Expression of the sodium potassium chloride cotransporter (NKCC1) and sodium chloride cotransporter (NCC) and their effects on rat lens transparency.
OBJECTIVE
To characterize the expression patterns of the Na+-K+-Cl(-) cotransporter (NKCC) 1 and NKCC2, and the Na+-Cl(-) cotransporter (NCC) in the rat lens and to determine if they play a role in regulating lens volume and transparency.


METHODS
RT-PCR was performed on RNA extracted from fiber cells to identify sodium dependent cotransporters expressed in the rat lens. Western blotting and immunohistochemistry, using NKCC1, NKCC2, and NCC antibodies, were used to verify expression at the protein level and to localize transporter expression. Organ cultured rat lenses were incubated in Artificial Aqueous Humor (AAH) of varying osmolarities or isotonic AAH that contained either the NKCC specific inhibitor bumetanide, or the NCC specific inhibitor thiazide for up to 18 h. Lens transparency was monitored with dark field microscopy, while tissue morphology and antibody labeling patterns were recorded using a confocal microscope.


RESULTS
Molecular experiments showed that NKCC1 and NCC were expressed in the lens at both the transcript and protein levels, but NKCC2 was not. Immunohistochemistry showed that both NKCC1 and NCC were expressed in the lens cortex, but NCC expression was also found in the lens core. In the lens cortex the majority of labeling for both transporters was cytoplasmic in nature, while in the lens core, NCC labeling was associated with the membrane. Exposure of lenses to either hypotonic or hypertonic AAH had no noticeable effects on the predominantly cytoplasmic location of either transporter in the lens cortex. Incubation of lenses in isotonic AAH plus the NKCC inhibitor bumetanide for 18 h induced a cortical opacity that was initiated by a shrinkage of peripheral fiber cells and the dilation of the extracellular space between fiber cells in a deeper zone located some approximately 150 microm in from the capsule. In contrast, lenses incubated in isotonic AAH and the NCC inhibitor thiazide maintained both their transparency and their regular fiber cell morphology.


CONCLUSIONS
We have confirmed the expression of NKCC1 in the rat lens and report for the first time the expression of NCC in lens fiber cells. The expression patterns of the two transporters and the differential effects of their specific inhibitors on fiber cell morphology indicate that these transporters play distinct roles in the lens. NKCC1 appears to mediate ion influx in the lens cortex while NCC may play a role in the lens nucleus.
Chee
K N
KN
New Zealand National Eye Centre, University of Auckland, Auckland, New Zealand.
Vorontsova
I
I
Lim
J C
JC
Kistler
J
J
Donaldson
P J
PJ
D000818:Animals; D015153:Blotting, Western; D002034:Bumetanide / Q000494:pharmacology; D006706:Homeostasis / Q000502:physiology; D007150:Immunohistochemistry; D007904:Lens Cortex, Crystalline / Q000378:metabolism; D007908:Lens, Crystalline / Q000166:cytology / Q000502:physiology*; D009924:Organ Culture Techniques; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D049992:Sodium Chloride Symporters / Q000187:drug effects / Q000378:metabolism*; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters / Q000187:drug effects / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1; D064506:Solute Carrier Family 12, Member 2; D049971:Thiazides / Q000494:pharmacology; D014018:Tissue Distribution
C578716:Slc12a1 protein, rat; C578879:Slc12a2 protein, rat; D049992:Sodium Chloride Symporters; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D064506:Solute Carrier Family 12, Member 2; D049971:Thiazides; D002034:Bumetanide
Exp Eye Res. 1969 Oct;8(4):421-8
5358238
J Exp Biol. 1998 Jul;201(Pt 14):2091-102
9639584
J Bioenerg Biomembr. 1998 Apr;30(2):161-72
9672238
Exp Eye Res. 2006 Aug;83(2):447-55
16635486
Histochem Cell Biol. 2009 Jun;131(6):667-80
19288125
J Biol Chem. 1994 Jul 1;269(26):17713-22
8021284
Mol Vis. 2009 Nov 24;15:2448-63
19956408
Invest Ophthalmol Vis Sci. 2004 Dec;45(12):4400-8
15557448
Ophthalmic Res. 1999;31(4):317-20
10325548
Cell Physiol Biochem. 2000;10(5-6):341-54
11125215
Cell Physiol Biochem. 2008;21(5-6):335-46
18453742
Invest Ophthalmol Vis Sci. 2007 Nov;48(11):5142-51
17962467
J Physiol. 2004 Sep 15;559(Pt 3):883-98
15272034
Exp Eye Res. 1981 Feb;32(2):151-62
7238617
Invest Ophthalmol Vis Sci. 2005 Aug;46(8):2869-77
16043861
Exp Eye Res. 2003 Jan;76(1):61-70
12589776
Am J Physiol Cell Physiol. 2008 May;294(5):C1133-45
18367590
Clin Exp Pharmacol Physiol. 2004 Dec;31(12):890-5
15659055
J Biol Chem. 1997 Sep 26;272(39):24556-62
9305921
Invest Ophthalmol Vis Sci. 2000 Sep;41(10):3049-55
10967063
Physiol Rev. 2005 Apr;85(2):423-93
15788703
Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2749-53
8464884
Mol Pharmacol. 1988 Jun;33(6):696-701
3380083
Exp Eye Res. 2003 Nov;77(5):567-74
14550398
Biophys J. 1985 Sep;48(3):423-34
3876116
J Gen Physiol. 1991 Dec;98(6):1085-103
1783895
Invest Ophthalmol. 1976 Oct;15(10):866-9
977257
Physiol Rev. 2009 Jan;89(1):193-277
19126758
Physiol Rev. 2000 Jan;80(1):211-76
10617769
Exp Eye Res. 1994 Apr;58(4):479-90
7925684
Prog Retin Eye Res. 1999 Mar;18(2):223-33
9932284
Exp Eye Res. 2009 Feb;88(2):144-50
19091312
Invest Ophthalmol Vis Sci. 2009 Aug;50(8):3808-18
19279312
Exp Eye Res. 2010 Feb;90(2):300-7
19941852
Annu Rev Physiol. 2000;62:515-34
10845101
Microsc Res Tech. 2003 Sep 1;62(1):83-91
12938120
Invest Ophthalmol Vis Sci. 2002 Sep;43(9):3008-15
12202523
Exp Eye Res. 2001 Nov;73(5):669-80
11747367
Invest Ophthalmol Vis Sci. 2006 Feb;47(2):673-82
16431967