The changing face of the Na+/H+ exchanger, NHE1: structure, regulation, and cellular actions.

The NHE family of ion exchangers includes six isoforms (NHE1-NHE6) that function in an electroneutral exchange of intracellular H(+) for extracellular Na(+). This review focuses on the only ubiquitously expressed isoform, NHE1, which is localized at the plasma membrane where it plays a critical role in intracellular pH (pHi) and cell volume homeostasis. All NHE isoforms share a similar topology: an N-terminus of 12 transmembrane (TM) alpha-helices that collectively function in ion exchange, and a C-terminal cytoplasmic regulatory domain that modulates transport activity by the TM domain. Extracellular signals, mediated by diverse classes of cell-surface receptors, regulate NHE1 activity through distinct signaling networks that converge to directly modify the C-terminal regulatory domain. Modifications in the C-terminus, including phosphorylation and the binding of regulatory proteins, control transport activity by altering the affinity of the TM domain for intracellular H(+). Recently, it was determined that NHE1 also functions as a membrane anchor for the actin-based cytoskeleton, independently of its role in ion translocation. Through its effects on pHi homeostasis, cell volume, and the actin cortical network, NHE1 regulates a number of cell behaviors, including adhesion, shape determination, migration, and proliferation.

Quantitation of Na+-K+-2Cl- cotransport splice variants in human tissues using kinetic polymerase chain reaction.

A kinetic reverse transcription-polymerase chain reaction (RT-PCR)-based assay is described that can discriminate and quantitate differentially spliced mRNAs. This assay should be generally applicable for high-throughput quantitation of differentially spliced transcripts. The utility of this method was assessed for spliced transcripts encoded by the human Na+-K+-2Cl- cotransporter gene hNKCC1. Evidence is presented that the NKCC1 isoform of the human Na+-K+-2Cl- cotransporter is differentially spliced analogous to that recently described for the mouse Na+-K+-2Cl- cotransporter gene BSC2. The nucleotide sequences of the two human splice variants predict Na+-K+-2Cl- cotransporter proteins differing only in length. Stable transfectants expressing these human splice variants, designated NKCC1a or NKCC1b, were constructed. Both splice variants produce functional Na+-K+-2Cl- cotransporters in vivo. The abundance of NKCC1 mRNA and patterns of differential splicing in 10 different tissue types and three cell lines were quantitated using the kRT-PCR assay. The results showed that the total amount of NKCC1 mRNA varied by more than 30-fold in the human tissues and cell lines examined. The ratio of NKCC1a/NKCC1b varied nearly 70-fold among these same tissues and cell lines suggesting that differential splicing of the NKCC1 transcript may play a regulatory role in human tissues.

Intracellular Cl regulates Na-K-Cl cotransport activity in human trabecular meshwork cells.

The trabecular meshwork (TM) of the eye plays a central role in modulating intraocular pressure by regulating aqueous humor outflow, although the mechanisms are largely unknown. We and others have shown previously that aqueous humor outflow facility is modulated by conditions that alter TM cell volume. We have also shown that the Na-K-Cl cotransport system is a primary regulator of TM cell volume and that its activity appears to be coordinated with net efflux pathways to maintain steady-state volume. However, the cellular mechanisms that regulate cotransport activity and cell volume in TM cells have yet to be elucidated. The present study was conducted to investigate the hypothesis that intracellular Cl concentration ([Cl]i) acts to regulate TM cell Na-K-Cl cotransport activity, as has been shown previously for some other cell types. We demonstrate here that the human TM cell Na-K-Cl cotransporter is highly sensitive to changes in [Cl]i. Our findings reveal a marked stimulation of Na-K-Cl cotransport activity, assessed as ouabain-insensitive, bumetanide-sensitive K influx, in TM cells following preincubation of cells with Cl-free medium as a means of reducing [Cl]i. In contrast, preincubation of cells with media containing elevated K concentrations as a means of increasing [Cl]i results in inhibition of Na-K-Cl cotransport activity. The effects of reducing [Cl]i, as well as elevating [Cl]i, on Na-K-Cl cotransport activity are concentration dependent. Furthermore, the stimulatory effect of reduced [Cl]i is additive with cell-shrinkage-induced stimulation of the cotransporter. Our studies also show that TM cell Na-K-Cl cotransport activity is altered by a variety of Cl channel modulators, presumably through changes in [Cl]i. These findings support the hypothesis that regulation of Na-K-Cl cotransport activity, and thus cell volume, by [Cl]i may participate in modulating outflow facility across the TM.

