Trifluoperazine inhibition of calmodulin-sensitive Ca2+ -ATPase and calmodulin insensitive (Na+ +K+)- and Mg2+ -ATPase activities of human and rat red blood cells.

Trifluoperazine dihydrochloride-induced inhibition of calmodulin-activated Ca2+ -ATPase and calmodulin-insensitive (Na+ +K+)- and Mg2+ -ATPase activities of rat and human red cell lysates and their isolated membranes was studied. Trifluoperazine inhibited both calmodulin-sensitive and calmodulin-insensitive ATPase activities in these systems. The concentration of trifluoperazine required to produce 50% inhibition of calmodulin-sensitive Ca2+ -ATPase was found to be slightly lower than that required to produce the same level of inhibition of other ATPase activities. Drug concentrations which inhibited calmodulin-sensitive ATPase completely, produced significant reduction in calmodulin-insensitive ATPases as well. The data presented in this report suggest that trifluoperazine is slightly selective towards calmodulin-sensitive Ca2+ -ATPase but that it is also capable of inhibiting calmodulin-insensitive (Na+ +K+)-ATPase and Mg2+ -ATPase activities of red cells at relatively low concentrations. Thus the action of the drug is not due entirely to its interaction with calmodulin-mediated processes, and trifluoperazine cannot be assumed to be a selective inhibitor of calmodulin interactions under all circumstances.

Effects of calcium and soluble cytoplasmic activator protein (calmodulin) on various states of (Ca2+ + Mg2+)-ATPase activity in isolated membranes of human red cells.

(Ca2+ + Mg2+)-ATPase activity of red cells and their isolated membranes was investigated in the presence of various Ca2+ concentrations and cytoplasmic activator protein. Red cell ATPase activity was high at low Ca2+ concentrations, and low at moderate and high concentrations of Ca2+. In the case of isolated membranes, both low and moderate ca2+ concentrations produced higher (Ca2+ + Mg2+)-ATPase activity than high Ca2+ concentration. Membrane-free hemolysate containing soluble activator of (Ca2+ + Mg2+)-ATPase produced a significant increase in (Ca2+ + Mg2+)-ATPase activity only at low ca2+ concentration. Regardless of Ca2+ and activator concentrations, the enzyme activity in the membrane was lower than lysed red cells. The low level of (Ca2+ + Mg2+)-ATPase activity seen at high Ca2+ concentration can be augmented by lowering the Ca2+ concentration of EGTA in the assay medium. However, once the membrane was exposed to a high Ca2+ concentration, the activator could no longer exert it maximum stimulation at the low Ca2+ concentration brought about by addition of EGTA. This loss of activation was not attributable to the Ca2+-induced denaturation of activator protein but rather related to the alteration of (Ca2+ + Mg2+)-ATPase states in the membrane. On the basis of these data, it is suggested that only a small portion of (Ca2+ + Mg2+)-ATPase activity of isolated membranes can be stimulated by the soluble activator and that (ca2+ + Mg2+)ATPase most likely exists in various states depending upon ca2+ concentration and the presence of activator. The enzyme state exhibiting the high degree of stimulation by activator may undergo irreversible damage in the presence of high Ca2+ concentrations.

(Ca2+ + Mg2+)-ATPase of density-separated human red cells. Effects of calcium and a soluble cytoplasmic activator (calmodulin).

