K-p-Nitrophenylphosphatase activity,Na and K content,Na permeability and membrane lipid composition in rabbit myocardium after cholesterol rich diet

Summary The aim of the present study was to investigate the effects of a cholesterol-rich diet on membrane function and lipid composition in rabbit myocardium. The activity and the ouabain sensitivity of the K-p-nitrophenylphosphatase (K-pNPPase), a partial reaction of the Na, K-ATPase, were diminished after a cholesterol/oil or pure cholesterol diet. The content of cholesterol, cholesterol esters and of several classes of phospholipids was enhanced in microsomes. A causal relationship is assumed between cholesterol accumulation and a decrease in membrane fluidity as well as in Na, K-ATPase activity. The intracellular Na content and the Na-Li-exchange rate were higher after the cholesterol diet. The increase in the Na content is supposed to be induced by a lower Na transport and a higher Na permeability. An enhanced Ca flux via the sarcolemma could be the consequence.To whom reprints should be addressed     

Failure of calcium to stimulate Na,K-ATPase in the presence of EDTA

The effect of calcium on Na,K-ATPase activity of rat brain homogenates and its modification by the chelating agent EDTA has been investigated. In the absence of EDTA, free calcium (approximately 10(-6) mol/l) stimulates Na,K-ATPase activity; in the presence of EDTA the same concentration of free calcium is without effect on the enzyme. In the absence of EDTA the stimulation by calcium of Na,K-ATPase activity is enhanced by the additional presence of calmodulin but in the presence of EDTA, even when calmodulin is added to excess, calcium still fails to stimulate the enzyme. The possibility that EDTA interferes with an interaction between a calcium-calmodulin complex and Na,K-ATPase is discussed.     

Failure of calcium to stimulate Na,K-ATPase in the presence of EDTA

Summary The effect of calcium on Na, K-ATPase activity of rat brain homogenates and its modification by the chelating agent EDTA has been investigated. In the absence of EDTA, free calcium (approximately 10^–6mol/l) stimulates Na,K-ATPase activity; in the presence of EDTA the same concentration of free calcium is without effect on the enzyme. In the absence of EDTA the stimulation by calcium of Na,-K-ATPase activity is enhanced by the additional presence of calmodulin but in the presence of EDTA, even when calmodulin is added to excess, calcium still fails to stimulate the enzyme. The possibility that EDTA interferes with an interaction between a calcium-calmodulin complex and Na,K-ATPase is discussed.The expert technical assistance of Mrs Paula Jarvie is gratefully acknowledged. Thanks are due also to Professor Philip Kuchel for assistance with the calculations to determine the concentrations of metal-ligand complexes in the experimental media.     

Identification of organ-specific glycosylation of a membrane protein in two tissues using lectins

Since glycosylation of proteins is performed by the host cell, and variable sugar groupings can confer heterogeneity on the same polypeptide, we wished to see whether membrane proteins, in particular the ubiquitous transmembrane Na, K-ATPase, could be glycosylated differently in different organs. Using a highly sensitive enzyme-linked antibody detection system of bound digoxigenin-labelled lectins on nitrocellulose sheets containing electroblotted and subunits of kidney and brain Na,K-ATPase, isolated from various rat strains, in combination with isoform-specific immunoblots, we discovered that brain Na,K-ATPase was highly mannosylated in contrast to renal Na,K-ATPase. Thus, we describe the existence of organ-related glycoforms of an integral ubiquitous membrane protein, i.e. diversification of the same polypeptide by organ-typical sugars. At the same time, the presence of the same glycosylation pattern can make distinct protein isoforms occurring in a same organ more homogeneous. Such organ-related glycoforms may serve for tissue identification and as tissue-specific receptors.     

Effect of cholesterol oxidation on (Na+, K+) ATPase activity of erythrocyte membranes.

By incubation of human erythrocyte ghosts with cholesterol oxidase (EC 1.1.3.6) part of the cholesterol of the membrane is replaced by 4-cholesten-3-one. This alteration in the sterol composition is accompanied by an inhibition of the (Na+, K+) ATPase of the erythrocyte membrane.     

