ATP-driven, Na(+)-independent inward Cl- pumping in neuroblastoma cells.
In immature neurones, the steady-state intracellular Cl- concentration [Cl-](i) is generally higher than expected for passive distribution, and this is believed to be due to Na(+)-K(+)-2Cl(-) co-transport. Here, we show that N2a neuroblastoma cells, incubated in HEPES-buffered NaCl medium maintain a [Cl-](i) around 60 mm, two- to threefold higher than expected for passive distribution at a membrane potential of - 49 mV. When the cells were transferred to a Cl(-) -free medium, [Cl-](i) decreased quickly (t(1/2) < 5 min), suggesting a high Cl- permeability. When the intracellular ATP concentration was reduced to less than 1 mm by metabolic inhibitors, the initial rate of (36) Cl- uptake was strongly inhibited (60-65%) while steady-state [Cl-](i) decreased to 24 mm, close to the value predicted from the Nernst equilibrium. Moreover, after reduction of [ATP](i) and [Cl-](i) by rotenone, the subsequent addition of glucose led to a reaccumulation of Cl-, in parallel with ATP recovery. Internal bicarbonate did not affect Cl- pumping, suggesting that Cl-/HCO(3)(-) exchange does not significantly contribute to active transport. Likewise, Na(+) -K(+) -2Cl(-) co-transport also appeared to play a minor role: although mRNA for the NKCC1 form of the co-transporter was detected in N2a cells, neither the initial rate of (36)Cl- uptake nor steady-state [Cl-](i) were appreciably decreased by 10 microm bumetanide or replacement of external Na(+) by choline. These results suggest that a highly active ATP-dependent mechanism, distinct from Na(+) -K(+) -2Cl(-) co-transport, is responsible for most of the inward Cl- pumping in N2a cells.
Bettendorff
Lucien
L
Center for Cellular and Molecular Neurobiology, University of Liège, 17 place Delcour, B-4020 Liège, Belgium. L.Bettendorf@ulg.ac.be
Lakaye
Bernard
B
Margineanu
Ilca
I
Grisar
Thierry
T
Wins
Pierre
P
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002463:Cell Membrane Permeability / Q000502:physiology; D002712:Chlorides / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D007424:Intracellular Fluid / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D007476:Ionophores / Q000494:pharmacology; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D012402:Rotenone / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism; D064506:Solute Carrier Family 12, Member 2; D014407:Tumor Cells, Cultured; D014475:Uncoupling Agents / Q000494:pharmacology
D002712:Chlorides; D004791:Enzyme Inhibitors; D007476:Ionophores; D012333:RNA, Messenger; C578878:Slc12a2 protein, mouse; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D014475:Uncoupling Agents; D012402:Rotenone; D000255:Adenosine Triphosphate; D012964:Sodium; D005947:Glucose; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid