A bicarbonate-dependent process inhibitable by disulfonic stilbenes and a Na+/H+ exchange mediate 22Na+ uptake into cultured bovine corneal endothelium.
22Na+ uptake into confluent monolayers of cultured bovine corneal endothelial cells was studied in the presence of ouabain (10(-4)M) to inhibit active sodium extrusion. In bicarbonate saline, uptake was reduced to a similar degree either by amiloride (10(-3)M) or by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) (10(-3)M). A further reduction was obtained with SITS-pretreated cells in the presence of amiloride. SITS-sensitive uptake was further characterized in saline containing both ouabain (10(-4)M) and amiloride (10(-3)M). It was absolutely dependent on bicarbonate, which could not be substituted by other plasma membrane permeable buffers (50 mM acetate or 25 mM glycodiazine). It was a saturable function of both bicarbonate and sodium concentration. Half-maximal fluxes occurred between 3 and 7 mM HCO3 (at 151 mM Na) and between 35 and 60 mM Na (at 28 mM HCO3). Uptake into sodium-depleted cells was reduced as opposed to sodium-rich cells, and SITS-sensitive 22Na+ efflux out of 22Na+-loaded cells into sodium-free medium was less than efflux into sodium saline, indicating trans-stimulation by sodium. The amiloride-sensitive pathway was studied in the absence of bicarbonate to inhibit uptake via the SITS-sensitive pathway. 22Na+ uptake into sodium-depleted cells increased steeply with extracellular pH in the range between pH 6 and 8 and could be largely blocked by 10(-3), but not by 10(-5) M amiloride. It is concluded that bovine corneal endothelial cells possess at least two distinct pathways for sodium uptake, amiloride sensitive 22Na+ fluxes being mediated by a Na+/H+ antiport, while the SITS-sensitive process is probably identical to a bicarbonate-sodium cotransport system postulated earlier from electrophysiological studies.
Jentsch
T J
TJ
Stahlknecht
T R
TR
Hollwede
H
H
Fischer
D G
DG
Keller
S K
SK
Wiederholt
M
M
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology*; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D003315:Cornea / Q000187:drug effects / Q000378:metabolism*; D004727:Endothelium / Q000187:drug effects / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010042:Ouabain / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers; D013267:Stilbenes / Q000494:pharmacology*
D001639:Bicarbonates; D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D013267:Stilbenes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D010042:Ouabain; D000584:Amiloride; D012964:Sodium