The effect of calcium and a soluble cytoplasmic activator on (Ca2+ + Mg2+)-ATPase of density-separated human red cells was investigated. At all calcium concentrations tested, dense (old) lysed cells and their isolated membranes displayed lower activities as compared to the light (young) cells and their membranes. Isolated membranes from all density red cell fractions showed two distinct (Ca2+ + Mg2+)-ATPase activities; one at low calcium and another at moderate calcium concentrations. At high calcium concentration, (Ca2+ + Mg2+)-ATPase activity of isolated membranes was low in all cell fractions. In contrast to the isolated membranes, lysed cells from all density fractions had a maximum (ca2+ + Mg2+)-ATPase activity only at a low concentration of calcium, while moderate and high calcium concentrations produced low activity. Upon isolation of membranes, a substantial loss of (Ca2+ + Mg2+)-ATPase activity took place from all density cell fractions. Upon membrane isolation, the relative loss of (Ca2+ + Mg2+)-ATPase activity at low Ca2+ concentration was greater in older cells. The extent of stimulation of (Ca2+ + Mg2+)-ATPase by the activator at low calcium concentration was 3-4-fold greater in older cell membranes than in the young ones. These data suggest that the lower (Ca2+ + Mg2+)-ATPase activity in old cells could be accounted for by a selective loss of (Ca2+ + Mg2+)-ATPase activity at low Ca2+ concentration presumably due to reduced affinity of old cell membranes to activator protein.

Studies on an activator of the (Ca2+ plus Mg2+)-ATPase of human erythrocyte membranes.

1. An activator of the (Ca2+ plus Mg2+)-stimulated ATPase present in the human erythrocytes (membrane) has been isolated in soluble form from hemolysates of these cells. Partial purification has been achieved through use of carboxymethyl-Sephadex chromatography. The resulting activator fraction contained no hemoglobin and only 0.3% of the total adenylate kinase activity of the cell. 2. Whereas the activator was released from erythrocytes subjected to hemolysis in 20 miosM buffer at pH 7.6 or at pH 5.8, only the membranes prepared at pH 7.6 were affected by it. 2. Whereas the activator was released from erythrocytes subjected to hemolysis in 20 miosM buffer at pH 7.6 or at pH 5.8, only the membranes prepared at pH 7.6 were affected by it. 3. When (Ca2+ plus Mg2+)-ATPase activity was measured by 32Pi release from (gamma-32P)ATP, freeze-thawed erythrocytes, as well as membranes prepared at pH 5.8 and at pH 7.6, expressed lower values than noted by assay for total Pi release. When ADP instead of ATP was used as substrate, significant amount of Pi were released by these erythrocyte preparations. Further study revealed (a) production of ATP and AMP from ADP with membranes and hemolysate alone, and (b) exchange of the gamma-and B-position phosphate on (gama-32P)ATP in the presence of membranes plus hemolysates. These observations established the presence of adenylate kinase activity in the (membrane-free) hemolysates and in membranes. It further supports the conclusion that Pi release from ADP by human erythrocytes (freeze-thawed) and by their isolated membranes is due to formation of ATP by adenylate kinase and hydrolysis of this generated ATP by (Ca2+ plus Mg2+)-ATPase. 4. The following points were also established: (a) absence of an ADPase in human erythrocytes; (b) the (Ca2+ plus Mg2+)-ATPase activator enhanced cleavage only of the gama-position of ATP and (c) the (Ca2+ plus Mg2+)-ATPase activator is neither adenylate kinase nor hemoglobin.

Calmodulin. An activator of human erythrocyte (Ca2+ + Mg2+)ATPase phosphorylation.

The effect of purified calmodulin on the calcium-dependent phosphorylation of human erythrocyte membranes was studied. Under the conditions employed, only one major peak of phosphorylation was observed when solubilized membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of this phosphorylated protein band was estimated to be 130000 and in the presence of purified red blood cell calmodulin, the rate of phosphorylation of this band was increased. These data suggest that calmodulin activation of (Ca2+ + Mg2+)-ATPase could be a partial reflection of an increased rate of phosphorylation of the (Ca2+ + Mg2+)-ATPase of human erythrocyte membranes.

Effects of Tris and histidine on human erythrocytes and conditions influencing their mode of action.

