scholarly journals Comparison of the side-dependent effects of Na and K on orthophosphate-, UTP-, and ATP-promoted ouabain binding to reconstituted human red blood cell ghosts.

1976 ◽  
Vol 67 (5) ◽  
pp. 527-545 ◽  
Author(s):  
H H Bodemann ◽  
J F Hoffman
Keyword(s):  
Blood Cell ◽  
Adenosine Triphosphate ◽  
Mechanism Of Action ◽  
Red Blood Cell ◽  
Ouabain Binding ◽  
Uridine Triphosphate ◽  
Binding Rate ◽  
External K

This paper is concerned with analyzing the sidedness of action of various determinants which alter the rate of ouabain binding to human red blood cell ghosts. Thus, ouabain binding promoted by orthophosphate (Pi) and its inhibition by Na are shown to be due to inside Pi and inside Na. External K inhibits Pi-promoted ouabain binding and Nao acts to decrease the effectiveness of Ko. Similarly, inside uridine triphosphate (UTPi) stimulates the rate of ouabain binding which can be antagonized by either Nai or Ko acting alone. The actions of Nai and Ko are different when ouabain binding is promoted by Pi and UTPi compared to inside adenosine triphosphate (ATPi). With ATPi, the ouabain binding rate is only affected when Nai and Ko are both present. Possible differences in the mechanism of action of K and Na on Pi-and UTP-promoted binding are discussed in the light of their sidedness of action.

scholarly journals Interaction of External K, Na, and Cardioactive Steroids with the Na-K Pump of the Human Red Blood Cell

1974 ◽  
Vol 63 (2) ◽  
pp. 123-143 ◽  
Author(s):  
John R. Sachs
Keyword(s):  
Blood Cell ◽  
Red Blood Cell ◽  
Ouabain Binding ◽  
Long Period ◽  
Steady State Kinetic ◽  
Straight Line ◽  
Cardioactive Steroids ◽  
Simple Kinetic Model ◽  
State Kinetic ◽  
External K

The interaction of extracellular Na (Nao), K (Ko), and strophanthidin with the Na-K pump of the human red blood cell has been investigated. Inhibition by submaximal concentrations of strophanthidin rapidly reaches a level which does not increase further over a relatively long period of time. Under these circumstances, it is possible to apply a steady-state kinetic analysis to the interaction of Nao, Ko, and strophanthidin with the pump. In Na-free solutions, strophanthidin increases the apparent K1/2 of the pump for Ko, but does not change the form of the relation between the reciprocal of the active K influx (iMKP–1) and the reciprocal of [Ko] ([Ko]–1); the relation both in the presence and absence of strophanthidin is adequately described by a straight line. In solutions containing Na, strophanthidin changes the form of the curve describing the relation between iMKP–1 vs. [Ko]–1; the curve becomes more parabolic in solutions containing strophanthidin. The rate of ouabain binding to K-free cells has also been measured; in the absence of K, the rate of binding is unaffected by Nao. The data are considered in terms of a simple kinetic model. The findings can be explained if it is supposed that at low external K the form of the pump combined with one Nao is more likely to combine with strophanthidin than is the uncombined form of the pump. The uncombined form of the pump is more likely to combine with K even at very low Ko than with strophanthidin.

scholarly journals Effects of Mg and Ca on the side dependencies of Na and K on ouabain binding to red blood cell ghosts and the control of Na transport by internal Mg.

1976 ◽  
Vol 67 (5) ◽  
pp. 547-561 ◽  
Author(s):  
H H Bodemann ◽  
J F Hoffman
Keyword(s):  
Blood Cell ◽  
Red Blood Cell ◽  
Inhibitory Action ◽  
External Surface ◽  
The Other ◽  
Na Transport ◽  
Ouabain Binding ◽  
Conformational States ◽  
Binding Rate ◽  
Relative Affinity

The effect of alteration in the concentration of internal Mg on the rate of ouabain binding to reconstituted human red blood cell ghosts has been evaluated as well as the effect of Mgi on Na:Na compared to Na:K exchange. It was found that the dependence of the rate of ATP-promoted ouabain binding on the combined presence of Nai and Ko which occurs at high [Mg]i is lost when the concentration of Mgi is lowered. The sensitivity of the external surface for Ko is also changed since Ko can now inhibit the ouabain binding rate in the absence of Nai; on the other hand Nao at low [Mg]i can stimulate ouabain binding indicating that the relative affinity of the outside surface for Nao has either increased or that for Ko has decreased or both. Thus the effects of changes in [Mg]i result in a change in the side-dependent actions of Na and K and emphasize the possible difficulties of interpreting results obtained on systems lacking sidedness. Mgi was found to be required for Pi-promoted ouabain binding and that the inhibitory action of Nai increased as [Mg]i was increased. In addition, Ca was found to be most effective in inhibiting the rate of ATP-promoted ouabain binding when Na and K were present together than when either was present alone. Na:K exchange was found to be more sensitive to the concentration of Mgi than Na:Na exchange; at low [Mg]i Na:K exchange could be stimulated without changing the extent of Na:Na exchange. These results are consistent with the idea that conformational states of the pump complex are directly influenced by [Mg]i.

scholarly journals Side-dependent effects of internal versus external Na and K on ouabain binding to reconstituted human red blood cell ghosts.

