Stobadine: bellwether of a broader view of drug actions.

Stobadine was recognized early in its development as having antioxidant properties. A number of laboratories found associations between the antioxidant properties of stobadine and its potential beneficial effects. We found that stobadine acted as an antioxidant in a modification of an oxygen radical absorbance capacity (ORAC) assay. Similar results were observed with other drugs, including tirilazad and pramipexole. We suggest that stobadine and certain other drugs exhibit antioxidant properties in both hydrophilic and hydrophobic environments. Other drugs have been developed for their antioxidant properties and some currently marketed drugs have antioxidant properties. Although they may not have been explicitly sought during development, at least some of the beneficial effects may be related to antioxidant properties and/or scavenging of free radicals. Because stobadine was one of the first drugs for which useful properties were associated with its antioxidant actions, stobadine may be seen as a bellwether of a broader view of pharmacological actions--a view that encompasses antioxidant properties as a useful basis of therapeutic effects.

Inhibition of Ca2+-pump ATPase and the Na+/K+-pump ATPase by iron-generated free radicals. Protection by 6,7-dimethyl-2,4-DI-1- pyrrolidinyl-7H-pyrrolo[2,3-d] pyrimidine sulfate (U-89843D), a potent, novel, antioxidant/free radical scavenger.

Preincubation of red blood cell (RBC) membranes with a model system known to generate reactive oxygen species (ROS) and free radicals (200 microM ferrous sulfate and 200 microM EDTA, Fe2+/EDTA) resulted inhibition of the Na+/K+ -pump ATPases was also associated with membrane protein crosslinking and lipid peroxidation, the latter as monitored by the formation of thiobarbituric acid reactive substances (TBARS). Inhibition of the ion transport ATPases, protein cross-linking and formation of TBARS were prevented by U-89843D in a concentration-dependent manner, with half-maximal protection seen at 0.3 microM. U-89843D was more potent than the classical antioxidant butylated hydroxytoluene. Neither U-89843D nor the solvent DMSO had any effect on the assay of TBARS. U-89843D exerted only minimal inhibitory activity on ATPase activities. Thus, U-89843D was potent in vitro in preventing a variety of membrane-damaging reactions mediated by ROS. It is suggested that protection of membranes from ROS-mediated damage is of potential usefulness in the prevention and treatment of certain disease processes.

Inhibition by activated neutrophils of the Ca2+ pump ATPase of intact red blood cells.

Human neutrophils, activated by phorbol myristate acetate in the presence of intact red blood cells (RBCs), caused inhibition of the Ca2+ pump ATPase of the RBCs and fragmentation of the enzyme as well as other membrane proteins. Inhibition of the Ca2+ pump ATPase of intact RBCs was directly related to the neutrophil concentration and the time of incubation. Ca2+ pump ATPase activity was partially protected by the addition of exogenous glutathione-glutathione peroxidase, but not by superoxide dismutase. The addition of sodium azide, a potent inhibitor of endogenous RBC catalase, enhanced inhibition of the Ca2+ pump ATPase of intact RBCs. Examination by SDS-polyacrylamide gel electrophoresis of membrane proteins isolated from RBCs preincubated with activated neutrophils showed gross changes in banding patterns as compared to controls. Thus, a significant amount of methemoglobin appeared to be associated with the membrane proteins, and, in general, protein bands appeared to be more diffuse and less defined than proteins in control lanes. In addition, there was an increase in the low molecular weight protein bands. Using a monoclonal antibody to the Ca2+ pump ATPase, it was shown that the 140 kDa band representing the Ca2+ pump ATPase decreased, with concomitant appearance of two low molecular weight bands running at 8.2 and 6.8 kDa in the membrane proteins from RBCs preincubated with activated neutrophils. The data are interpreted to suggest that inhibition of the Ca2+ pump ATPase in intact RBCs under these conditions occurred as a result of: neutrophil-derived superoxide, dismutation of superoxide, to H2O2, diffusion of H2O2 into RBCs, a Fenton type reaction between oxyhemoglobin, and H2O2 producing hydroxyl radical and/or a ferryl radical capable of promoting protein fragmentation of RBC membrane proteins, including the plasma membrane Ca2+ pump ATPase.

Inhibition of the Ca pump of intact red blood cells by t-butyl hydroperoxide: importance of glutathione peroxidase.