1. The incubation of human erythrocytes in 0.172 M Tris - HCl, pH 7.6 buffer at 37 degrees leads to (1) a pronounced cellular volume increase, (2) a preferential release of Na+, and (3) if continued sufficiently long, hemolysis. These effects are pH dependent and also are influenced to a considerable degree by such diverse reagents as NaC glucose, and histidine. In each instance, increasing levels of the latter compounds in a Tris - HCl incubation mixture led to diminished cellular volume increase and prolonged time of onset of hemolysis. 2. Histidine solutions of 0.31 M, pH 7.5 caused a rapid and dramatic decrease in cellular volume of human erythrocytes and a concomitant rapid exit of cations. However, in a prolonged incubation, human erythrocytes slowly regained their cell volume as a result of histidine entry into the cell. Of considerable interest: Tris swollen cells undergo immediate shrinkage to far below the initial cell volume when incubated in histidine at 37 degrees C. Through repetition of this process two additional times, as much as 90-95% of the total cellular Na+ and K+ was removed without hemolysis. 3. Human erythrocytes washed in 0.12 M MgCl2 and then suspended in 0.31 M histidine, pH 7.5, lost upwards of 60% of their total Na+ and 30% of their total K+ after a 40 min incubation at 37 degrees C. However, when increasing amounts of 0.172 M Tris - HCl, pH 7.6 were added to the histidine suspension of cells, the release of K+ was reduced to 5% but the release of Na+ decreased only to 40% of the total cellular level. On the basis of these observations, it is evident that Tris exerts a preferential activity towards the efflux of Na+ from the human erythrocyte, whereas histidine results in high efflux of K+ and Na+ from the cell.

Observations on the (Ca2+ plus Mg2+)-ATPase activator found in various mammalian erythrocytes.

1. A soluble activator of membrane (Ca2+ plus Mg2)-ATPase is present in hemolysates of the newborn calf and cow, the new born and adult pig as well as human erythrocytes. 2. The activator is also found in reticulocytes of the adult pig. 3. The activator obtained from any of the above species is capable of stimulating the membrane (Ca2+ plus Mg2+)-ATPases of the other species, regardless of the age of the animals. 4. The results obtained from density fractionation of human erythrocytes revealed that the soluble factor has little simulatory effect on membranes of young erythrocytes from which it is derived but caused a marked stimulation on (Ca2+ plus Mg2+)-ATPase activity of the intermediate aged and old erythrocyte membranes. 5. The above observations support the following conclusions: (a) the extremely low levels of (Ca2+ plus Mg2+)-ATPase in cow erythrocytes is not due to the lack of a (Ca2+ plus Mg2+)-ATPase activator; (b) the distribution of (Ca2+ plus Mg2+)-atpase activator is not species specific and the differences in the level of membrane (Ca2+ plus Mg2+)-ATPase activity in various species of cells is an inherent property of that particular membrane (c) the (Ca2+ plus Mg2+)-ATPase activator is present at least from the time of reticulocyte formation and remain during tthe life span of the erythrocyte.

Biochemical characterization of density-separated human erythrocytes.

A simple, reproducible method for the separation of human erythrocytes, described recently (Murphy, J. R. (1973) J. Lab. Clin. Med. 82, 334-341) has been utilized for the purpose of obtaining a wide range of biochemical data on these cells. Using phthalate ester density centrifugation of the fractions obtained by Murphy's method, we established that the cells were separated exclusively on the basis of their densities. Data on a wide range of biochemical and hematological parameters, when compared with previously reported density separation procedures showed that this simple technique can be used to fractionate the cells according to their densities (age) in their own plasma. Cells of increasing density consistently and reproducibly exhibited an increase in hemoglobin concentration, a moderate elevation in Na+ and a decrease in the following: K+, acetylcholinesterase, sialic acid, membrane protein, 2,3-diphosphoglycerate, ATP, cholesterol, phospholipid, mean corpuscular volume and critical hemolytic volume, However, no change in mean corpuscular hemoglobin was evident. The observed differences were not artifacts of the centrifugation process. This was determined in recentrifuged top fractions from which new top and bottom cells were obtained. The latter cells resembled the top fraction from which they were obtained, rather than the original bottom fraction. Whereas the parameters mentioned above exhibited consistency and reproducibility, such was not the case with the ATPase values. Depending on the cell density group examined and/or buffer as well as other conditions, significant variability in the activity levels of the ouabain sensitive, as well as the Ca2+ -stimulated ATPase, was observed. Use of these enzyme activities as indicators of cell age must be viewed with caution.