Na-K-Cl cotransport in normal and glaucomatous human trabecular meshwork cells.

PURPOSE: Previous results from this laboratory showed that intracellular volume of trabecular meshwork (TM) cells is regulated by the Na-K-Cl cotransport system. Other studies suggest that TM cell volume, in turn, is a determinant of permeability across the TM. Given that a decrease in outflow facility across the TM is thought to be the primary cause of elevated intraocular pressure in primary open-angle glaucoma, the present study was conducted to investigate the possibility that Na-K-Cl cotransport function may be altered in glaucomatous TM cells compared with normal TM cells. METHODS: Normal and glaucomatous human TM cells were cultured from donor eyes and trabeculectomy specimens, respectively. Trabecular meshwork cell monolayers were evaluated for Na-K-Cl cotransport activity, assessed as ouabain-insensitive, bumetanide-sensitive K influx using 86Rb as a tracer for K. Cotransporter protein expression was determined by western blot analysis, and intracellular volume was determined radioisotopically using [14C]urea and [14C]sucrose as markers of total and extracellular water space, respectively. RESULTS: Na-K-Cl cotransport activity of glaucomatous TM cells was found to be reduced by 32% +/- 2% compared with that of normal TM cells, whereas western blot analyses showed that cotransporter protein expression in glaucomatous TM cells was reduced by 64% +/- 14% compared with expression in normal TM cells. Also, exposure of normal TM cells to 10 microM norepinephrine or 50 microM 8-bromo-3',5'-cyclic adenosine monophosphate was found to diminish Na-K-Cl cotransport activity, whereas these agents were without effect on glaucomatous TM cell cotransport. Finally, resting cell volume of glaucomatous TM cells was found to be increased compared with that of normal TM cells, whereas intracellular volume of both cell types was reduced after exposure to 10 microM benzmetanide or 10 microM bumetanide. CONCLUSIONS: These findings indicate that Na-K-Cl cotransport function and regulation are altered in glaucomatous TM cells compared with that of normal TM cells. However, the observation that cell volume of glaucomatous TM cells is greater than that of normal TM cells, despite reduced Na-K-Cl cotransport activity, suggests that other volume-regulatory ion flux pathways may be involved in the reduced outflow of glaucoma.

Effects of dexamethasone on sodium-potassium-chloride cotransport in trabecular meshwork cells.

PURPOSE: Previous studies in the authors' laboratory have shown that bovine and human trabecular meshwork (TM) cells possess a robust sodium-potassium-chloride (Na-K-Cl) cotransport system that functions in regulating intracellular volume and may play a central role in modulating outflow facility across the TM. Dexamethasone, which can induce ocular hypertension, has been found to increase resistance to aqueous outflow across the TM. The current study was conducted to investigate the hypothesis that alteration of TM cell Na-K-Cl cotransport function, regulation, or both may be an underlying factor in steroid-induced glaucoma. To this end, the authors evaluated the effects of dexamethasone treatment of TM cells on Na-K-Cl cotransport activity and cotransporter protein expression. METHODS: Cultured bovine and human TM cell monolayers were exposed to dexamethasone (10(-9) to 10(-6) M) for varying times, then evaluated for Na-K-Cl cotransport activity or harvested for cellular membrane proteins. Cotransport activity was assessed as bumetanide-sensitive K influx. Cotransport protein expression was evaluated by Western blot analysis of cellular proteins using a monoclonal antibody to the human colonic T84 epithelial cell Na-K-Cl cotransporter. RESULTS: The authors found that 24- and 48-hour exposures of human and bovine TM cells to dexamethasone stimulates Na-K-Cl cotransport activity (10(-8) to 10(-6) M dexamethasone in human cells; 10(-8) and 10(-7) M in bovine cells). The authors also found that dexamethasone (10(-8) M) stimulates Na-K-Cl cotransport activity of TM cells with exposure times as early as 12 hours and up to 5 days. In addition, the authors found that the level of Na-K-Cl cotransport protein expressed in TM cells is modulated by dexamethasone. When bovine or human TM cells are exposed to 10(-8) or 10(-6) M dexamethasone for 2 to 5 days, cotransporter protein expression is increased. With longer exposures, however, cotransporter protein levels decrease below control levels. Finally, the authors found that TM cells exposed to dexamethasone become unresponsive to regulation by hypertonicity and vasopressin. CONCLUSIONS: The authors' findings suggest that dexamethasone may be exerting its effect, at least in part, through altering Na-K-Cl cotransport function and regulation in TM cells.