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Storage of human erythrocytes in CPD produced a lesion of the erythrocyte membrane manifested by abnormal endocytosis in resealed ghosts and in intact erythrocytes. Endocytosis produced by resealing Ca, Mg, and ATP into ghosts was impaired by five weeks of storage and this defect was promptly reversed by the prior regeneration of ATP in the stored erythrocytes. Drug-induced endocytosis was studied in intact stored erythrocytes. Vinblastine endocytosis was not affected by storage. Chlorpromazine endocytosis was not affected by storage. Chlorpromazine endocytosis was variably but never completely inhibited by storage. Chlorpromazine endocytosis was variably but never completely inhibited by storage, and restoration of ATP occasionally resulted in complete restoration of chlorpromazine endocytosis to base line values. However, primaquine endocytosis was usually totally inhibited after three to four weeks of storage at a time when residual ATP levels were 30 to 50 per cent of base line values. Restoration of ATP levels to at least base line values did not completely primaquine endocytosis to control values. Study of primaquine endocytosis provides an opportunity for defining an erythrocyte membrane storage lesion.

In order to study 45Ca distribution within erythrocytes, a method was devised that had minimal deleterious effects on the treated erythrocytes. It was observed that newly introduced 45Ca was predominantly recoverable from the cytosol and exchanged relatively slowly with membrane-associated Ca. Younger erythrocytes appeared to have relatively more 45Ca in membrane-associated sites. Erythrocytes from patients with sickle cell anemia had significantly more 45Ca in membrane-associated sites than did normal controls or patients with reticuloctosis due to a variety of disorders. There are theoretical reasons for considering the possibility that the distribution of 45Ca between cytosol and membrane-associated sites could modulate some of the properties of the erythrocyte membrane.

When amphipathic cationic drugs are added to intact human RBCs, the RBCs first undergo a stomatocytic shape change and then, if relatively large amounts of drug are added and if the metabolic state of the RBC is appropriate, endocytic vacuoles form. Vanadate has a structural similarity to the transition state of phosphate, which presumably accounts for its ability to inhibit phosphohydrolases, although other actions of vanadate have been described. Vanadate inhibited three forms of drug-induced endocytosis in intact RBCs despite the fact that the three drugs chosen (primaquine, chlorpromazine, and vinblastine) are known to have differing requirements for RBC ATP. Vanadate also inhibited the stomatocytic shape change produced by primaquine, chlorpromazine, and vinblastine, but not the stomatocytosis produced by low pH. Vanadate had no effect on RBC echinocytosis produced by lysophosphatidylcholine. In studying endocytosis in hypotonic, leaky, “white” ghosts, we discovered that vanadate inhibited only the endocytosis produced by Mg-ATP and not the endocytosis produced by manipulations that directly attack the cytoskeletal proteins. These findings suggest that ATP hydrolysis has a role in some forms of amphipathic cation-induced stomatocytosis and endocytosis in intact RBCs. In addition, studies in ghosts support the idea that Mg-ATP does indeed produce “energized” endocytosis dependent on utilization or hydrolysis of ATP.

Summary. There is impaired drug-induced vacuole formation in red cells from patients with hereditary spherocytosis (HS). The spherocytic shape per se accounts in part for the decreased vacuole formation seen, but there are constraints in HS red cell vacuole formation above that explicable by spheroidicity. Electron-microscopy indicates that the vacuoles produced in HS have one-third normal diameter. We propose that the decreased vacuole formation in HS is a result of a genetically determined abnormality of the membrane which limits its ability to invaginate and deform. The variability in vacuole formation from one HS family to another, but relative consistency within families, suggests that hereditary spherocytosis comprises a syndrome of related erythrocyte membrane disorders. Vacuole fromation in ghosts in normal in HS. This unanticipated finding suggests that the phenomenon of ghost endocytic vacuole formation may have requiements that by-pass the defect in the HS membrane.

The erythrocytes of the newborn infant have many properties that distinguish them from those of adults, and their membranes are also different from those of adult erythrocytes. We compared the ability of adult and neonatal RBCs to undergo endocytosis on exposure to drugs. Using a quantitative method, we showed that neonatal erythrocytes undergo a greater degree of endocytosis than do adult RBCs in response to primaquine, vinblastine, and chlorpromazine, and are sensitive to lower concentrations of the drugs. Some forms of drug-induced endocytosis are red cell age-dependent; when RBCs were separated by density gradient centrifugation, the membranes of the younger, less dense populations of both the neonatal and adult RBCs were capable of more extensive internalization than those of the denser, older RBCs. Neonatal RBCs of a given density undergo more endocytosis than do adult RBCs of the same density, suggesting that the membrane of the neonatal RBC is less stable and capable of more of the reorganization reflected in endocytosis than is the adult RBC membrane.

The ability of human RBC and their ghosts to undergo reversible shape changes as well as endocytosis and exocytosis raised the likelihood that contractile proteins like actomyosin might be present. Actin, a component of actomyosin, is a constituent of the red cell membrane. Two generally accepted criteria for identifying a myosin include the ability of purified preparations to form arrowhead complexes with F actin fibers visible in the electron microscope, and the presence of an unusual enzyme activity where a low level of Mg++-ATPase is markedly enhanced by addition of F actin, a reaction reflecting the physiologic interaction of actin and myosin. Our studies on a RBC myosin were therefore based on the use of the actin-activated ATPase (AA-ATPase) as a reliable marker of a myosin-like protein. RBC contain AA-ATPase, all of which was recoverable in the ghost or membrane fraction prepared by either hypotonic or isotonic lysis. AA-ATPase was optimally stimulated by human erythrocyte F actin as compared to rabbit muscle F actin or human erythrocyte G actin. In common with the myosin AA-ATPase of macrophages there was no saturation effect seen upon F actin addition. Myosin AA-ATPase of platelets and macrophages is inhibited when the protein is dephosphorylated. Dephosphorylation of the RBC membrane resulted in almost complete inhibition of AA-ATPase. Another myosin enzyme marker is a K+, EDTA-ATPase, which is also detectable in the red cell membrane. The AA-ATPase does not appear to be related to the other RBC membrane ATPases. Attempts to solubilize, isolate, and purify the AA-ATPase have thus far been unsuccessful; however, the AA-ATPase is not a peripheral membrane protein and no AA-ATPase is found in purified spectrin preparations. The presence of AA-ATPase and K+, EDTA-ATPase in the red cell membrane as well as several distinguishing characteristics of activation and inactivation of the AA-ATPase presumptively establishes the likelihood that there is a myosin-like protein (which is not spectrin) in the red cell membrane.

In order to study 45Ca movements within erythrocytes, a method was devised that had minimal deleterious effect on the treated erythrocytes. Agents that induce endocytosis in intact erythrocytes (primaquine, vinblastine, and chlorpromazine) caused a prompt movement of 45Ca from cytosol to membrane-associated sites. This drug-induced movement of 45Ca to membrane sites was blocked by depleting erythrocytes of adenosine triphosphate (ATP) or by incubating them with known inhibitors of endocytosis, NaF of N-ethylmaleimide (NEM). It appears that endocytosis in intact human erythrocytes involves the movement and redistribution of 45Ca from cytosol to membrane-associated sites. Therefore, in the erythrocyte, as in perhaps other cells, movement of Ca from one site to another may modulate important cellular biologic functions.