Phosphate from the phosphointermediate (EP) of the human red blood cell Na/K pump is coeffluxed with Na, in the absence of external K.

Abstract

A B ST RA C T This study is concerned with Na/K pump-mediated phosphate effiux that occurs during uncoupled Na efflux in human red blood cells. Uncoupled Na efflux is known to be a ouabain-sensitive mode of the Na/K pump that occurs in the absence of external Nao and Ko. Because this efflux (measured with ZZNa) is also inhibited by 5 mM Nao, the efflux can be separated into a Nao-sensitive and a Nao-insensitive component. Previous work established that the Nao-sensitive effiux is actually comprised of an electroneutral coefflux of Na with cellular anions, such as SO4 (as ~5SO4). The present work focuses on the Nao-insensitive component in which the principal finding is that orthophosphate (Pi) is coeffluxed with Na in a ouabain-sensitive manner. This Pi efl:lux can be seen to occur, in the absence of Ko, in both DIDS-treated intact cells and resealed red cell ghosts. This effiux of Pi was shown to be derived directly from the pump's substrate, ATP, by the use of resealed ghosts made to contain both ATP and Pi in which either the ATP or the Pi were labeled with, respectively, [3~-~2p]ATP or [32p]H3PO 4. (These resealed ghosts also contained Na, Mg, Pi, SO4, Ap5A, as well as an arginine kinase/creatine kinase nucleotide regenerating system for the control of ATP and ADP concentrations, and were suspended usually in (NMG)2SO4 at pH 7.4.) It was found that szp was only coeffluxed with Na when the 32p was contained in [~/-~P]ATP and not in [s2p]H3PO 4. This result implies that the s2p that is released comes from ATP via the pump's phosphointermediate (EP) without commingling with the cellular pool of Pi. Ko (as K2804) inhibits this 32p efflux as well as the Nao-sensitive 35SO 4 efflux, with a K0.5 of 0.3-0.4 mM. The K05 for inhibition of Pi efflux by Ko is not influenced by Nao, nor can Nao act as a congenor for Ko in any of the flux reactions involving Ko. The stoichiometry of Na to SO4 and Na to Pi efl]ux is ~ 2:1 under circumstances where the stoichiometry of Na effluxed to ATP utilized is 3:1. From these and other results reported, it is suggested that there are two types of uncoupled Na efflux that differ Dr. Marin's permanent address is Instituto Venezolano de lnvestigaciones Cientificas, CBB. Aptdo.

The Effect of Ionic Strength and Specific Anions on Substrate Binding and Hydrolytic Activities of Na,K-ATPase

“…Pertinent to the reaction of anions with the Na,K-pump are also the three papers ( Dissing and Hoffman, 1990 ; Marín and Hoffman, 1994 a , b ) describing a coefflux of Na + and SO 4 2− or HPO 4 2− (P i ) in red blood cells. The transport is ouabain inhibitable and intimately connected to the so-called uncoupled Na + efflux through the Na,K-pump.…”

ADP+orthophosphate (P(i)) stimulates an Na/K pump-mediated coefflux of P(i) and Na in human red blood cell ghosts.

“…The studies reported in this paper utilize resealed human red blood cell ghosts that had been prepared to contain different added constituents. The procurement of blood and the initial hemolysis and washing of the ghosts up to the stage where the ghosts were concentrated, before being transferred to the reversal medium, were carried out following the same protocol as described in Marin and Hoffman (1994). For the present purposes the composition of the reversal solution unless otherwise specified, was as follows: 12.5 mM Na~SO4, 5 mM MgSO4, 25 mM Yris2SO4, from 0 to 10 mM Tris H2PO4, from 0 to 500 ~M Tris2-ADP, 50 mM glucose, 70 U Hexokinase, 50 IzM Trypan blue, 40 ~M PI,PS-di(adenosine-5')pentaphosphate (Ap5A), together with sufficient (NMG)2SO4 (N-methyl glucamine sulfate) to bring the osmolarity of the solution to 300 mosM and adjustment of the pH to 7.5 at 0~ When K was present it was added as K2SO4 in substitution for an equivalent concentration of (NMG)2SO 4.…”

“…This effect of Ko in type II uncoupled Na efflux is analogous to its actions in type I or ATP-dependent uncoupled Na efflux. In type I, without Ko, cellular anions are also cotransported with Na out of the cell, but the addition of Ko activates in this instance the pumped exchange of Nai for Ko, which pari passu inhibits completely the efflux of anions (Dissing and Hoffman, 1990;Marin and Hoffman, 1994. ) The stoichiometry of Na/Pi cotransport can be estimated from experiments where ~176 and ~176 ""Na "'Pi were measured on ghosts prepared to contain the same concentrations of Pi (10 mM) and ADP (500 ~M) and incubated in media free of Nao and Ko.…”

“…All reagents other than the following were purchased from Sigma Chemical Co. (St. Louis, MO) (see Marin and Hoffman, 1994…”

“…Because cellular anions were found to be cotransported with Na in type I or ATP-dependent uncoupled Na efflux (Dissing and Hoffman, 1990;Marin and Hoffman, 1994) it was of interest to study the extent to which anion transport may occur in type II or [ADP + Pi]-promoted uncoupled Na efflux. Ghosts were thus labeled with either 32p i or 35804 during entrapment of 10 mM Pi and 50 mM SO4 in their normal preparation before exposure to DIDS and before resealing at 37~ The results of these studies are presented in Table VI where it is clear that, in the absence Resealed ghosts were prepared in accordance with the protocol described before (legend to Table I, Methods section), in the presence of 10 mM Pi + 500 p,M ADP together with the other constituents listed.…”