Sodium- and Potassium-Activated ATPase in Uptake of Fe59 By the Rabbit Reticulocyte.

Abstract

Iron enters the reticulocyte during a phase of its maturation to an erythrocyte. There is evidence that the mechanism is dependent upon metabolic energy-producing processes and is blocked by low concentrations of metabolic inhibitors, such as 2,4-dinitrophe-no1 and iodoacetic acid ( 1 ) . Furthermore, the transfer does not seem to be linked to heme synthesis, and uptake of Fe59 by human reticulocytes is moderately increased by glucose.Dowdle, Schacter and Schenker studied the transport of Fe59 by everted segments of rat duodenum( 2 ) . They reported that transport took place from the mucosal to the serosal surface against a concentration gradient in vitro, and was dependent on oxidative metabolism and the generation of phosphate bond energy.Sodium-and potassium-activated adenosine triphosphatase activity in human erythrocytes is inhibited by a relatively low concentration of ouabain( 3 ) . Other investigations indicated a relationship between Na-K activated ATPase and the active movement of both electrolytes (Na+ and K f ) (4) and non-electrolytes (glucose and its analogs) ( 5) against their respective concentration qradients.In light of these findings, evidence was sought and found which suggested that there is a link between sodium-and potassiumactivated ATPase and uptake of Fe59 by the rabbit reticulocyte.Methods. Reticulocytes were obtained from phenylhydrazine-treated rabbits. New Zealand white rabbits were given l ml of a 2.5% aqueous phenylhydrazine solution intraperitoneally for 4 consecutive days. Blood was obtained by cardiac puncture on the seventh day, using heparin as the anticoagulant. After centrifugation, the packed cells were washed with 2 volumes of isotonic NaCl solution and *This work was supported by grant 0.23559 from Nat. Science Foundation.recentrifuged. Five ml of packed cells, containing approximately 601% reticulocytes, were suspended in 5 ml of isotonic NaCl solution and mixed with 20 ml NaCl solution containing 9 X mole of Fe59,t 50 ml Eagle's MEM growth medium,$ and 10 ml commercial rabbit serum.$ This suspension was separated into 3 equal aliquots, and duplicate samples were immediately taken from each aliquot. The average radioactive counts of all the duplicate samples, after processing, were assumed to represent instantaneouslybound membrane iron, released by the cells upon hemolysis, and this count was subtracted from each subsequent count. The aliquots were then incubated at 37°C in a water bath shaker. After 30, 60, 90, 1201, and 180 minutes, duplicate samples were taken from each aliquot and processed. After 60 minutes' incubation, 0.5 ml 6 x lW3 M ouabain was added to one flask, 0.5 ml 6 X l0-l5M ouabain was added to the second, and 0.5 ml isotonic NaCl solution was added to the third, which served as control.Each sample was processed as follows. The cells were washed with isotonic NaCl solution containing non-radioactive FeCls (1 X M ) to minimize cellular iron loss; after centrifugation, the cells were hemolyzed with water and the supernatant was assayed for radioactivity. ...

Accumulation of Iron in the Rabbit Erythroid Cell As Affected by Ouabain, Sodium and Potassium Ions, and Temperature

“…Crane postulated that a mobile carrier was capable only of equilibrium. T h e asymmetry required to achieve uphill accumulation of the substance was attributed to a gradient of N a + concentration downhill into the T h e findings in the present experiments and those reported from this l a b o r a t o r y earlier (5,15,16) constitute supporting evidence that the N a -K A T P a s e influences only a small p a r t of iron transport, a n d that the N a -K A T P a s e appears to be i m p o r t a n t in the m o v e m e n t of iron from the cell m e mb r a n e (stroma) to the cell interior.…”

“…physical absorption, and second, the development of a stronger union dependent on cell metabolism (4). Preliminary experiments indicate that ouabain restricts accumulation of SgFe in the rabbit reticulocyte, raising speculation as to whether the sodiumpotassium-activated ATPase enzyme system may be involved in some manner in iron transport (5). Recent studies indicate the possible participation of a Na+-K+-activated ATPase system in the transport scheme of a number of substrates, as well as transport of Na + and K +.…”

“…FIGURE5. Lineweaver-Burk plots obtained from supernatant 6°Fe counts when reticulocytes were incubated for 0-6 min with and without Na + (K+-free).…”