Effects of inhibitors of ion-motive ATPases on the plasma membrane potential of murine erythroleukemia cells.
The membrane electric effects of N,N'-dicyclohexyl-carbodiimide (DCCD) and vanadate were studied in murine erythroleukemia cells (MELC), comparing the patch-clamp technique and the accumulation ratio (ARexp) of [3H]-tetraphenylphosphonium (TPP+). Electrophysiological measurements showed that both these inhibitors produce, at micromolar concentrations, a 20-30 mV hyperpolarization of resting potential (delta psi p) of MELC, which is abolished when the electrochemical equilibrium potential of K+ (EK) is brought close to zero. DCCD and vanadate turned out to have distinct targets on the plasma membrane of MELC (an H+ pump and the Na+,K(+)-ATPase, respectively). Measurements of ARexp showed that: (i) patch-clamp measurements of delta psi p were equivalent to those based on ARexp of antimycin-pretreated cells (ARANT); (ii) DCCD produced a strong increase in ARANT, that was antagonized by carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) and diethylstilbestrol (DES); (iii) vanadate determined a marked increase in ARANT that was insensitive to FCCP, but antagonized by ouabain; (iv) incubation in high K+ medium (HK) brought ARANT to 1.0 in the controls, but did not lower this ratio below 3.0 in the presence of DCCD or vanadate; (v) the total amount of TPP+ taken up by the cells was in any case water extractable by a freezing and thawing procedure. On the whole, our data indicate that DCCD and vanadate hyperpolarize the MELC by increasing the K+ conductance and, at the same time, enhance the TPP+ binding, probably by changing the electrostatic potential profile of the plasma membrane. These effects seem to involve functional modifications of the target pumps, apparently related to the ion-occluding state of these enzymes.
Arcangeli
A
A
Institute of General Pathology, University of Florence, Italy.
Del Bene
M R
MR
Becchetti
A
A
Wanke
E
E
Olivotto
M
M
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology / Q000502:physiology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D004054:Diethylstilbestrol / Q000494:pharmacology; D004915:Leukemia, Erythroblastic, Acute; D008564:Membrane Potentials; D051379:Mice; D009840:Oligomycins / Q000494:pharmacology; D009861:Onium Compounds / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology*
D009840:Oligomycins; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D014638:Vanadates; D004024:Dicyclohexylcarbodiimide; D010042:Ouabain; D004054:Diethylstilbestrol; D000251:Adenosine Triphosphatases; D011188:Potassium; C013289:tetraphenylphosphonium