Sulfhydryl reducing agents and shape regulation in human erythrocytes

Abstract

Metabolic crenation of red cells is reversible; on addition of nutrients, echinocytes recover the normal discoid shape. When the shape recovery takes place in the presence of reducing agents such as dithiothreitol (DTT), morphological change continues until the cells are stomatocytic. The degree of stomatocytosis varies, depending on the cell morphology when the nutrients and reducing agent are added. DTT has minimal effect on the shape of normal discocytes, but in its presence, mildly echinocytic cells become slightly cupped and advanced- stage echinocytes become severely stomatocytic. DTT must be present continuously for development and retention of stomatocytosis; echinocytes preincubated with or metabolically depleted in DTT do not become stomatocytic when supplemented in the absence of DTT, and DTT- induced stomatocytes revert to discocytes when the reducing agent is removed. DTT has no effect on adenosine triphosphate synthesis or equilibrium cell glutathione levels, and the induced stomatocytosis is not inhibited by excluding oxygen from cells during depletion. Spectrin phosphorylation and phosphate turnover are not affected by DTT. The echinocyte-to-discocyte transformation coincides with phosphorylation of membrane inner monolayer lipids (diacylglycerol to phosphatidic acid and phosphatidylinositol to phosphatidylinositol-4,5-bisphosphate). Overphosphorylation of these phospholipids is not responsible for the exaggerated shape recovery seen with reducing agents; phosphorylation of inner monolayer lipids proceeds identically in the presence and absence of DTT.

The basis of echinocytosis of the erythrocyte by glucose depletion

“…It is generally believed that the biconcave disc shape of the erythrocyte is maintained by adenosine triphosphate (ATP) on the basis of observations made more than 5 decades ago on the disc‐sphere transformation of erythrocytes, via echinocytosis, induced by the glycolytic inhibitor sodium fluoride (NaF; 20 mmol·l –1 ) in a physiological saline or by storage of acid citric dextrose blood at 4 °C over a long period, and on the shape transformation of round ghost cells obtained from long‐stored erythrocytes after the addition of ATP . However, the process by which ATP depletion leads to the disc‐sphere transformation still remains unknown in spite of continuous investigations over the years in which the disc‐sphere transformation was generally induced by glucose depletion in buffered saline . Indeed, it appears unrelated to any of the following: (i) active transport of Na + and K + by ouabain‐sensitive membrane Na + , K + ‐ATPase, opposed by Na + and K + leakage; (ii) Ca 2+ uptake, opposed by membrane Ca 2+ ‐ATPase; (iii) formation of lysophosphatidylcholine by a fatty acid deacylation of phosphatidylcholine, opposed by a ATP‐dependent fatty acid acylation; (iv) phosphorylation state of long and flexible filamentous spectrin, constituting 60% of the total proteins of the two‐dimensional reticulated membrane skeleton; (v) phosphorylation state of the major transmembrane protein Band 3 (AE1), exchanging anion chloride (Cl – ), and bicarbonate (HCO 3 – ) and binding to filamentous spectrin near its center by intermediary of ankyrin R; (vi) metabolism of phosphatidylinositides, constituting 1–2% of the total membrane phospholipids; (vii) contraction of the erythrocyte by myosin present at a relatively low cell number copies (~6000), presumably interacting with actin protofilament cross‐linking spectrin at its ends with Band 4.1R binding to glycophorin C .…”

“…Indeed, it appears unrelated to any of the following: (i) active transport of Na + and K + by ouabain‐sensitive membrane Na + , K + ‐ATPase, opposed by Na + and K + leakage; (ii) Ca 2+ uptake, opposed by membrane Ca 2+ ‐ATPase; (iii) formation of lysophosphatidylcholine by a fatty acid deacylation of phosphatidylcholine, opposed by a ATP‐dependent fatty acid acylation; (iv) phosphorylation state of long and flexible filamentous spectrin, constituting 60% of the total proteins of the two‐dimensional reticulated membrane skeleton; (v) phosphorylation state of the major transmembrane protein Band 3 (AE1), exchanging anion chloride (Cl – ), and bicarbonate (HCO 3 – ) and binding to filamentous spectrin near its center by intermediary of ankyrin R; (vi) metabolism of phosphatidylinositides, constituting 1–2% of the total membrane phospholipids; (vii) contraction of the erythrocyte by myosin present at a relatively low cell number copies (~6000), presumably interacting with actin protofilament cross‐linking spectrin at its ends with Band 4.1R binding to glycophorin C . Moreover, there are several observations suggesting that ATP is not the only determinant of the erythrocyte shape . It has been previously suggested that inorganic phosphate (P i ) also is a determinant of the erythrocyte shape .…”

“…The rationale of suggesting this process of echinocytosis is the following: (i) glucose depletion increases the Donnan ratio as a result of the catabolism of 2, 3‐BPG . (ii) Echinocytes begin to be observed only after a degradation of a large fraction of ATP (~50%), whereas ATP synthesis by metabolic substrates, accompanied by 2,3‐BPG synthesis, concomitantly reverses echinocytosis . (iii) The faster echinocytosis induced by the glycolytic inhibitor NaF in a physiological saline is uniquely caused by the cyclic process because NaF preferentially inhibits enolase, which reversibly transforms 2‐phosphoglycerate (2‐PG) to phosphoenolpyruvate, the substrate of pyruvate kinase, and 2‐PG is reversibly transformed to 3‐PG by phosphoglycerate mutase by the intermediary of coenzyme 2,3‐BPG.…”

“…These agents activate the pentose phosphate pathway by increasing the ratio of oxidized and reduced forms of coenzyme of nicotinamide adenine dinucleotide phosphate (NADP + /NADPH) of glucose‐6‐phosphate dehydrogenase, which catalyses the first and rate‐limiting step of this pathway. It is likely that the pentose phosphate pathway also contributes in the reversal by metabolic substrates of echinocytosis induced by glucose depletion because oxidized glutathione formed in echincocytosis can be reduced by glutathione reductase with the concomitant oxidation of the coenzyme NADPH to NADP + . Glutathione reductase also can reduce blood component lipoid acid .…”

“…Glutathione reductase also can reduce blood component lipoid acid . It may be that the acceleration of the reversal of echinocytosis or the reversal of echinocytosis to stomatocytosis by the addition of the strong reducing agent dithiothreitol (10 mmol·l –1 ) to the medium supplemented by metabolic substrates is caused by the reduction of the oxidized form of this agent by glutathione reductase. (viii) The suggested echinocytosis process also is not contradicted by the observation that echinocytosis of erythrocyte ghost membranes induced by buffered saline is reversed by Mg 2+ –ATP because ATP is hydrolysed to ADP, P i and H + by a vanadate‐sensitive membrane Mg 2+ –ATPase, which would promote an outward transport of P i by Band 3.…”