Incubation of human red blood cells (RBCs) with t-butyl hydroperoxide (tBHP) resulted in inhibition of the Ca-pump ATPase. This was demonstrated using an assay of the Ca-pump ATPase activity in intact RBCs. In this assay, activity of the Ca-pump ATPase is expressed as the rate constant of the initial loss of ATP in RBCs exposed to Ca and A23187. Pseudo-first-order rate constants (Ca-pump ATPase rate constants) were lower in the presence of tBHP versus controls. Incubation of RBCs with tBHP resulted in both a time- and concentration-dependent inhibition of the Ca-pump ATPase (IC50 approximately 1 mM). Incubation of RBCs with tBHP also resulted in decreased oxyhemoglobin, increased methemoglobin and increased thiobarbituric acid reactive substances (TBARS). GSH levels were significantly lower in the presence of tBHP. GSH fell from a control value of 2.2 mmol/l RBC to 0.46 mmol/l RBC after incubation with 0.25 mM tBHP for 15 min. Both butylated hydroxytoluene and stobadine prevented the formation of TBARS and were partially effective in protecting the Ca-pump ATPase from tBHP-induced inhibition. Dithiothreitol was completely effective in preventing the tBHP-induced formation of TBARS as well as inhibition of the Ca-pump ATPase. However, when added after exposure to tBHP, dithiothreitol was unable to restore Ca-pump ATPase activity completely. An activity of dithiothreitol independent of enzymic thiol group reduction was apparent. In the presence of mercaptosuccinate, a potent inhibitor of glutathione peroxidase, the ability of dithiothreitol to protect the Ca-pump ATPase from tBHP-induced inhibition was abolished. Therefore, protection by dithiothreitol may be afforded by its ability to replenish GSH from oxidized glutathione, thus allowing glutathione peroxidase to metabolize tBHP. These results may be interpreted to suggest that inhibition of the Ca-pump ATPase in intact RBCs occurs as a result of tBHP-induced oxidant stress and subsequent lipid peroxidation which can be prevented by certain antioxidants including butylated hydroxytoluene, stobadine, and thiol-containing compounds such as dithiothreitol. These findings provide further insight into the mode of action of hydroperoxides and certain reactive oxygen species that have been implicated in oxidative stress associated with various pathological conditions. The importance of the GSH/glutathione peroxidase system in metabolizing organic hydroperoxides is also demonstrated.

Simultaneous measurement of multiple membrane ATPases in microtiter plates.

An assay for activities of erythrocyte membrane-bound ATPases adapted from the autoanalyzer method of Raess and Vincenzi (1980a) is described in detail. Mg2+ ATPase, Na+/K(+)-ATPase, Ca2+ ATPase, and calmodulin- (CaM) activated Ca2+ ATPases were determined in microtiter plates. Total volume was 100 microL. Ten microliters of 0.75 mg/mL of red blood cells membranes were added to appropriate buffer in microtiter plates. Plates were preincubated at 37 degrees C for 10 min, reactions were started by the addition of ATP, and plates were incubated for an additional 60 min at 37 degrees C. Reactions were stopped by sodium dodecyl sulfate (SDS). Inorganic phosphate (P(i)) was measured by modifications of the method by Fiske and Subbarow, (Fiske and Subbarow, 1925) in the same plate using a plate reader. The P(i) assay range was between 0 and 250 nm/mL. Results obtained for intraassay precision, (n = 7) are as follows: Mg2+ ATPase = 4.39 +/- 0.25 (5.7% CV); Na+/K(+)-ATPase = 7.33 +/- 0.40 (5.4% CV); Ca2+ ATPase = 15.86 +/- 0.76 (4.8% CV); and CaM-activated Ca2+ ATPase = 74.12 +/- 2.34 (3.2% CV) (nmole P(i)/mg protein/min.). This is a rapid, simple, and nonisotopic method for the determination of membrane-bound ATPases activities. All steps are performed in the same microtiter plate, thus reducing handling and associated errors.

Increased cytosolic free calcium in red blood cells is associated with essential hypertension in humans.

