Studies on the effect of vanadate on endocytosis and shape changes in human red blood cells and ghosts.

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.

Endo- and exovesiculation and the structure of the human red cell membrane.

Experiments were designed to study functional associations of proteins in human red cell membranes as the membranes are induced to undergo the critical membrane events of invagination or evagination followed by constriction and fusion. Three examples were chosen for study: the inside-out vesicle (IOV) produced in white ghosts by hypotonic removal of cytoskeletal proteins; the endocytic vacuole produced in white ghosts by incubation with Mg-adenosine triphosphate; and the exocytic vesicle produced by metabolic depletion of intact red blood cells. The resulting particles were harvested, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for membrane protein content and by enzymic analysis to detect the presence or absence of the exofacial enzyme acetylcholinesterase (ACE), the cytosol facing enzyme glyceraldehyde phosphate dehydrogenase (GAPD), and the integral protein adenosine triphosphatases (ATPases). Each of the vesicles is variably depleted of spectrin and actin, and each retains the exofacial enzyme ACE as well as Mg-ATPase. These findings suggest that there must be local partial depletion of cytoskeletal proteins before invagination or evagination occurs and that in each case part of the exoface of the membrane containing ACE is carried along into the resulting vesicle. The two forms of endocytosis differ with regard to their ATPase content with the energized endocytic vacuole retaining Ca-Mg-ATPase and actin-activated ATPase. The large amount of hemoglobin present in the exocytic vesicle is best explained by trapping of free cytosol and probably reflects a direct interaction of cytosolic components containing hemoglobin with the phospholipid bilayer.

Drug-induced endocytosis of neonatal erythrocytes.

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.

Red blood cell membrane abnormalities during storage: correlation with in vivo survival.

Experiments with citrate-phosphate-dextrose supplemented with adenine, 0.5 mM final concentration (CPD-A2), provided an opportunity for study of red blood cells (RBC) membrane storage lesions which could limit extension of storage. Impairment in drug-induced endocytosis in intact RBC occurred with storage, but the changes produced did not quantitatively predict or reflect the 24-hour in vivo 51Cr-labelled RBC survival. Abnormalities in RBC membrane protein did appear after storage, but these alterations neither paralleled nor predicted the in vivo 24-hour 51Cr-labelled RBC survival. The RBC membrance protein changes were not reminiscent of those produced by either acute adenosine triphosphate depletion, oxidative attack, or calcium accumulation. Therefore, while storage produces significant alterations in RBC membrane protein and function, the changes detected were not those that determined in vivo RBC survival.

Calcium distribution within human erythrocytes.

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.

Calcium distribution within human erythrocytes during endocytosis.

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.

Analysis of human erythrocyte membrane vesicles produced by shearing.

Shearing of ghosts in a French pressure cell produces three classes of microvesicles that differ from endocytic vacuoles, exocytic vacuoles, and inside-out vesicles. It was thought that an analysis of these vesicles might provide some clues about the assembly of proteins within the human erythrocyte membrane. The microvesicles were separated into three visible bands, labeled top, middle, and bottom, and assayed for activity of Mg++-ATPase, Na+,K+-ATPase, acetylcholinesterase, glyceraldehyde-phosphate dehydrogenase, and NADH oxidoreductase. Their proteins were also characterized by polyacrylamide gel electrophoresis with both Coomassie blue staining, to assess total protein content and distribution, and PAS-staining, to characterize sialoglycopeptides. In order to minimize problems inherent in ghost preparation, Dodge or hypotonic ghosts and glycol or isotonic ghosts were used in all studies. Middle membrane vesicles most resembled intact ghosts. Top vesicles had reduced levels of NADH oxidoreductase and more PAS-2 at the expense of PAS-1. The bottom vesicle class was very much enriched with PAS-1 at the expense of PAS-2, and PAS-3 was completely absent. In addition bottom vesicles had highest NADH oxidoreductase activity but lowest activity of all the other enzymes measured. These vesicle classes could not have been produced by tangential shearing through the membrane, nor could radial shearing through a membrane in which all proteins were free to move laterally have accounted for the three discrete vesicle classes or for their different patterns of enzymes and proteins. The analysis of the microvesicles produced by shearing is most consistent with radial shearing through membranes where there may be fixed domains superimposed on the basic fluid-mosaic structure.

