Mineralocorticoid Hormone Signaling Regulates the ‘Epithelial Sodium Channel’ in Fibroblasts from Human Cornea

Abstract

We investigated the regulation of sodium absorption by steroid hormones in embryologically diverse cells from the human eye. A cell extract from human corneal fibroblasts was positive for both the epithelial sodium channel (ENaC) and the mineralocorticoid receptor (MCR) as 82- to 85-kD and 102-kD bands, respectively, by the Western blot technique. In fluorescent, confocal and electron microscopy, the MCR was revealed as a nucleocytoplasmic protein, whereas the ENaC was almost exclusively membrane bound; both appeared aligned along actin filaments of corneal keratocytes, and both were widely colocalized in various cell types of human cornea in situ. Following reverse transcription and amplification of total RNA isolated from corneal fibroblasts, the ENaC and MCR genes in the PCR product were evident as predicted bands of 520 and 843 bp, respectively, whose sequence exhibited 100% identity with those from known human sources. The multiplication of corneal fibroblasts was influenced by both the MCR-specific antagonist RU 26752 and the natural hormone aldosterone, and these steroids also stimulated protein phosphorylation. In quantitative PCR, both the basal and aldosterone-induced levels of ENaC were diminished by the MCR-specific antagonist ZK 91587. Consequently, the ocular sodium channel appears to be regulated by steroid signalling in cells of diverse embryological origins, contrary to the existing notions where (a) this process would be limited exclusively to the epithelial cells and (b) ocular sodium transport would be regulated via the Na⁺-K⁺-ATPase in the basolateral membrane.

Four Subunits (αβγδ) of the Epithelial Sodium Channel (ENaC) Are Expressed in the Human Eye in Various Locations

“…55 This conclusion is further supported by molecular evidence of ENaC expression in corneal epithelial cells. [32][33][34]36,56 ENaC expression has also been reported in rabbit and human conjunctival tissue. 30 Interestingly, in the latter study, topical application of amiloride was shown to increase the quantity of The present study shows ␣-, ␤-, and ␥-, but not ␦-, ENaC mRNA expression in the human cornea.…”

“…12,29,33,63,67 ENaC has been detected in Müller glial cells 12,28,37 and RPE, 29 in which the mineralocorticoid receptor may colocalize with ENaC. Moreover, aldosterone has been shown to upregulate ENaC expression in Müller glial cells 12 and in the RPE.…”

“…12,[23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] Ocular ENaC expression does not seem to be limited to epithelial cells but has been observed in fibroblasts, 33 retinal neurons, glial cells, 12,24 and photoreceptors. 28,29 In the human eye, ENaC expression has been detected in the cornea, 30,33,36 ciliary body, 26,34,35 and retina. 34 Little is known about the (patho-)physiological role of ENaC in the eye, but ENaC may contribute to aqueous humor formation, 26 corneal endothelial sodium and fluid transport, 31 and the pathophysiology of certain forms of glaucoma.…”

On the mechanism of fluid transport across corneal endothelium and epithelia in general

“…However, we found (Kuang et al,'93) that the most efficient inhibitor of this entry is phenamil, a compound that is specific for an epithelial-type Na þ channel (ENaC). The presence in rat (Mirshahi et al,'99) and human (Mirshahi et al, 2001) corneal endothelium has been established by immunocytochemistry. Preliminary immunocytochemical work in our laboratory is consistent with an apical location for EnaC.…”

Could Mineralocorticoids Play a Role in the Pathophysiology of Open Angle Glaucoma?

“…The presence of MR and HSD2 in the trabecular meshwork is described controversially [22–24]. Mineralocorticoid effects are thought to be mediated by epithelial sodium channels (EnaC) [38, 39], which are also present at numerous places in the anterior eye segment [40–43]. These channels might serve two different functions: one is fluid secretion from the ciliary epithelium (increase of aqueous humour formation), and the other is regulation of the trabecular meshwork resistance by volume regulation of the trabecular meshwork cells.…”