Intracellular free calcium (Cai2+) was measured in human red blood cells (RBCs) using the Ca(2+)-sensitive fluorescent probe, fluo-3. Fresh RBCs were loaded with the acetoxymethyl ester of fluo-3 (fluo-3-AM) in vitro. Following incubation at 37 degrees C for 60 min, cells were separated from nonincorporated fluo-3-AM and fluo-3. Fluorescence was quantified with excitation and emission wavelengths of 506 nm and 526 nm, respectively. The relatively long wavelength characteristics of fluo-3 avoid much of the interference from hemoglobin that makes agents such as quin-2 and fura-2 of limited value in RBCs. A protocol with corrections for quenching and for extracellular fluo-3 was employed for each sample. Cai2+ in red blood cells from 15 normotensive volunteers averaged 134 +/- 12 nmol/L. In RBCs from 30 hypertensive patients who were not treated pharmacologically, Cai2+ was 317 +/- 32 nmol/L (P < .05). In a group of 27 hypertensives who were treated with various drugs, RBC Cai2+ was 221 +/- 27 nmol/L. Measurements were also performed in RBCs from 16 patients before and at least 2 weeks following treatment with one or more antihypertensive agents. Reduction of RBC Cai2+ from 342 +/- 47 to 252 +/- 39 (P < .001) was observed in 16 patients whose blood pressure fell with treatment. Overall, there was a correlation between RBC Cai2+ and diastolic blood pressure (r = 0.413, P < .01, d.f. = 71). Thus, the present results confirm an association between elevated RBC Ca2+ and essential hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Ion transport ATPases as targets for free radical damage. Protection by an aminosteroid of the Ca2+ pump ATPase and Na+/K+ pump ATPase of human red blood cell membranes.

Preincubation of red blood cell membranes in the presence of ferrous sulfate and EDTA resulted in both a concentration- and time-dependent inhibition of the Na+/K+ pump ATPase, basal Ca2+ pump ATPase, and the calmodulin- (CaM) activated Ca2+ pump ATPase. The IC50 for all three ATPases was approximately 2.5 x 10(-5) M iron. The addition to membranes of ferrous iron and EDTA in an approximately 1:1 ratio resulted in conversion to the ferric iron form in several minutes. However, inhibition of the ion pump ATPases and cross-linking of membrane proteins occurred over the course of several hours. The time course of formation of thiobarbituric acid-reactive substances (TBARS) closely paralleled inhibition of the ion pump ATPases. Inhibition of the ion pump ATPases was prevented by the addition of deferoxamine or superoxide dismutase but not by mannitol, or catalase. Both butylated hydroxytoluene and tirilazad mesylate (U74006F) prevented the formation of TBARS, limited the inhibition of the ion pump ATPases, and reduced cross-linking of membrane proteins. These data may be interpreted to suggest that inhibition of ion pump ATPases in plasma membranes may occur as a result of iron-promoted formation of superoxide and subsequent lipid peroxidation, which can be prevented by free-radical scavengers including butylated hydroxytoluene and U74006F.

Osmotic stress in red blood cells from beagles with hemolytic anemia.

Red blood cell populations separated by density centrifugation were compared in a dynamic assay of osmotic stress. Red blood cells from Beagles genotypically normal and nonanemic (nonaffected), Beagles with inherited hemolytic anemia (anemic), and Beagles presumed to be carriers of the anemia trait (trait carriers) were examined for rate and extent of swelling after exposure to the ionophore A23187 in a medium containing calcium and potassium chloride. Comparisons were made between RBC populations separated on the basis of density. Significant differences were observed in the rates of cell swelling in RBC populations separated by density between nonaffected and anemic Beagles. The response of RBC from Beagles presumed to carry the anemia trait was similar to that of RBC from nonaffected dogs. One phenotypic expression of this inherited abnormality of RBC in Beagles was an accelerated rate of RBC swelling under osmotic stress, and this swelling response diminished with increasing RBC density.

Assay of the Ca pump ATPase activity of intact red blood cells.

An assay for the Ca pump ATPase of intact human red blood cells (RBCs) was developed. The assay utilized a small volume (typically 10 microliters) of packed RBCs in 1 ml of a buffer of known composition. The assay was based on the exposure of intact RBCs to the ionophore, A23187, in the presence of Ca. Such exposure caused a rapid degradation of ATP in RBCs. This degradation process is modeled in a numerical simulation in a companion paper (Vincenzi, F. F. and Hinds, T. R. (1992) Biochim. Biophys. Acta 1105, 63-70). The loss of ATP followed pseudo-first-order kinetics, and the rate constants for ATP degradation was taken as a measure of the capacity of the Ca pump ATPase. A number of variables were examined to optimize the activity of the ATPase. These variables included the concentrations of Ca and A23187. Because A23187 can promote loss of cellular Mg, it was necessary to include MgCl2 in the incubation medium to optimize ATPase activity. Likewise, it was determined that inclusion of iodoacetic acid optimized the rate of ATP loss, presumably by preventing the resynthesis of ATP from ADP and inorganic phosphate. Cobalt inhibited the ionophore-dependent loss of ATP by apparent competition with Ca for binding to A23187. Results of many assays demonstrated substantial differences in the rate constant for ATP loss in RBCs from different individuals. RBCs were selected according to density. Density associated loss of Ca pump ATPase activity was observed both by the intact RBC assay, and by assay of Ca pump ATPase activity in saponin lysates of RBCs. The correlation coefficient between the two assays was 0.93. It is suggested that the rate constant for ATP loss in intact RBCs exposed to A23187 and Ca can be taken as a measure of the Ca pump ATPase activity. This may be useful when isolated membrane ATPase assays fail (e.g., dog RBCs). The intact cell assay can also be carried out on very small volumes of cells and may be of particular value when RBC volumes are limited.