1976 ◽  
Vol 67 (5) ◽  
pp. 497-525 ◽  
Author(s):  
H H Bodemann ◽  
J F Hoffman
Keyword(s):  
Blood Cell ◽  
Binding Site ◽  
Red Blood Cell ◽  
Red Cell ◽  
Ouabain Binding ◽  
Binding Properties ◽  
Conformational States ◽  
Binding Rate ◽  
Lack Of Control

The side-dependent effects of internal and external Na and K on the ouabain binding rate, as promoted by inside MgATP, has been evaluated utilizing reconstituted human red blood cell ghosts. Such ghost systems provide the situation where [Na]i, [K]i, [Na]o, and [K]o can each be varied under conditions in which the others are either absent or fixed at constant concentrations. It was found that, in the presence of Ko, increasing either [Na]i or [K]i resulted in decreasing the rate at which ouabain was bound. Changes in [Na]i or [K]i in the absence of Ko were without effect on the ouabain binding rate. Thus, the ouabain binding rate was found to vary inversely with the rate of Na:K and K:K exchange but was independent of the rate of Na:Na exchange. The effect of Ko in antagonizing ouabain binding, as well as the influence of Nao on this interaction, were found to require the presence of either Nai or Ki. The results are interpreted in terms of a model relating the availability of the ouabain binding site to different conformational states of the pump complex. Differences were observed in the ouabain binding properties of red cell ghosts compared to microsomal preparations but it is not known whether the basis for the differences resides in the different preparations studied or in the lack of control of sidedness in the microsomal systems.

Use of the red blood cell as a simple drug target and diagnostic by manipulating and monitoring its ability to release adenosine triphosphate (ATP)

2010 ◽  
Vol 82 (8) ◽  
pp. 1623-1634 ◽  
Author(s):  
Nicole V. Tolan ◽  
Jennifer A. Meyer ◽  
Chia-Jui Ku ◽  
Welivitiya Karunarathne ◽  
Dana M. Spence
Keyword(s):  
Blood Cell ◽  
Adenosine Triphosphate ◽  
Red Blood Cell ◽  
Drug Targets ◽  
Oxygen Carrier ◽  
Disease Onset ◽  
Diagnostic Tools ◽  
Molecular Evidence ◽  
Vascular Regulation

Without question, one of the main tasks of the red blood cell (RBC) is to deliver oxygen to various tissues and organs in vivo. However, due to the lack of a nucleus and mito-chondria, the RBC is typically not thought to be a determinant in many diseases or abnormal physiological conditions. Recent efforts by many labs world-wide are resulting in a body of evidence, suggesting that the RBC may serve many other roles in vivo besides that of an oxygen carrier. If so, the RBC may eventually emerge as one of the simplest drug targets and diagnostic tools available. Here, molecular evidence is provided, suggesting that the RBC, via its ability (or inability) to maintain proper levels of adenosine triphosphate (ATP) release in the circulation, may be a major factor in vascular regulation. Moreover, due to the RBC’s response to slight modifications in its normal environment, the use of the RBC as an important diagnostic for early prediction of disease onset is discussed.

scholarly journals On the functional use of the membrane compartmentalized pool of ATP by the Na+ and Ca++ pumps in human red blood cell ghosts

2009 ◽  
Vol 134 (4) ◽  
pp. 351-361 ◽  
Author(s):  
Joseph F. Hoffman ◽  
Alicia Dodson ◽  
Fulgencio Proverbio
Keyword(s):  
Energy Source ◽  
Blood Cell ◽  
Pyruvate Kinase ◽  
Adenosine Triphosphate ◽  
Red Blood Cell ◽  
Red Cell ◽  
Previous Evidence ◽  
Caged Atp ◽  
Na Pump ◽  
Membrane Bound

