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.

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 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.

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.

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+.

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.

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.

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.

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.

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.

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.

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+.

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.

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.

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.

ATP-dependent activation of adenylate cyclase.

Incubation of rat liver plasma membranes with MgCl2, ATP, and an ATP-regenerating system at 4 degrees C provides a 4-7-fold persistent activation of adenylate cyclase. Enzyme activation is time-dependent and 48 h of incubation is usually required to achieve maximal stimulation of adenylate cyclase activity. The activation described is not affected by GTP, cAMP, or cGMP, and does not occur when ATP is replaced by a nonphosphorylating analogue, adenyl-5'-imidodiphosphate.

Activation of adenylate cyclase by molybdate.

The effect of molybdate on adenylate cyclase (EC 4.6.1.