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Biomedical subjects

N I Swislocki

Publications and source records attributed to N I Swislocki.

At least 19 recordsLinked to original sources
PubMed1992

The effects of pentoxifylline on the plasma membrane Ca2+ ATPase in age-separated rat and human erythrocytes.

Pentoxifylline, a dimethyl xanthine derivative given to patients with peripheral vascular disorders, increases erythrocyte deformability, diminishes Ca2+ entry, inhibits Ca(2+)-dependent transglutaminase activity and elevates ATP levels. The present study examined the effects of pentoxifylline on the Ca2+ pump ATPase, an enzyme which regulates intracellular Ca2+ levels. Studies were carried out with inside-out vesicles (IOVs) prepared from young (Ey) and old (Eo) human and rat erythrocytes. The pumping of Ca2+ depends on the concomitant hydrolysis of ATP; the two processes were measured using radiolabeled substrates. The catalytic properties of IOVs from young and old erythrocytes were stimulated by pentoxifylline when added directly to the assay medium. Pentoxifylline (0.5 to 5.0 mM) significantly activated the rates of Ca2+ dependent ATP hydrolysis in Ey and Eo IOVs of rat erythrocytes. The percent of activation was greater in the IOVs from older erythrocytes. The Ca2+ translocation was also affected by pentoxifylline. The early burst of Ca2+ uptake into IOVs decreased in the presence of pentoxifylline in Ey IOVs prepared from either species whereas steady state rates of Ca2+ transport only declined at 5.0 mM pentoxifylline. This pattern was not evident in the corresponding Eo IOVs. The response of Ey and Eo IOVs to pentoxifylline may be the basis of the difference in sensitivity of young and old erythrocytes to the drug in regulating intracellular Ca2+ concentrations.

Animals↗
PubMed1991

A 20 kDa erythrocyte membrane phosphoprotein.

A small (approximately 20 kDa) protein associated with the human erythrocyte membrane undergoes phosphorylation that is potentiated by the addition of phosphatidylinositol. It is indicated that phosphatidylinositol 4.5-bisphosphate, generated in situ during the protein phosphorylation reaction. is the effective agent. Phosphorylation of the small protein is also potentiated when isolated membranes are exposed to conditions that promote the phosphorylation of the phosphatidylinositols. It is suggested that the 20 kDa membrane protein participates in biochemical signaling in the erythrocyte.

Erythrocyte Membrane↗
PubMed1991

Ca2+ transport activities of inside-out vesicles prepared from density-separated erythrocytes from rat and human.

The plasma membrane Ca2+ ATPase in erythrocytes is vital for the maintenance of intracellular Ca2+ levels. Since the cytoplasmic Ca2+ concentration is elevated in older erythrocytes, the properties of the Ca2+ transport ATPase were examined during cell aging using inside-out vesicles (IOVs) prepared from density-separated, young (less dense, Ey) and old (more dense, Eo) rat and human erythrocytes. The transport of Ca2+ and the coupled hydrolysis of ATP were measured using radiolabeled substrates. The calmodulin-independent Ca2+ transport activity (Ey, 38.8 vs. Eo, 23.3 nmols/min/mg IOV protein) and the Ca2+ dependent ATP phosphohydrolase activity (Ey, 53.5 vs. Eo, 48.8 nmols/min/mg protein) were greater in IOVs prepared from younger (less dense) rat erythrocytes. The calmodulin-independent Ca2+ transport activity in IOVs from human erythrocytes was 12.9 nmols/min/mg IOV protein for Ey and 10.7 nmols/min/mg IOV protein for Eo. Inside-out vesicles from older (more dense) cells exhibited a lower pumping efficiency as determined by the calculated stoichiometry, molecule of Ca2+ transported per molecule of ATP hydrolyzed (rat: Ey, 0.74 vs. Eo, 0.49; human: Ey, 1.22 vs. Eo, 0.77). The enzymatic activity of rat and human Ey IOVs was labile. The Ca2+ transport activity in Ey but not Eo IOVs rapidly declined during cold storage (4 degrees C). The decrease in Ca2+ transport activity during aging may accentuate the age-related decline in several erythrocytic properties.

Acetylcholinesterase↗
PubMed1991

A p-nitrophenylphosphatase activity associated with the human erythrocyte membrane.

A p-nitrophenylphosphatase activity has been identified as a component of the human erythrocyte membrane. This activity is distinct from that associated with the cell's Na(+)+K(+)-dependent ATPase, Ca(2+)-dependent ATPase, or spectrin phosphatase. The activity described here is stimulated by Mn2+ but not by Ca2+ with or without calmodulin. A potential erythrocyte membrane substrate for this activity is a 95 kDa phosphoprotein that can be shown to undergo Mn(2+)-stimulated but not Mg(2+)-stimulated dephosphorylation.

