Standardized method for the determination of human erythrocyte membrane adenosine triphosphatases

Bitensky

Proc. Natl. Acad. Sci. U.S.A.

et al. 1989

We have found a defect in the ouabain-sensitive Na+,K+-ATPase (Na+ pump, EC 3.6.1.37) oferythrocytes from streptozotocin diabetic rats. This defect was accompanied by an increase in cell volume and osmotic fragility and a decrease in the cytosolic K+/Na+ ratio. There was also a doubling in the time needed for diabetic erythrocytes to pass through 4.7-jim channels in a polycarbonate filter. Our data are consistent with a primary defect in the erythrocyte Na+ pump and secondary changes in cell volume, osmotic fragility, K+/Na+ ratio, and cell ifiterability. All were reversed or prevented in vivo by insulin or the aldose reductase inhibitor Sorbinil. Protein kinase C agonists (phorbol ester and diacylglycerol) and agonist precursor (myoinositol) reversed the Na+ pump lesion, suggesting that protein kinase C-dependent phosphorylation of the 100-kDa subunit regulates Na+ pump activity and that insulin can influence erythrocyte protein kinase C activity. Ouabain inhibition of the erythrocyte Na+ pump also produced increases in cell size and reductions in rates of ifitration. Theoretical treatment of the volume changes also predicts reduction in ifiterability as a consequence of cell swelling. We suggest that enlarged erythrocytes could play a role in the evolution of the microvascular changes of diabetes mellitus.A decrease in ouabain-sensitive Na',K+-ATPase (Na' pump, EC 3.6.1.37) activity has been found in diabetic lens, nerve, and glomerulus (1-3). This Na' pump impairment was corrected by aldose reductase inhibitors or by normalization of blood sugar with insulin. In contrast, Na' pump activity in diabetic erythrocytes (RBCs) is a subject of controversy, with reports of increases (4, 5) and decreases (6, 7). We therefore initiated a study of the Na' pump in erythrocytes of rats rendered diabetic with streptozotocin. The streptozotocin diabetic rat provided a reliable source ofRBCs whose time-integrated exposure to hyperglycemia was determined by measurement of glycated albumin. Since nondiabetic RBCs must deform considerably to pass through small capillaries, we were especially interested in learning whether such a putative Na' pump lesion would influence the volume and hence filterability of affected RBCs. MATERIALS

“…Such substrate inhibition has been previously reported in the Na' transport system (9). In the case of ATP, Vm.…”

supporting

confidence: 77%

mentioning

confidence: 99%

Reversible sodium pump defect and swelling in the diabetic rat erythrocyte: effects on filterability and implications for microangiopathy.

Bitensky

Proc. Natl. Acad. Sci. U.S.A.

et al. 1989

“…These values are almost 1000 times higher than the activities reported by Katz and Emery (30) but similar to those reported by Foder et al (18). The intersubject variability is also comparable to other reports (36). The Ca-ATPase activities for an individual control subject studied 8 times over a 4-month period using the same conditions of membrane preparation and ATPase assays were: basal Ca-ATPase activity, 29.5 + 5.9 nmol/ min/mg protein, range = 25-40; and calmodulin-stimulated CaATPase activity, 74.8 & 11.3 nmol/min/mg protein, range = 60-89.…”

Section: Agesupporting

confidence: 92%

Calcium-ATPase Activity in Cystic Fibrosis Erythrocyte Membranes: Decreased Activity in Patients with Pancreatic Insufficiency

et al. 1984

Summary modulin, of the pancreatic sufficient patients (34.3 f 4.2 and 75.9 f 6.9 nmol/min/mg) was indistinguishable from that of The activity of Ca-ATPase (Ca2+,Mg2+-ATPase, ATP phos-controls (35.8 2.6 and 84.3 f 4.7 nmol/min/mg), while both phohydrolase, EC 3.6.1.3) was measured in erythrocyte mem-activities of patients with pancreatic insufficiency were signifibrane preparations from 37 cystic fibrosis patients, 27 with cantly decreased (28.9 + 1.3, < 0.02; 65.2 + 3.0, < 0.001) pancreatic insufficiency and 10 with pancreatic sufficiency, and to the control group. Similarly, the mean erythrocyte from 24 healthy controls. The mean maximal calcium-stimulated membrane (Na + K)ATPase activity was decreased only for specific activities, in the absence and presence of purified cal-those patients with a history of steatorrhea and who clinically Received August 18, 1983; accepted March 12, 1984. required pancreatic enzyme therapy and had low immunoreactive

“…Plasma membranes of erythrocytes obtained from normal human volunteers were prepared as described by Reinila et al [12]. Pro tein was estimated by the procedure of Lowry et al [ 13] using bovine serum albumin as a standard.…”

Section: Methodsmentioning

confidence: 99%

Conversion of Adrenaline to Indolic Derivatives by the Human Erythrocyte Plasma Membrane

Marques

Duarte²,

Moura

et al. 1996

Neurosignals

The conversion of adrenaline to aminochromes by the human erythrocyte plasma membranes at pH 9.5 was shown to be a complex reaction that proceeded at least by two distinct phases. The first one, corresponding to the formation of adrenochrome, is catalyzed in the presence of the membranes, suggesting the involvement of an enzyme-mediated process. Active oxygen species were identified as intermediates during this phase. Oxygen radical scavengers (catalase and superoxide dismutase) suggested H₂O₂ and O₂ involvement. Adrenochrome formation was stimulated by NADH indicating the participation of another enzyme (NADH dehydrogenase) which is known to be present in the human erythrocyte plasma membrane. The second phase, corresponding to the disappearance of adrenochrome, is also stimulated by NADH and inhibited in the presence of the membranes. In this reaction, adrenochrome is converted to aminochromes via adrenochrome semiquinone. The formation of radical species is demonstrated by EPR spectroscopy. The results led to the proposal of a mechanism for the formation of adrenochrome and other oxidation products from adrenaline.