Contribution of the Na+/K+-pump to the membrane potential

The inward movement of sodium ions and the outward movement of potassium ions are passive and the reverse movements against the electrochemical gradients require the activity of a metabolism-driven Na+/K+-pump. The activity of the Na+/K+-pump influences the membrane potential directly and indirectly. Thus, the maintenance of a normal electrical function requires that the Na+/K+-pump maintain normal ionic concentrations within the cell. The activity of the Na+/K+-pump also influences the membrane potential directly by generating an outward sodium current that is larger when the Na+/K+-pump activity is greater. The activity of the Na+/K+-pump is regulated by several factors including the intracellular sodium concentration and the neuromediators norepinephrine and acetylcholine. The inhibition of the Na+/K+-pump can lead indirectly to the development of inward currents that may cause repetitive activity. Therefore, the Na+/K+-pump modifies the membrane potential in different ways both under normal and abnormal conditions and influences in an essential way many cardiac functions, including automaticity, conduction and contraction. Key words. Active transport of ions; cardiac tissues; electroneutral and electrogenic Na+/K/-pump; control of Na+/K+-pump; normal and abnormal electrical events.     

Na, K ATPase activity during early postnatal development of the rat submandibular gland

The activity of the ouabain-sensitive Na, K ATPase was measured in membrane fractions of the submandibular gland of 1-, 7-, 14- and 21-day-old rats. This activity increased with age and reached adult levels by 21 days.     

Na,K ATPase activity during early postnatal development of the rat submandibular gland

Summary The activity of the ouabain-sensitive Na, K ATPase was measured in membrane fractions of the submandibular gland of 1-, 7-, 14- and 21-day-old rats. This activity increased with age and reached adult levels by 21 days.     

The effect of physostigmine on (Na+ + K+)-ATPase activity in different rat brain regions

The activity of (Na+ + K+)-ATPase and acetylcholine esterase were followed in rat brain cerebral cortex, caudate, thalamus, hippocampus and medulla after i.v. administration of physostigmine. Both enzymes were found to be inhibited in a dose-dependent manner. The most pronounced inhibition of (Na+ + K+)-ATPase was found in caudate, where the highest activity of acetylcholine esterase is found.     

Sex-dependent action of prenatal fractionated X-irradiation in the mouse. Biochemical findings.

X-irradiation of pregnant NMRI-mice on gestational days 11-13 with 3 x 10.5 Gy increased postnatal mortality of the female offspring only. Weights, protein content and acetylcholinesterase, as well as Na,K-ATPase activities in the brains of all treated offspring, were changed. There were, however, no differences between females and males with respect to these parameters.     

Differential effects of prostaglandins and isoproterenol on cAMP content and Na, K pump activity in rat submandibular acini

The beta-adrenergic agonist isoproterenol and prostaglandins E1 and E2 (but not F2 alpha) increased the cAMP content of rat submandibular acini in vitro, but only isoproterenol enhanced ouabain-sensitive 86Rb (K) uptake. These findings suggest that cAMP is not involved in the activation of the Na, K pump in salivary cells.     

Further evidence for the dissociation of digoxin-like immunoreactivity from Na+,K(+)-ATPase inhibitory activity

The effects of adrenalectomy or nephrectomy, carried out one hour previously, on the levels of endogenous digitalis-like factors were determined in rat plasma. Factors were assayed by digoxin-like immunoreactivity and direct Na+,K(+)-ATPase inhibitory activity. Digoxin-like immunoreactivity significantly decreased one hour after bilateral ablation of adrenals, while Na+,K(+)-ATPase inhibitory activity remained unaltered. There were no changes in either activity one hour after bilateral nephrectomy. These results suggest that digoxin-like immunoreactivity may be derived from the adrenal gland or under adrenal control and the major substances detected by digoxin-like immunoreactivity and direct Na+,K(+)-ATPase inhibitory activity may be different.     

Characterization of the (Na+-K+)-ATPase from endometrial plasma membranes of immature mammals]

The (Na+-K+)-ATPase in plasma membrane from Mammiferous endometrium is characterized by the Mg/ATP ratio equal to one, and by a distinct affinity for Na+ (1.3 mM) and K+ (2 mM). The activity is maximum for pH 7.4-7.5 in presence of Mg++ 2mM and ATP 2 mM, Na+ 140 mM and K+ 10 mM.     

Effect of free fatty acids and cholesterol in vitro on liver plasma membrane-bound enzymes

The effect of cholesterol and fatty acid treatment in vitro was tested on rat liver plasma membrane-bound enzymes and lipid fluidity. The observed alterations of membrane fluidity affect both (Na+-K+)-ATPase and Mg2+-ATPase activities but not 5'-nucleotidase; basal adenylate cyclase as well as its hormonal sensitivity were differentially affected by changes of membrane microenvironment.     