A symbiotic relationship of energy metabolism between a 'non-glycolytic' mammalian red cell and the liver.

The red cell of newborn pig loses the ability to carry out glycolysis within a month after birth. The metabolic energy source for this 'non-glycolytic' mammalian red cell is unknown. Hepatectomy of an adult pig results in the loss of red cell ATP with a characteristic half-time of 7--8 h which is identical to the rate with which ATP disappears in the pig cells under in vitro substrate-free incubation. Exposure of pig red cells with either normal or depleted levels of ATP to isolated hepatocytes causes a net synthesis of red cell ATP during a 12 h incubation. These findings suggest that a symbiotic relationship of energy metabolism may exist between the red cell and the liver of the pig.

Pig reticulocytes. III. Glucose permeability in naturally occurring reticulocytes and red cells from newborn piglets.

The loss of facilitated glucose transport of red cells occurring in the newborn pig was monitored in 11 density-separated cells from birth to a 4 wk of age. At birth there was a threefold increase in glucose permeability from the lightest cells to the most dense, suggesting that cells having progressively less glucose permeability are released into the circulation as gestation proceeds. Because of extraordinary stimulation of erythropoietic activity, the uppermost top fraction constituting 2-3 percent of the total cells is composed purely of reticulocytes in the growing animal. The glucose permeability of these reticulocytes which at birth has a slow but significant rate of 3.7 mumol/ml cell x min at 25 degrees C is rapidly decreased within 3-4 days to the level of reticulocytes produced in the adult in response to phenylhydrazine assault. Moreover, reticulocytes themselves discard their membrane permeability to glucose in the course of maturation to red cells. Thus, even though reticulocytes at birth are permeable to glucose, they will become red cells practically impervious to glucose within a few days. These findings suggest that the transition from a glucose- permeable fetal state to a glucose-impermeable postnatal state is brought about by two mechanisms: (a) dilution of fetal cells by glucose-impervious cells produced coincidentally with or shortly after birth; and (b) elimination of fetal cells, which have a shorter half-life, from the circulation.

Muscle glycolipids in inherited muscular dystrophy of chickens.

Gangliosides and neutral glycolipids of muscles from normal and dystrophic chickens were studied. Total glycolipid content of the degenerating muscles was higher than the normal muscles. In addition, the myopathic muscles contained a ganglioside which was absent in the unaffected muscles from normal and dystrophic chickens. Based on the thin-layer chromatographic mobility, treatment with neuraminidases from Vibrio cholerae and Arthrobacter ureafaciens, and reactivity of the asialo-derivative towards anti-ganglio-N-triaosylceramide antibody, the dystrophic-specific ganglioside was tentatively identified as GM2. Data obtained from young and old dystrophic chickens suggested a direct relationship of this ganglioside to muscular dystrophy.

Factors influencing osmotic fragility of red blood cells in Duchenne muscular dystrophy.

Factors affecting osmotic fragility were studied in red blood cells of patients with Duchenne muscular dystrophy. The mean osmotic fragility (MOF), operationally defined as the NaCl concentration for 50% hemolysis, was found to be higher by 3.63 +/- 0.51 mM in Duchenne cells than in normal cells having an MOF of 60.1 +/- 0.5 mM NaCl buffered with 10 mM sodium phosphate at pH 7.0. However, about 20% of Duchenne patients had red cells indistinguishable from their age- and sex-matched controls. Temperature, pH, preincubation in plasma, and proteolytic digestion all affected Duchenne and normal cells to the same extent. However, after salt loss, induced either by preincubation in isotonic nonelectrolyte solutions or by exposure to ionophore A23187, Duchenne cells showed a greater change in MOF. Osmotic fragility of Duchenne cells was increased even in younger blood cells, suggesting that the membrane was abnormal in the early stages of red cell maturation.

