PMID- 9845552
OWN - NLM
STAT- MEDLINE
DCOM- 19990205
LR  - 20231213
IS  - 0006-4971 (Print)
IS  - 0006-4971 (Linking)
VI  - 92
IP  - 12
DP  - 1998 Dec 15
TI  - Two distinct pathways mediate the formation of intermediate density cells and 
      hyperdense cells from normal density sickle red blood cells.
PG  - 4844-55
AB  - In sickle cell anemia (SS), some red blood cells dehydrate, forming a hyperdense 
      (HD) cell fraction (>1.114 g/mL; mean corpuscular hemoglobin concentration 
      [MCHC], >46 g/dL) that contains many irreversibly sickled cells (ISCs), whereas 
      other SS red blood cells dehydrate to an intermediate density (ID; 1.090 to 1.114 
      g/mL; MCHC, 36 to 46 g/dL). This study asks if the potassium-chloride 
      cotransporter (K:Cl) and the calcium-dependent potassium channel [K(Ca2+)] are 
      participants in the formation of one or both types of dense SS red blood cells. 
      We induced sickling by exposing normal density (ND; 1.080 to 1.090 g/mL; MCHC, 32 
      to 36 g/dL) SS discocytes to repetitive oxygenation-deoxygenation (O-D) cycles in 
      vitro. At physiologic Na+, K+, and Cl-, and 0.5 to 2 mmol/L Ca2+, the appearance 
      of dense cells was time- and pH-dependent. O-D cycling at pH 7.4 in 5% 
      CO2-equilibrated buffer generated only ID cells, whereas O-D cycling at pH 6.8 in 
      5% CO2-equilibrated buffer generated both ID and HD cells, the latter taking more 
      than 8 hours to form. At 22 hours, 35% +/- 17% of the parent ND cells were 
      recovered in the ID fraction and 18% +/- 11% in the HD fraction. Continuous 
      deoxygenation (N2/5% CO2) at pH 6.8 generated both ID and HD cells, but many of 
      these cells had multiple projections, clearly different from the morphology of 
      endogenous dense cells and ISCs. Continuous oxygenation (air/5% CO2) at pH 6.8 
      resulted in less than 10% dense cell (ID + HD) formation. ATP depletion 
      substantially increased HD cell formation and moderately decreased ID cell 
      formation. HD cells formed after 22 hours of O-D cycling at pH 6.8 contained 
      fewer F cells than did ID cells, suggesting that HD cell formation is 
      particularly dependent on HbS polymerization. EGTA chelation of buffer Ca2+ 
      inhibited HD but not ID cell formation, and increasing buffer Ca2+ from 0.5 to 2 
      mmol/L promoted HD but not ID cell formation in some SS patients. Substitution of 
      nitrate for Cl- inhibited ID cell formation, as did inhibitors of the K:Cl 
      cotransporter, okadaic acid, and [(dihydroindenyl) oxy]alkanoic acid (DIOA). 
      Conversely, inhibitors of K(Ca2+), charybdotoxin and clotrimazole, inhibited HD 
      cell formation. The combined use of K(Ca2+) and K:Cl inhibitors nearly eliminated 
      dense cell (ID + HD cell) formation. In summary, dense cells formed by O-D 
      cycling for 22 hours at pH 7.4 cycling are predominately the ID type, whereas 
      dense cells formed by O-D cycling for 22 hours at pH 6.8 are both the ID and HD 
      type, with the latter low in HbF, suggesting that HD cell formation has a greater 
      dependency on HbS polymerization. A combination of K:Cl cotransport and the 
      K(Ca2+) activities account for the majority of dense cells formed, and these 
      pathways can be driven independently. We propose a model in which reversible 
      sickling-induced K+ loss by K:Cl primarily generates ID cells and K+ loss by the 
      K(Ca2+) channel primarily generates HD cells. These results imply that both 
      pathways must be inhibited to completely prevent dense SS cell formation and have 
      potential therapeutic implications.
FAU - Schwartz, R S
AU  - Schwartz RS
AD  - The Albert Einstein College of Medicine and Montefiore Medical Center, Bronx 
      Comprehensive Sickle Cell Center and Division of Hematology, Bronx, NY, USA. 
      rschwart@worldnet.att.net
FAU - Musto, S
AU  - Musto S
FAU - Fabry, M E
AU  - Fabry ME
FAU - Nagel, R L
AU  - Nagel RL
LA  - eng
GR  - HL38655/HL/NHLBI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Blood
JT  - Blood
JID - 7603509
RN  - 0 (Buffers)
RN  - 0 (Carrier Proteins)
RN  - 0 (Chlorides)
RN  - 0 (Enzyme Inhibitors)
RN  - 0 (Symporters)
RN  - 142M471B3J (Carbon Dioxide)
RN  - 8L70Q75FXE (Adenosine Triphosphate)
RN  - 9034-63-3 (Fetal Hemoglobin)
RN  - RWP5GA015D (Potassium)
RN  - S88TT14065 (Oxygen)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Adenosine Triphosphate/deficiency/physiology
MH  - Anemia, Sickle Cell/*pathology
MH  - Buffers
MH  - Calcium/pharmacology
MH  - Carbon Dioxide/metabolism
MH  - Carrier Proteins/physiology
MH  - Centrifugation, Density Gradient
MH  - Chlorides/physiology
MH  - Enzyme Inhibitors/pharmacology
MH  - Erythrocytes/drug effects/metabolism/*pathology
MH  - Fetal Hemoglobin/analysis
MH  - Humans
MH  - Hydrogen-Ion Concentration
MH  - Ion Transport/drug effects
MH  - Oxygen/metabolism
MH  - Potassium/physiology
MH  - Specific Gravity
MH  - *Symporters
MH  - Time Factors
MH  - K Cl- Cotransporters
EDAT- 1998/12/09 00:00
MHDA- 1998/12/09 00:01
CRDT- 1998/12/09 00:00
PHST- 1998/12/09 00:00 [pubmed]
PHST- 1998/12/09 00:01 [medline]
PHST- 1998/12/09 00:00 [entrez]
AID - S0006-4971(20)57713-6 [pii]
PST - ppublish
SO  - Blood. 1998 Dec 15;92(12):4844-55.