Previous evidence established that a sequestered form of adenosine triphosphate (ATP pools) resides in the membrane/cytoskeletal complex of red cell porous ghosts. Here, we further characterize the roles these ATP pools can perform in the operation of the membrane's Na+ and Ca2+ pumps. The formation of the Na+- and Ca2+-dependent phosphointermediates of both types of pumps (ENa-P and ECa-P) that conventionally can be labeled with trace amounts of [γ-3P]ATP cannot occur when the pools contain unlabeled ATP, presumably because of dilution of the [γ-3P]ATP in the pool. Running the pumps forward with either Na+ or Ca2+ removes pool ATP and allows the normal formation of labeled ENa-P or ECa-P, indicating that both types of pumps can share the same pools of ATP. We also show that the halftime for loading the pools with bulk ATP is 10–15 minutes. We observed that when unlabeled “caged ATP” is entrapped in the membrane pools, it is inactive until nascent ATP is photoreleased, thereby blocking the labeled formation of ENa-P. We also demonstrate that ATP generated by the membrane-bound pyruvate kinase fills the membrane pools. Other results show that pool ATP alone, like bulk ATP, can promote the binding of ouabain to the membrane. In addition, we found that pool ATP alone functions together with bulk Na+ (without Mg2+) to release prebound ouabain. Curiously, ouabain was found to block bulk ATP from entering the pools. Finally, we show, with red cell inside-outside vesicles, that pool ATP alone supports the uptake of 45Ca by the Ca2+ pump, analogous to the Na+ pump uptake of 22Na in this circumstance. Although the membrane locus of the ATP pools within the membrane/cytoskeletal complex is unknown, it appears that pool ATP functions as the proximate energy source for the Na+ and Ca2+ pumps.

scholarly journals Effects of altering the ATP/ADP ratio on pump-mediated Na/K and Na/Na exchanges in resealed human red blood cell ghosts.

1986 ◽  
Vol 87 (1) ◽  
pp. 47-72 ◽  
Author(s):  
B G Kennedy ◽  
G Lunn ◽  
J F Hoffman
Keyword(s):  
Blood Cell ◽  
Conformational Change ◽  
Red Blood Cell ◽  
Forward Direction ◽  
Nucleotide Composition ◽  
Reaction Cycle ◽  
High Affinity ◽  
High Affinity Site ◽  
Na Efflux ◽  
External K

Resealed human red blood cell ghosts were prepared to contain a range of ADP concentrations at fixed ATP concentrations and vice versa. ATP/ADP ratios ranging from approximately 0.2 to 50 were set and maintained (for up to 45 min) in this system. ATP and ADP concentrations were controlled by the addition of either a phosphoarginine- or phosphocreatine-based regenerating system. Ouabain-sensitive unidirectional Na efflux was determined in the presence and absence of 15 mM external K as a function of the nucleotide composition. Na/K exchange was found to increase to saturation with ATP (K 1/2 approximately equal to 250 microM), whereas Na/Na exchange (measured in K-free solutions) was a saturating function of ADP (K 1/2 approximately equal to 350 microM). The elevation of ATP from approximately 100 to 1,800 microM did not appreciably affect Na/Na exchange. In the presence of external Na and a saturating concentration of external K, increasing the ADP concentration at constant ATP was found to decrease ouabain-sensitive Na/K exchange. The decreased Na/K exchange that still remained when the ADP/ATP ratio was high was stimulated by removal of external Na. Assuming that under normal substrate conditions the reaction cycle of the Na/K pump is rate-limited by the conformational change associated with the release of occluded K [E2 X (K) X ATP----E1 X ATP + K], increasing ADP inhibits the rate of these transformations by competition with ATP for the E2(K) form. A less likely alternative is that inhibition is due to competition with ATP at the high-affinity site (E1). The acceleration of the Na/K pump that occurs upon removing external Na at high levels of ADP evidently results from a shift in the forward direction of the transformation of the intermediates involved with the release of occluded Na from E1P X (Na). Thus, the nucleotide composition and the Na gradient can modulate the rate at which the Na/K pump operates.

scholarly journals Targeting of Hematin by the Antimalarial Pyronaridine

2006 ◽  
Vol 50 (6) ◽  
pp. 2197-2200 ◽  
Author(s):  
Saranya Auparakkitanon ◽  
Soebsakul Chapoomram ◽  
Kannika Kuaha ◽  
Thamrong Chirachariyavej ◽  
Prapon Wilairat
Keyword(s):  
Plasmodium Falciparum ◽  
Blood Cell ◽  
Mechanism Of Action ◽  
Red Blood Cell ◽  
Mannich Base ◽  
Cell Lysis ◽  
Clinical Testing ◽  
Growth Studies

ABSTRACT Pyronaridine, 2-methoxy-7-chloro-10[3′,5′-bis(pyrrolidinyl-1-methyl-)4′hydroxyphenyl]aminobenzyl-(b)-1,5-naphthyridine, a new Mannich base schizontocide originally developed in China and structurally related to the aminoacridine drug quinacrine, is currently undergoing clinical testing. We now show that pyronaridine targets hematin, as demonstrated by its ability to inhibit in vitro β-hematin formation (at a concentration equal to that of chloroquine), to form a complex with hematin with a stoichiometry of 1:2, to enhance hematin-induced red blood cell lysis (but at 1/100 of the chloroquine concentration), and to inhibit glutathione-dependent degradation of hematin. Our observations that pyronaridine exerted this mechanism of action in situ, based on growth studies of Plasmodium falciparum K1 in culture showing antagonism of pyronaridine in combination with antimalarials (chloroquine, mefloquine, and quinine) that inhibit β-hematin formation, were equivocal.

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