4-Nitrophenylphosphatase↗
PubMed1991

Prevention by pentoxifylline of transient Ca2+ accumulation and transglutaminase activation in rat erythrocytes.

The authors have examined the effects of pentoxifylline, a drug used to improve peripheral blood flow in patients with vascular disorders, on shear-induced periodic Ca2+ entry and its consequences in the rat erythrocyte. To study the effects of periodic Ca2+ entry on Ca2+ dependent processes, erythrocytes, with and without pentoxifylline, were subjected to rotational shear produced by swirling-cell suspensions in an isosmotic medium for 5-second intervals. Pulses of increasing duration from 5-30 seconds promoted increased accumulation of 45Ca2+; intermittent 5 sec pulses, at 10-minute intervals, produced a stepwise accumulation of 45Ca2+. Ca2+ accumulation was accompanied by elevated Ca2+ dependent transglutaminase. Shear induced Ca2+ entry and the increase in transglutaminase activity was reduced in cells treated with pentoxifylline. These findings suggest that pentoxifylline can reduce shear induced periodic Ca2+ entry that leads to transient activation of Ca2+ dependent transglutaminase, accumulation of crosslinked proteins, and loss of erythrocyte deformability.

Animals↗
PubMed1989

The incidence of decreased red blood cell deformability in sepsis and the association with oxygen free radical damage and multiple-system organ failure.

We studied the incidence of decreased red blood cell deformability (RBCD) in sepsis and the association between decreased RBCD and oxygen free radical generation (as measured by malonyldialdehyde) and the occurrence of multiple-system organ failure (MSOF). Patients were divided into three groups: septic (n = 28), nonseptic (n = 15), and control (n = 5). Serial measurements of deformability index, malonyldialdehyde, and MSOF were made. The deformability index for the septic group (0.23 +/- 0.17) was significantly less than that for the nonseptic (1.12 +/- 0.48) and control (1.11 +/- 0.25) groups. The malonyldialdehyde levels for the septic group (4.5 +/- 1.0 nmol/mL) were significantly greater than those for the nonseptic (2.7 +/- 0.9 nmol/mL) and control (2.6 +/- 0.8 nmol/mL) groups. The MSOF index for the septic group (10.1 +/- 2.5) was significantly greater than that for the nonseptic (7.6 +/- 1.7) and control (6.0 +/- 0.0) groups. An inverse correlation existed between malonyldialdehyde and deformability index ( = .501, less than .001, n = 40) and between deformability index and MSOF index (= .350, less than .05, n = 61). We conclude that RBCD is decreased during human sepsis; free radicals generated during sepsis may play a role in the decrease in RBCD. Decreased RBCD may contribute to the MSOF that occurs during sepsis.

Adult↗
PubMed1989

Different sensitivities of rat and human red cells to exogenous Ca2+.

During an examination of the effects of shear and of the Ca2+ ionophore A23187 on Ca2+ entry into erythrocytes of rats and humans, we noted that rat erythrocytes were much more sensitive to Ca2+-induced hemolysis than the human cells. An examination of the effect of Ca2+ on transglutaminase, a cytosolic enzyme in the erythrocyte which cross-links membrane proteins and renders cells less deformable, demonstrated a correlation between enzyme activity and Ca2+-induced hemolysis. Both rat and human cells subjected to shear-induced Ca2+ entry exhibited increased enzyme activity and altered membrane protein SDS-PAGE patterns. Twenty micromolar A23187 with Ca2+ at concentrations above 80 microM caused hemolysis of rat erythrocytes. In contrast to human erythrocytes, under these conditions no membranes were recoverable from rat erythrocytes. At lower concentrations of Ca2+ (25 and 50 microM), however, rat erythrocytes maintained integrity, and exhibited enhanced transglutaminase activity and cross-linking of membrane proteins. The rat enzyme can be activated 30% by 10 microM Ca2+, while 50 microM Ca2+ was necessary to achieve a similar activation of the enzyme from human red blood cells. In studies of shear-stimulated Ca2+ uptake by erythrocytes the rat red cell enzyme was more readily activated. The SDS-PAGE pattern of rat red cell membranes after a 30 sec shear showed specific changes in protein banding, including the appearance of bands greater than 330 kDa. Changes in protein banding were also apparent in cytosolic proteins. This work supports the view that shear-induced Ca2+ entry activates transglutaminase that leads to cross-linking of membrane components, a loss of cell integrity, and eventual cell death.

Animals↗
PubMed1989

Effects of pentoxifylline on Ca2+-dependent transglutaminase in rat erythrocytes.

