enriched in selected neuronal populations and is unique its ability to act as a lactate-glycerol channel supplying neurons with alternative fuel under ischaemic conditions. AQP9 was detected in RGC-5 cells, a retinal ganglion cell-line, primary RGCs, and retina by Western blotting, real-time PCR (RT-PCR) and immunohistochemistry. RGC-5 cells subjected to a hypotonic stress increased their cell volume that was blocked by known inhibitor of AQP9 (phloretin (40 μM)). RGC-5 cells subjected to hypoxia, showed an up-regulation in AQP9 expression as judged by Western blotting and RT-PCR. Similarly, hypotonic shock (50%) increased AQP9 expression as determined by RT-PCR. AQP9 is involved in energy balance as a glycerol–lactate channel and also appears to regulate cell volume in retinal ganglion neurons. This water channel may play a key role in retinal ganglion pathology.

Introduction

Abnormalities in water balance play an important role in the pathophysiology of a variety of neurological disorders. The discovery of aquaporins (AQPs) has provided a molecular basis for understanding water transport in a number of tissues, including the ocular system [1]. The AQPs are a family of homologous water channel proteins (numbering at least 12 in mammals) that provide the major route for water movement across plasma membranes in a variety of cell types. AQPs are small hydrophobic membrane proteins (∼30 KDa monomer) that assemble in homotetramers and facilitate bi-directional water transport across the plasma membrane in response to osmotic gradients created by solute movement. AQPs 1, 2, 4, 5 and 8 function primarily as water selective transporters; AQPs 3, 7, 9 and 10 (referred to as aquaglyceroporins) also transport small solutes such as glycerol [1].

A number of AQPs are expressed in the eye: AQP0 (MIP) in lens fiber, AQP1 in cornea endothelium, ciliary and lens epithelia, trabecular meshwork and retinal photoreceptor cells, AQP3 in conjunctiva, AQP4 in ciliary epithelium and retinal Muller cells, and AQP5 in corneal and lacrimal gland epithelia [2]. Aquaporins have received attention after reports showing that mice lacking AQP1 have reduced IOP [3] and impaired corneal transparency after swelling [4]. Mice lacking AQP4 have reduced light-evoked potentials by electroretinography. Retinal function and cell survival were significantly improved in AQP4-deficient mice in both inbred (C57/bl6) and outbred (CD1) genetic backgrounds [5]. Therefore, it is suggested that AQP4 deletion in mice is neuroprotective in a transient ischaemia model of retinal injury. There is evidence for impaired cellular processing of AQP5 in lacrimal glands of humans with Sjogren's syndrome [6].

APQ9 was first isolated from adipose tissue [7]. AQP9 mRNA was also detected in leukocytes, testis, liver (highest expression), brain, epididymis duct cells and Leydig cells of rat testes [8] and goblet cells in small intestine [9]. In the rodent brain, AQP9 expression was observed in glial cells (tanycytes and astrocytes) [10], [11], [12], endothelial cells [11] and neurons [11], [13]. Neuronal AQP9 expression was found predominantly in the catecholaminergic neurons [11].

AQP9 possesses general features of a water channel, but in addition is permeable to lactate [14] and a wide variety of non-charged solutes such as: β-hydroxybutyrate, glycerol, carbamides, purines (adenine), pyrimidines (uracil and chemotherapeutic agent 5-fluorouracil), urea, mannitol, and sorbitol but impermeable to cyclic sugars (d-glucose, d-mannose and myo-inositol), the nucleoside uridine, glutamine and glycine [14]. AQP9 shows high homology to AQP3 and AQP7, but less homology with AQP1 [15]. AQP9 has a cysteine at position 213, three residues prior to the second NPA sequence that may explain inhibition by mercury similar to AQP1 [15]. AQP9 protein has protein kinase C and casein kinase II phosphorylation sites [15]. AQP9 mRNA and protein were down-regulated by stimulation of the PKC pathway [16]. By contrast, activation of cAMP-dependent protein kinase (PKA) by dibutyryl cAMP induces an increase in AQP9 mRNA and protein expression in astrocytic cultures [17]. P38 MAP-kinase has been also shown to be involved in increasing AQP4 and AQP9 expression after an osmotic stress [18].

The rodent AQP9 gene contains a negative insulin response element [19] which explains its down regulation by increased insulin levels while up-regulation in diabetic rats [19], [20]. In the liver, AQP9 functions as a glycerol channel allowing glycerol entry then participation in the neoglucogenesis during fasting periods [20]. Glycerol-mediated hepatic glucogenesis accounts for 90% of glucose production in the prolonged fasting state in rodents [21]. In human, it is estimated that ∼20% of the glucogenesis is mediated by glycerol after 60 h fasting [22]. Neuronal AQP9 also plays an important role in osmoregulation as well as energy balance by functioning as a glycerol–lactate channel. Both glycerol and lactate can serve as fuel for neurons and enhanced the recovery of neurons after ischaemic insults [23], [24], [25], [26], [27], [28].

A number of studies have implicated changes in AQP9 in the pathology of neurological diseases. Hypoxia, ischaemia and hyper-osmotic stress are associated with changes in the densities of AQP9 expression. Interestingly, such insults are key risk factors in the development of glaucomatous optic nerve neuropathy. The presence of neuronal AQP9 in the retinal ganglion cells was therefore investigated. AQP9 is present in RGC-5 cells, primary cultures of RGC cells as well as the retinal ganglion cell layer in rats. It is suggested that AQP9 acting as a water channel or a channel for glycerol and lactate (or a neuronal energy sensor) plays a key role following insults or stress that leads to optic nerve degeneration as seen in glaucoma or other diseases associated with optic nerve neuropathy.

Section snippets

Tissue culture

RGC-5 cells, a rat retinal ganglion cell-line, were maintained in DMEM low glucose in T-150 culture flasks (Gibco, Grand Island, NY) supplemented with 44 mM NaHCO3, 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) and antibiotics (Gibco) as described [29]. Cell passages 7–20 were used in this study. Primary RGC cultures were isolated by papain digestion and cell populations were plated on collagen-coated coverslips in neurobasal media (Gibco 10888-022) containing BDNF (50 ng ml−1), CNTF (10

Results

The simultaneous recording of cell volume changes in single cells technique is based on measurements of changes in the concentration of intracellularly fluorescent-trapped dyes such as fura 2 at its isosbestic point. Since dye concentration is inversely proportional to cell volume, measurement of changes in fluorescence intensity at the isosbestic point should report the dynamic changes in cell volume [33]. Cells bathed in isotonic solution then switched a hypotonic solution (20%) exhibited

Conclusion

The current study is first to report the existence of neuronal AQP9 in a retinal ganglion cell-line RGC-5 cells, primary RGC cultures, as well as the retinal ganglion cell layer in rats. AQP9 importance in retinal ganglion cells is suggested by at least two reactive mechanisms. The first is by acting as a lactate-glycerol channel allowing the uptake of excess lactate produced by astrocytes following stress (e.g., ischaemia) and thus providing an alternative fuel for neurons. Second, AQP9 plays

Acknowledgments

This research was supported in part by a grant from the glaucoma foundation to A.D. and an NEI 11179 grant to T.Y.

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