Numerical simulation of assay of the calcium pump of intact red blood cells.

An assay of the Ca pump ATPase of intact human RBCs is described in a companion paper (Wu, L., Hinds, T. R. and Vincenzi, F. F. (1992) Biochim. Biophys. Acta 1106, 56-62). The assay is based on the rapid loss of ATP in RBCs that occurs when the cells are exposed to the ionophore, A23187, in the presence of Ca. An unexpected finding was that the initial loss of ATP follows pseudo-first-order kinetics. This was unexpected because the ATP content of RBCs is somewhat higher than the Km of the Ca pump for ATP. Thus, the initial loss of ATP would be expected to follow zero-order kinetics; at least if the Ca pump ATPase operated with Michaelis kinetics. We performed a series of computer simulations of the Ca pump ATPase to investigate the possible cause of the unexpected pseudo-first-order behavior. The results confirmed that the data can not be accounted for by Michaelis kinetics of the Ca pump ATPase. Possible effects of adenylate kinase were tested and were also not found to account for the pseudo-first-order behavior of an ATPase operating with Michaelis kinetics. The enzymatic properties of the Ca pump ATPase were re-examined. It was found that the Ca pump ATPase exhibits positive cooperativity toward ATP. The apparent cooperativity was 1.91. In simulations it was found that positive cooperativity of the Ca pump ATPase in the range of 1.5 to 2.0 could account for the pseudo-first-order behavior. Excellent fit of the simulation data to first-order behavior was true with or without any contribution from adenylate kinase. Rate constants of ATP loss were thus examined using cooperativity of 2.0. Over a wide range the rate constant of the loss of ATP was directly proportional to the assumed Vmax of the Ca pump ATPase, but only if the data were limited to loss of less than 67% of the initial ATP. It is suggested, therefore, that the rate constant for the initial loss of ATP in intact RBCs, as stimulated by the ionophore A23187, can be taken as a measure of the capacity of the Ca pump ATPase.

Differential effects of arylating and oxidizing analogs of N-acetyl-p-benzoquinoneimine on red blood cell membrane proteins.

Incubation of human red blood cell membranes (white ghosts) with N-acetyl-p-benzoquinone imine (NAPQI), a toxic metabolite of acetaminophen, or with either an arylating or an oxidizing analog of NAPQI, resulted in the inhibition of membrane ion transporting systems and the modification of cytoskeletal proteins. NAPQI and 2,6-dimethyl-NAPQI, which primarily arylates protein thiols, inhibited the calmodulin-activated Ca pump ATPase activity, the basal (calmodulin-independent) Ca pump ATPase activity and the Na,K pump ATPase activity. In contrast, 3,5-dimethyl-NAPQI, which primarily oxidizes protein thiols, caused selective inhibition of the calmodulin-activated Ca pump ATPase activity. Sodium dodecyl sulfate gel electrophoresis of red blood cell (RBC) membrane proteins revealed that NAPQI and 2,6-dimethyl-NAPQI, but not 3,5-dimethyl-NAPQI, decreased the intensity of band 3 corresponding to the anion transporter, whereas NAPQI as well as 2,6-dimethyl-NAPQI, and to a lesser extent 3,5-dimethyl-NAPQI, caused a decrease of cytoskeletal protein bands, including spectrin, actin, and bands 4.1 and 4.2. These modifications were associated with increased formation of high molecular weight protein aggregates that did not enter the gel. Treatment of 3,5-dimethyl-NAPQI-exposed ghosts with the reducing agent dithiothreitol (DTT), resulted in the recovery of the affected cytoskeletal protein bands. Conversely, the modifications caused by NAPQI and 2,6-dimethyl-NAPQI were only partially reversed by DTT treatment. Taken together our results suggest that NAPQI and its two analogs modified ion transporting systems and cytoskeletal proteins by reacting with protein thiols. Both oxidation and arylation of protein thiols can alter the functional properties of important RBC membrane proteins. Of the two reactions, arylation appeared to be the less specific and more damaging event.