Sex-dependent action of prenatal fractionated X-irradiation in the mouse. Biochemical findings

Summary X-Irradiation of pregnant NMRI-mice on gestational days 11–13 with 3×1.05 Gy increased postnatal mortality of the female offspring only. Weights, protein content and acetylcholinesterase, as well as Na, K-ATPase activities in the brains of all treated offspring, were changed. There were, however, no differences between females and males with respect to these parameters.We gratefully acknowledge the assistance of Miss C. Gutmann.     

MDR1, cholesterol certification and cell growth: a comparative study in normal and multidrug-resistant KB cell lines

The product of the MDR1 gene (P-gp) has been implicated in the transport of cholesterol from plasma membrane to endoplasmic reticulum for esterification. In previous studies on leukemia cell lines, we suggested that cholesterol esterification may regulate the rate of cell growth and that the MDR1 gene might be involved in this process by modulating intracellular cholesterol esters levels. To further investigate this matter, the rate of cell growth, cholesterol metabolism, expression of the MDR1 gene, and P-gp activity were compared in KB cell lines displaying differences in expression and function of P-gp (drug-sensitive phenotype versus MDR phenotype). The rate of cell growth correlated with cholesterol esterification in all KB cell lines, whereas the over-expression of MDR1 observed in the MDR cell lines was not always associated with an increased capacity of cells to esterify cholesterol. Two known inhibitors of P-gp activity, progesterone and verapamil, strongly inhibited both cholesterol esterification and cell proliferation in all KB cell lines, but they affected intracellular accumulation of labeled vinblastine only in MDR cell lines. These results further support a role for cholesterol esters in the regulation of cell growth and suggest that the P-gp expressed in MDR KB cells is not involved in the general process leading to cholesterol esterification. Received 14 February 2000; received after revision 10 April 2000; accepted 8 May 2000     

Cholesterol feeding to rats does not modulate the expression of binding sites for HDL on liver membranes

Summary The binding of HDL, Apo-E-free, was studied in rats fed a cholesterol rich diet for 2, 4 and 7 days. Plasma cholesterol increased up to 16-fold (from 55 to 900 mg/dl); liver cholesterol was also raised, from 0.5 to 16 mg/g of tissue. The HDL binding to membrane preparations was not affected while the binding of VLDL was reduced to about 50% of the controls. These data show, therefore, that liver binding sites for HDL are refractory to regulation by dietary cholesterol.     

Dantrolene: evidence for effects on Na permeability properties of the nodal membrane

The effects of dantrolene on myelinated frog nerve fibers were studied in voltage clamp experiments. Dantrolene shifted the potential-dependent parameters describing Na+ permeability towards more negative membrane potentials. The findings are interpreted as a change in the negative surface charge of the membrane.     

Differential effects of prostaglandins and isoproterenol on cAMP content and Na,K pump activity in rat submandibular acini

Summary The -adrenergic agonist isoproterenol and prostaglandins E₁ and E₂ (but not F₂) increased the cAMP content of rat submandibular acini in vitro, but only isoproterenol enhanced ouabain-sensitive⁸⁶Rb (K) uptake. These findings suggest that cAMP is not involved in the activation of the Na, K pump in salivary cells.     

The Na/K pump,resting potential and selective permeability in canine Purkinje fibres at physiologic and room temperatures

All mammalian cells maintain a resting potential generated by ions moving down concentration gradients. In excitable cells, the inside potential is negative relative to outside. In order to maintain this electrochemical gradient, the sodium potassium (Na/K) pump actively transports out three sodium ions for every two potassium ions it brings in. This process generates a net outward current and thus hyperpolaizes the resting potential. I employed dihydroouabain (DHO) to inhibit the Na/K pump and thus measure its contribution to the resting potential. It contributed 9.0 mV at 34°C and 3.8 mV at 25°C. The P_K/P_Na ratios were calculated at both temperatures before and after subtracting the Na/K pump contribution. These ratios also suggested a decreased contribution of the Na/K pump under hypothermia. Taken together, these results suggest that the pump contribution to the resting potential is more significant at physiologic temperatures (34°C) than at room temperature (25°C), and that estimates of selective permeability can only be accurately obtained after assessing and eliminating the Na/K pump contribution to the resting potential.