Red blood cells of a transgenic mouse expressing high levels of human hemoglobin S exhibit deoxy-stimulated cation flux.
Deoxy-stimulated cation fluxes have been implicated in the generation of the dense and irreversibly sickled red blood cells (RBCs) in patients homozygous for hemoglobin S (SS). We now report on the effect of short term deoxygenation on K+ and Na+ transport in RBCs from control mice (C57Bl/6J) and a transgenic (alphaHbetaS[betaMDD]) mouse line that expresses high levels of human alphaH and betaS-chains and has a small percent dense cells but does not exhibit anemia. In transgenic mouse RBCs (n = 5) under oxygenated conditions, K+ efflux was 0.22 +/- 0.01 mmol/L cell x min and Na+ influx was 0.17 +/- 0.02 mmol/L cell x min. Both fluxes were stimulated by 10 min deoxygenation in transgenic but not in control mice. The deoxy-stimulated K+ efflux from transgenic mouse RBCs was about 55% inhibited by 5 nm charybdotoxin (CTX), a blocker of the calcium activated K+-channel. To compare the fluxes between human and mouse RBCs, we measured the area of mouse RBCs and normalized values to area per liter of cells. The deoxy-simulated CTX-sensitive K+ efflux was larger than the CTX-sensitive K+ efflux observed in RBCs from SS patients. These results suggest that in transgenic mice, deoxygenation increases cytosolic Ca2+ to levels which open Ca2+-activated K+ channels. The presence of these channels was confirmed in both control and transgenic mice by clamping intracellular Ca2+ at 10 microM with the ionophore A23187 and measuring Ca2+-activated K+ efflux. Both types of mouse had similar maximal rates of CTX-sensitive, Ca2+-activated K+ efflux that were similar to those in human SS cells. The capacity of the mouse red cell membrane to regulate cytosolic Ca2+ levels was examined by measurements of the maximal rate of calmodulin activated Ca2+-ATPase activity. This activity was 3-fold greater than that observed in human RBCs thus indicating that mouse RBC membranes have more capacity to regulate cytosolic Ca2+ levels. In summary, transgenic mouse RBCs exhibit larger values of deoxy-stimulated K+ efflux and Na+ influx when compared to human SS cells. They have a similar Ca2+-activated K+ channel activity to human SS cells while expressing a very high Ca2+ pump activity. These properties may contribute to the smaller percent of very dense cells and to the lack of adult anemia in this animal model.
Romero
J R
JR
Endocrine-Hypertension Division, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, 221 Longwood Avenue, Boston, MA 02115, USA.
Fabry
M E
ME
Suzuka
S
S
Nagel
R L
RL
Canessa
M
M
D000328:Adult; D000755:Anemia, Sickle Cell / Q000097:blood*; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000097:blood; D018999:Charybdotoxin / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism*; D006451:Hemoglobin, Sickle / Q000378:metabolism*; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D011188:Potassium / Q000097:blood*; D026902:Potassium Channel Blockers; D012964:Sodium / Q000097:blood*
D006451:Hemoglobin, Sickle; D007476:Ionophores; D026902:Potassium Channel Blockers; D018999:Charybdotoxin; D000001:Calcimycin; D012964:Sodium; D000252:Calcium-Transporting ATPases; D011188:Potassium