Stool diagnosis of giardiasis using a commercially available enzyme immunoassay to detect Giardia-specific antigen 65 (GSA 65).

A commercially available enzyme immunoassay for the diagnosis of giardiasis was evaluated in a clinical trial. The ProSpecT/Giardia diagnostic test (Alexon, Inc., Mountain View, Calif.) was compared with the standard ova and parasite (O&P) microscopic examination. Additionally, several widely used stool fixatives and a commonly used transport medium were assessed for compatibility with the immunoassay. A total of 325 stool specimens were collected and used to evaluate assay performance. Of those, 93 specimens were collected from symptomatic Giardia O&P-positive patients and 232 specimens were randomly collected from patients as part of a routine health screening procedure. All 93 Giardia O&P-positive stool specimens were strongly positive by visual and spectrophotometric examination using the immunoassay. Of the 232 randomly collected specimens, 16 were positive by O&P examination and immunoassay, 6 were negative by O&P examination but positive by immunoassay, and 1 was positive by O&P examination and negative by immunoassay. There was substantial supportive evidence that indicated that the six immunoassay-positive, O&P-negative specimens were true-positives. When these six specimens were accepted as true-positives, the immunoassay detected almost 30% more cases of Giardia infection than did O&P examination. Its sensitivity and specificity were 96 and 100%, respectively, while the sensitivity and specificity of O&P examination were 74 and 100%, respectively. The immunoassay also performed well on specimens treated with 10% neutral Formalin, sodium acetate-Formalin fixative, and Cary-Blair transport medium. However, the test was not compatible with polyvinyl alcohol-treated specimens. Overall, the ProSpecT/Giardia test was a sensitive, specific immunoassay which was easy to run and interpret. It offers a simple solution to traditional difficulties encountered in diagnosing Giardia infection.

Inhibition of basal and calmodulin-activated Ca2+-pump ATPase by fractionated compound 48/80.

Compound 48/80 (48/80), a mixture of polycationic compounds was fractionated using affinity chromatography on calmodulin-Sepharose. Unfractionated 48/80 and various fractions were tested for their potential inhibitory effects on ATPase activities of isolated human red blood cell membranes. ATPase activities tested included: Mg2+-ATPase, the Na+/K+-pump ATPase, and the Ca2+-pump ATPase in both its basal (calmodulin-independent) and calmodulin-activated state. Neither 48/80 nor its various fractions were very potent or efficacious inhibitors of the Mg2+-ATPase or the Na+/K+-pump ATPase. In agreement with previous reports, 48/80 was found to be an inhibitor of the calmodulin-activated Ca2+-pump ATPase. By contrast, we found that unfractionated, as well as some fractionated, material inhibited both the basal (calmodulin-independent) and calmodulin-activated Ca2+-pump ATPase activity. A fraction designated as Fraction III bound to calmodulin-Sepharose in the presence of Ca2+ and low salt and was eluted in the absence of Ca2+ and 0.15 M NaCl. By gel filtration, Fraction III had an apparent average molecular weight of 2064 (1320 for unfractionated material). Fraction III was the most potent inhibitor of the Ca2+-pump ATPase with IC50 values for the basal and calmodulin-activated forms of the enzyme of 0.6 and 1.2 micrograms/ml, respectively. Inhibition by Fraction III was cooperative with n apparent values of 2.4 and 5.7, respectively, for the basal and calmodulin-activated forms of the enzyme. Thus, binding of 48/80 constituents to calmodulin can not fully account for the observed data. Direct interaction of 48/80 constituent(s) with the enzyme and/or the lipid portion of the membrane is suggested.

The activity of the red blood cell Ca pump is decreased in hemolytic anemia of the beagle dog.

A mild hereditary nonspherocytic anemia in Beagle dogs was studied. Compared to RBCs from normal dogs, RBCs from hemolytic Beagles were larger on average, contained more potassium, and exhibited an approximately 50% decrease in rate of loss of ATP induced by Ca and the ionophore, A23187. Under certain conditions, this rate of ATP loss can be taken as a measure of the Ca pump ATPase activity of intact RBCs. From RBC fractionation studies it appeared that the defective Ca pump ATPase was acquired during the relatively short life-span of the hemolytic RBC. Significant loss of Ca pump ATPase may be causally related to the hemolytic anemia. The mechanism(s) by which Ca pump ATPase activity is lost in this hemolytic anemia remain(s) to be determined.