Enhanced T1 differentiation between normal and dystrophic muscles.

Proton spin-lattice relaxation times (T1) of pectoralis major muscles from normal (Line 412) and homozygous dystrophic (Line 413) chicks was measured by FONAR QED 80 at 1.69 MHz. The T1 values of dystrophic muscles (216.8 +/- 17.3 ms) was two-fold higher than those of normal muscles (110.2 +/- 8.1 msec). When these values were compared with the T1 values obtained at high frequencies (20 MHz and 32 MHz), the T1 differentiation between normal and dystrophic muscles was considerably enhanced at 1.69 MHz. Based on these results, we suggest that the high resolution of T1 obtained at low frequency (1.69 MHz) could be effectively used to detect the degenerative processes in muscles by the NMR techniques.

Evaluation of muscle degeneration in inherited muscular dystrophy by nuclear magnetic resonance techniques.

Nuclear magnetic resonance (NMR) techniques were applied to study the muscular dystrophy in chicks. The water proton spin-lattice relaxation times (T1) of fast, slow, and mixed muscles and plasma were measured. The T1 values of dystrophic pectoralis major and posterior latissimus dorsi (PLD) were significantly higher than those of the normal pectoralis and PLD muscles. The present results establish a direct relationship between the differences in T1 values and the severity of muscle degeneration. Consistent with this conclusion, it was also found that the T1 values of muscles unaffected in muscular dystrophy, namely, the gastrocnemius, and anterior latissimus dorsi (ALD), were not different between the normal and dystrophic chicks. Although the affected muscles of dystrophic chicks contained higher percent water and fat than those of normal chicks, the results show that the higher T1 values in dystrophic muscles were not solely due to variations in their water content. The increase in the T1 values is principally a result of altered interaction between cellular water and macromolecules in the diseased muscles. These data also point out the potential use of NMR imaging in evaluating muscle degeneration.

MicroRNA-196a targets annexin A1: a microRNA-mediated mechanism of annexin A1 downregulation in cancers.

Suppression of annexin A1 (ANXA1), a mediator of apoptosis and inhibitor of cell proliferation, is well documented in various cancers but the underlying mechanism remains unknown. We investigated whether decreased ANXA1 expression was mediated by microRNAs (miRNAs), which are small, non-coding RNAs that negatively regulate gene expression. Using Sanger miRBase, we identified miR-584, miR-196a and miR-196b as potential miRNAs targeting ANXA1. Only miRNA-196a showed significant inverse correlation with ANXA1 mRNA levels in 12 cancer cell lines of esophageal, breast and endometrial origin (Pearson's correlation -0.66, P=0.019), identifying this as the candidate miRNA targeting ANXA1. Inverse correlation was also observed in 10 esophageal adenocarcinomas (Pearson's correlation -0.64, P=0.047). Analysis of paired normal/tumor tissues from additional 10 patients revealed an increase in miR-196a in the cancers (P=0.003), accompanied by a decrease in ANXA1 mRNA (P=0.004). Increasing miR-196a levels in cells by miR-196a mimics resulted in decreased ANXA1 mRNA and protein. In addition, miR-196a mimics inhibited luciferase expression in luciferase plasmid reporter that included predicted miR-196a recognition sequence from ANXA1 3'-untranslated region confirming that miR-196a directly targets ANXA1. miR-196a promoted cell proliferation, anchorage-independent growth and suppressed apoptosis, suggesting its oncogenic potential. This study demonstrated a novel mechanism of post-transcriptional regulation of ANXA1 expression and identified miR-196a as a marker of esophageal cancer.