We have examined the effects of pentoxifylline, a drug used to improve peripheral blood flow in patients with chronic vascular disorders, on shear-induced Ca2+ entry and Ca2+-dependent protein crosslinking in the erythrocyte. Cells were washed in isosmotic bicarbonate buffer, pH 7.5, and brought to a 20% suspension. 5 ml samples were sheared by swirling them for seconds; 45Ca2+ uptake, activation of Ca2+-dependent transglutaminase, and products of the crosslinking reaction were measured. A 5 second shear promoted 45Ca2+ entry, activation of Ca2+-dependent transglutaminase and crosslinking of proteins in the membrane-cytoskeletal fraction. Pentoxifylline, a drug that promotes erythrocyte deformability, diminished Ca2+ entry and inhibited activation of Ca2+-dependent transglutaminase, and had a prophylactic role on the effects of Ca2+ entry due to shear. Incubation of cells with 2.5 mM pentoxifylline before swirling minimized the effects of shear on 45Ca2+ entry, Ca2+-dependent transglutaminase and crosslinking of proteins. This study indicates that pentoxifylline promotes erythrocyte flexibility by minimizing shear-induced Ca2+ entry and Ca2+-dependent crosslinking.

Animals↗
PubMed1986

Contamination of commercial preparations of calmodulin by phospholipase A2.

In the course of studies of the possible regulation of cellular phospholipase A2 activities by calcium and calmodulin, it was observed that some of the commercial preparations of calmodulin contained significant phospholipase A2 activity. Six commercially available calmodulin sources were compared for the presence of contaminating phospholipase A2 activity, relative purity by SDS-gel electrophoresis, and relative biological activity in stimulating calmodulin-deficient phosphodiesterase. One of the commercial calmodulin sources contained a relatively high specific phospholipase A2 activity (1.30 +/- 0.11 nmol [1-14C]arachidonic acid released/mg protein per h) and yielded two major bands in SDS-gel electrophoresis. Two of the calmodulin sources tested were relatively free of phospholipase A2 activity, were quite pure (one band on SDS-gel) and had high biological activity in stimulating calmodulin-deficient phosphodiesterase. Thus, investigators using commercially available preparations of calmodulin should be aware of the contamination of some of these sources by phospholipase A2 activity. These findings may be of importance to investigators considering the role of calmodulin in activating a variety of calcium-dependent enzymes, including phospholipase A2.

Animals↗
PubMed1985

The effects of pentoxifylline on rat erythrocytes of different age.

Age-separated rat erythrocytes were exposed to pentoxifylline, a dimethylxanthine derivative which increases erythrocyte deformability. A comparison of drug-induced effects in young and old erythrocytes yielded age-specific alterations in: (1) accumulation of intracellular Ca2+; (2) membrane protein phosphorylation; (3) ATP concentrations; and (4) membrane associated protein kinase activity. The effect of Ca2+ accumulation and membrane protein phosphorylation appears to be biphasic. Low drug concentrations (0.5-2.5 mM) reduced intracellular Ca2+ and increased membrane protein phosphorylation, whereas higher concentrations (4.0-5.0 mM) increased Ca2+ levels and reduced membrane protein phosphorylation. Young cells exhibited increased ATP levels over the whole range of pentoxifylline tested; however, older erythrocytes demonstrated higher ATP levels at 5.0 mM drug only. Membrane-associated protein kinase activity was enhanced 10% in young erythrocytes at 1.0 mM pentoxifylline and decreased to 30% of control values at 4.0 and 5.0 mM drug. Protein kinase of old erythrocytes exhibited gradual inhibition over the entire drug concentration range. In general, younger erythrocytes appear to be more responsive to pentoxifylline exposure. Based on these studies, it appears that the ageing of the erythrocyte and loss of deformability in vivo may be a consequence of increased Ca2+ entry into the cell.

Animals↗
PubMed1984

Solute-excluded volumes near the Novikoff cell surface.

A differential centrifugation technique, in which all extracellular water except that intimately associated with the cell (pericellular domain) is removed, has been applied to isolated Novikoff hepatoma cells. The pericellular volumes accessible to albumin, inulin, raffinose, and sucrose were inversely related to the molecular weights of the test solutes. This phenomenon was not detectable in erythrocytes or in fat cells. Selective removal of cell surface components by enzymatic treatment produced proportional changes in the relative volumes of distribution accessible to the solutes. This discrimination in the volume accessible to each of the solutes is analogous to that obtained in gel chromatographic separation and represents, in effect, excluded volumes which are inversely related to solute size. This exclusion is associated with components of the Novikoff cell surface, including the surface coat and the microvilli that cover the Novikoff cell. These structures provide an additional level of discrimination for the Novikoff cell not seen in certain other cell types.

Animals↗
PubMed1983

Modulation of the activity of the (Ca2+ + Mg2+)-dependent adenosine triphosphatase of the human erythrocyte.

