Localization of a Na(+)-K(+)-2Cl(-) cotransporter in the rabbit lens
L J Alvarez1, O A Candia, H C Turner
1Department of Ophthalmology, Mount Sinai School of Medicine, 100th Street and 5th Avenue, New York, NY 10029-6574, USA.
Experimental Eye Research
|December 19, 2001
Summary
The Na-K-2Cl cotransporter (NKCC1) is present in the lens but its activity is low in intact organs. Conditions like cell shrinkage activate NKCC1, especially in cultured cells.
Area of Science:
- Ophthalmology
- Cell Physiology
- Molecular Biology
Background:
- Previous studies showed bumetanide-sensitive K+ uptake in cultured lens cells, but not intact lenses.
- The distribution of the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC1) in the lens requires further investigation.
Purpose of the Study:
- To re-examine the distribution and activity of the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC1) in intact rabbit lenses.
- To investigate the conditions that modulate NKCC1 activity in lens epithelial cells.
Main Methods:
- (86)Rb+ uptake experiments on intact rabbit lenses in a specialized chamber.
- Western blot analysis of lens membrane proteins to detect NKCC1.
- Uptake studies on cultured rabbit lens epithelial cells under varying osmotic conditions.
Main Results:
- K+ influx rates were low at intact lens surfaces and largely insensitive to bumetanide.
- Bumetanide-sensitive K+ influx was observed under hypertonic conditions at anterior and equatorial surfaces.
- NKCC1 protein was detected in epithelial and cortical equatorial fractions of the lens.
- Cultured lens epithelial cells showed significant bumetanide-sensitive K+ uptake, indicating upregulation.
Conclusions:
- NKCC1 is present in the lens, but its activity in intact organs is limited and context-dependent.
- Cell shrinkage or hypertonic conditions may activate NKCC1 in the intact lens.
- The cotransporter's activity is significantly upregulated in cultured lens epithelial cells, suggesting a response to culture conditions.
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