Localization of a Na+-K+-2Cl− Cotransporter in the Rabbit Lens

Alvarez, Lawrence J.; Candia, Oscar A.; Turner, Helen; Polikoff, Lee

doi:10.1006/exer.2001.1075

Cited by 25 publications

(23 citation statements)

References 42 publications

(53 reference statements)

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“…The presence of the NKCC1 cotransporter was demonstrated by the 12-transmembrane subunit, which showed a band at ϳ170 kDa. Bands at similar sizes were seen by Alvarez et al (1).…”

Section: Methodssupporting

confidence: 82%

Membrane proteins involved in potassium shifts during muscle activity and fatigue

Kristensen

Hansen²,

Juel³

2006

American Journal of Physiology-Regulatory, Integrative and Comp

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“…The presence of the NKCC1 cotransporter was demonstrated by the 12-transmembrane subunit, which showed a band at ϳ170 kDa. Bands at similar sizes were seen by Alvarez et al (1).…”

Section: Methodssupporting

confidence: 82%

Membrane proteins involved in potassium shifts during muscle activity and fatigue

Kristensen

Hansen²,

Juel³

2006

American Journal of Physiology-Regulatory, Integrative and Comp

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“…The molecular and immunochemical data of Figure 11 on the presence of the NKCC1 isform in hLECs is novel in regard to hLECs in culture and consistent with the flux data, confirming the independent demonstration of NKCC1 in animal lens tissues [25].…”

Section: Discussionsupporting

confidence: 71%

“…These molecular data establish for the first time that, in addition to NKCC1 predicted from a report in rabbit LECs [25], NCC is present in hLECs at both the mRNA and protein level matching the functional data of the existence of a K-independent and Cl-dependent Li flux through the NCC mechanism.…”

Section: Resultsmentioning

confidence: 66%

Lithium Fluxes Indicate Presence of Na-Cl Cotransport (NCC) in Human Lens Epithelial Cells

Lauf¹,

Chimote²,

Adragna

2008

Cell Physiol Biochem

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During regulatory volume decrease (RVD) of human lens epithelial cells (hLECs) by clotrimazole (CTZ)–sensitive K fluxes, Na-K-2Cl cotransport (NKCC) remains active and K-Cl cotransport (KCC) inactive. To determine whether such an abnormal behavior was caused by RVD-induced cell shrinkage, NKCC was measured in the presence of either CTZ or in high K media to prevent RVD. NKCC transports RbCl + NaCl, and LiCl + KCl; thus ouabain-insensitive, bumetanide-sensitive (BS) or Cl-dependent (ClD) Rb and Li fluxes were determined in hyposmotic high NaCl media with CTZ, or in high KCl media alone, or with sulfamate (Sf) or nitrate as Cl replacement at varying Rb, Li or Cl mol fractions (MF). Unexpectedly, NKCC was inhibited by 80% with CTZ (IC₅₀ = 31 µM). In isosmotic (300 mOsM) K, Li influx was ñ 1/3 of Rb influx in Na, 50% lower in Sf, and bumetanide-insensitive (BI). In hypotonic (200 mOsM) K, only the ClD but not BS Li fluxes were detected. At Li MFs from 0.1-1, Li fluxes fitted a bell-shaped curve maxing at ñ0.6 Li MF, with the BS fluxes equaling ñ¼ of the ClD-Li influx. The difference, i.e. the BI/ClD Li influx, saturated with increasing Li and Cl MFs, with K_ms for Li of 11 with, and 7 mM without K, and of ñ46 mM for Cl. Inhibition of this K-independent Li influx by thiazides was weak whilst furosemide (<100 µM) was ineffective. Reverse transcription polymerase chain reaction and Western blots verified presence of both NKCC1 and Na-Cl cotransport (NCC). In conclusion, in hyposmotic high K media, which prevents CTZ-sensitive K flux-mediated RVD in hLECs, NKCC1, though molecularly expressed, was functionally silent. However, a K-independent and moderately thiazide-sensitive ClD-Li flux, i.e. LiCC, likely occurring through NCC was detected operationally and molecularly.

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“…This transporter is reported to be involved in transport of Na, K and Cl ions into and out of cells in an electrically neutral manner, and is involved in maintenance of cell volume. 35,36 The reduced expression of NKCC2, in conjunction with lowered expression of the potassium voltage-gated channel in C3 lenses, might be expected to affect the osmotic balance leading to abnormal fiber swelling ( Figures 4-6) and vacuole accumulation as seen in C3 lenses (Figures 1 and 3).…”

Section: Discussionmentioning

confidence: 99%

Impaired cytoskeletal organization and membrane integrity in lens fibers of a Rho GTPase functional knockout transgenic mouse

Maddala

Pingping

Costello

et al. 2004

Laboratory Investigation

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To investigate the effects of Rho GTPase inactivation on lens fiber cell cytoskeletal and morphological integrity, a transgenic mouse model expressing C3-exoenzyme (a bacterial toxin) in a lens-specific manner was utilized. Cryosections of whole eyes from C3 transgenic mice and littermate controls were stained for F-actin with rhodamine-phalloidin or immunostained for b-catenin, aquaporin-0 or connexin-50, and confocal images were recorded. Lens fiber cell morphology was examined at both light and electron microscopic levels. To investigate the influence of Rho GTPase inactivation on the profiles of gene expression, cDNA libraries generated from transgenic and littermate control mouse lenses were screened by cDNA microarray analysis. In contrast to the wild-type lens, fiber cells of the transgenic lens were grossly swollen and disorganized, with abnormal membrane architecture. Staining of F-actin, b-catenin, aquaporin-0 and connexin-50 was reduced dramatically in the C3 transgenic lens as compared to controls. Western blot analysis and cDNA microarray analysis did not reveal any noticeable decreases in actin, b-catenin and aquaporin-0 protein levels or expression in C3 transgenic lenses, indicating that altered cytoskeletal organization in response to Rho GTPase inactivation might underlie the noted changes in staining for these proteins. Additionally, cDNA microarray analysis of C3 lens revealed altered expression (at least two-fold, compared to littermate controls) of 44 genes. These include genes encoding extracellular matrix and basement membrane proteins, cell survival and apoptotic pathways, and ion and protein transport. These data indicate that disruption of Rho GTPase function in the developing mouse lens results in abnormal cytoskeletal organization, fiber cell interactions, impaired lens fiber cell morphology and altered gene expression of cellular proteins involved in diverse functions. This work reveals that the morphological and cytoskeletal abnormalities triggered upon Rho GTPase inactivation in lens could be one of the important insults associated with cataract formation in C3 transgenic mouse lens.

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Localization of a Na+-K+-2Cl− Cotransporter in the Rabbit Lens

Cited by 25 publications

References 42 publications

Membrane proteins involved in potassium shifts during muscle activity and fatigue

Membrane proteins involved in potassium shifts during muscle activity and fatigue

Lithium Fluxes Indicate Presence of Na-Cl Cotransport (NCC) in Human Lens Epithelial Cells

Impaired cytoskeletal organization and membrane integrity in lens fibers of a Rho GTPase functional knockout transgenic mouse

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