We previously reported that the activity of the (Ca2+ + Mg2+)-dependent adenosine triphosphatase (ATPase) of the human erythrocyte membrane is inhibited by micromolar or nanomolar concentrations of cyclic AMP. Our further studies have now indicated that the inhibition of (Ca2+ + Mg2+)-dependent phosphohydrolase activity requires the participation of a membrane-associated cyclic AMP-dependent protein kinase and a membrane-associated protein substrate that is distinct from the ATPase itself. We have furthermore, identified a 20 kDa membrane protein which undergoes phosphorylation that is promoted by micromolar, but not millimolar, concentrations of cyclic AMP and which, when phosphorylated, undergoes dephosphorylation that is promoted by Ca2+. We suggest that this membrane component can participate in the modulation of the activity of the (Ca2+ + Mg2+)-dependent ATPase of the human erythrocyte.

Ca(2+) Mg(2+)-ATPase↗
PubMed1983

Spectrin phosphorylation in senescent rat erythrocytes.

The rates of phosphorylation and dephosphorylation of the erythrocyte cytoskeletal protein, spectrin, were analyzed in young and old rat erythrocytes. Endogenous membrane protein kinase activity was measured in age-separated rat erythrocytes, and was found to decrease as a function of cell age. Membranes prepared from young and old erythrocytes contained comparable levels of protein phosphatase activity. Spectrin phosphatase activity was readily observed in erythrocyte membranes. Partially purified spectrin kinase and spectrin were prepared from membranes obtained from young and old erythrocytes, and the phosphorylation of the spectrin fractions was measured with the isolated kinases. The kinases prepared from young or old cells phosphorylated spectrin from young cells to the same extent. When spectrin from old cells was used as the substrate, it was phosphorylated ten-fold less extensively by the spectrin kinase prepared from old cells than by the spectrin kinase from young cells. This finding indicated that the decreased phosphorylation of spectrin observed in membranes prepared from age-separated red cells was due to a structural alteration in the spectrin. A structural basis for the decreased phosphorylation of spectrin in older erythrocytes was sought. Treatment of erythrocyte membranes with malonyldialdehyde, a product of lipid peroxidation which accumulates in erythrocyte membranes during senescence, adversely affected spectrin phosphorylation. The results presented here indicate that intramolecular derivatization of spectrin was sufficient to impair its function as a substrate for protein kinase.

Animals↗
PubMed1982

Role of peroxidation in erythrocyte aging.

The role of membrane peroxidation in red cell aging was investigated by exposing rat erythrocytes to a peroxide-generating system of xanthine oxidase with hypoxanthine, and the resulting alterations were compared to those observed during in vivo aging. Erythrocyte incubation with peroxides decreased the activities of adenylate cyclase and protein kinase, enzymes which were found to be reliable markers reflecting differences between young and old cells. Membranes from age-separated erythrocytes were solubilized and subjected to electrophoresis on gradient polyacrylamide-sodium dodecyl sulfate gels. Electrophoretic patterns indicated that old erythrocytes contained higher amounts of several membrane proteins than young cells. These were proteins in bands 2.1 and 4.1, and proteins of molecular weight larger than 250,000. There was a relative increase in the amount of proteins in band 4.1 and the high molecular weight (greater than 250,000) proteins as a results of peroxidation. These studies demonstrate that cells incubated in vitro with a peroxide-generating system resemble senescent erythrocytes in membrane enzyme activities, cell density, and membrane protein composition. We propose that peroxidation plays an important role in red cell aging.

Adenylyl Cyclases↗
PubMed1981

Mechanism of molybdate activation of adenylate cyclase.

Molybdate activation of rat liver plasma membrane adenylate cyclase has been examined and compared with the effect of glucagon, Gpp(NH)p and fluoride. Glucagon does not stimulate the detergent solubilized enzyme though molybdate, fluoride, and Gpp(NH)p are effective in this regard. The stimulatory effects of either fluoride or molybdate are additive with those of GTP and do not require guanyl nucleotide to evoke their activation. Neither fluoride nor molybdate can substitute for GTP when glucagon is the activator of rat liver adenyl cyclase. The stimulatory effects of either ion on adenylate cyclase are additive with that produced by glucagon. Activation of adenylate cyclase by either molybdate or fluoride occurs by a mechanism distinct from that of glucagon or guanyl nucleotide. The data presented her suggest that fluoride and molybdate may act via in a similar mechanism of action. Neither ion displays a lag in activation of adenylate cyclase. The pH profiles of fluoride and molybdate-stimulated adenylate cyclase activity are similar, and distinct from guanyl nucleotide-stimulated activity. Cholera toxin treatment of adenylate cyclase blocks fluoride and molybdate stimulation of the enzyme to the same extent, while enhancing the activation obtained with GTP and hormones.

Adenylyl Cyclases↗