Red blood cell membrane storage lesion.

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.

Requirements of drug-induced endocytosis by intact human erythrocytes.

Study of drug-induced endocytosis in intact human erythrocytes continues to provide an opportunity for correlating membrane functions such as invagination and fusion with erythrocytic energetics and other determinants of plasma membrane function like Ca++. The studies reported indicate that high concentrations of vinblastine and chlorpromazine can produce endocytic vacuoles, albeit in reduced amounts, even in severely ATP depleted erythrocytes. In contrast, primaquine-induced endocytosis seems definitely dependent upon persistence of erythrocytic ATP stores. The ionophore mediated entry of Ca++ into erythrocytes potentiates primaquine endocytosis, inhibits vinblastine endocytosis, and has no regular effect on chlorpromazine endocytosis. Sodium lactate enhances primaquine endocytosis, probably by causing an increase in the entry of primaquine into erythrocytes. Cytochalasin B neither enhances nor inhibits erythrocytic endocytosis, thereby suggesting that microfibrils or analogues of microfibrils in erythrocytes are not involved in endocytosis. Cyclic nucleotide inhibition of endocytosis is confined to a very high concentration range of nucleotides in the medium. Primaquine and chlorpromazine endocytosis are inhibited by cyclic nucleotides as is vinblastine endocytosis.

Energized endocytosis in human erythrocyte ghosts.

The mechanism of endocytosis in resealed human erythrocyte ghosts was studied. The energy for endocytosis or micropinocytosis appears to be derived from Mg-ATP, and membrane internalization is preceded by activation of a membrane-associated Ca,Mg-ATPase and by the active efflux of Ca. Endocytosis, Ca,Mg-ATPase activity, and active Ca efflux all require the presence of Mg. Furthermore, these three phenomena, endocytosis, Ca,Mg-ATPase activity, and active Ca extrusion, all have a concentration dependence on Ca such that low concentrations stimulate and higher concentrations inhibit the phenomena. The optimal concentration of Ca is identical for endocytosis, active Ca efflux, and Ca,Mg-ATPase. Morphologic studies indicated that while active Ca efflux and activation of the Ca,Mg-ATPase activity occurred promptly upon onset of incubation, there was a significant time delay before endocytosis occurred, which suggests that endocytosis additionally involved a more slowly functioning mechanicochemical mechanism. Ruthenium red, a specific inhibitor of Ca,Mg-ATPase and Ca transport, inhibited endocytosis in a concentration-related manner. Prostaglandins E1 and E2 had no measurable effect on ghost endocytosis, active Ca efflux, or Ca,Mg-ATPase activity.

Characterization of erythrocyte membrane-associated enzymes (glyceraldehyde-3-phosphate dehydrogenase and phosphoglyceric kinase).

The purpose of our study was to determine why several of the glycolytic enzymes of the erythrocyte have the propensity for adhering to erythrocyte membranes while others do not. Two of these membrane-associated enzymes, glyceraldehyde phosphate dehydrogenase (GAPD) and phosphoglyceric kinase (PGK), have been shown to have controlling functions on intra-erythrocytic glycolysis and sodium-potassium transport. In order to test the hypothesis that the membrane-associated fraction of these enzymes consisted of isozymes with increased capacity to become membrane associated, the enzymes from cytosol or membrane sources were partially purified and characterized. Determination of molecular weight by gel filtration chromatography, measurement of certain kinetic parameters, and the curves relating to pH optimal activity indicated that there were no measurable differences between membrane and cytosol PGK and membrane and cytosol GAPD. It was possible to raise inhibitory antibodies to GAPD in certain species of mice, and these antibodies did not distinguish between GAPD isolated from either membrane or cytosol sources. GAPD was firmly bound to membranes and the membrane-associated fraction counted for approximately 60 per cent of total erythrocytic enzyme. Membrane-associated PGK was only loosely adherent, and accounted for only 1 per cent of the total erythrocytic enzyme. The reasons for membrane association have yet to be determined, but these inward facing membrane-associated enzymes appear to function by carrying the organization of the membrane into the cell interior.