Plasma membrane fluidity measurements in intact endothelial cells: effect of hyperoxia on fluorescence anisotropies of 1-[4-(trimethylamino)phenyl]-6-phenyl hexa-1,3,5-triene

Fluorescence anisotropy measurements are widely used as sensitive indicators of cell membrane fluidity. 1-[4-(trimethylamino)phenyl]-6-phenyl hexa-1,3,5-triene (TMA-DPH) is a cationic fluorescent aromatic hydrocarbon that anchors at the lipid-water interface of membrane lipid bilayers. Its uptake into porcine pulmonary artery and aortic endothelial cells was monitored and the probe remained specifically localized on the cell surface for at least 4 h. It can therefore be recommended for use for specific plasma membrane lipid fluidity measurements in these cells. The effect of hyperoxia on plasma membrane fluidity was measured by using TMA-DPH. In both cell types, hyperoxic damage resulted in decreases in plasma membrane fluidity. Recovery was achieved 48 h after a 42-h hyperoxic exposure. These results indicate that TMA-DPH is a sensitive probe of plasma membrane lipid domains of pulmonary artery and aortic endothelial cells and that hyperoxia causes reversible changes in the physical state of superficial lipid domains of the plasma membrane of these cells.     

The effect of oxidant gases on membrane fluidity and function in pulmonary endothelial cells

Free radicals and oxidant gases, such as oxygen (O2) and nitrogen dioxide (NO2), are injurious to mammalian lung cells. One of the postulated mechanisms for the cellular injury associated with these gases and free radicals involves peroxidative cleavage of membrane lipids. We have hypothesized that oxidant-related alterations in membrane lipids may result in disordering of the plasma membrane lipid bilayer, leading to derangements in membrane-dependent functions. To test this hypothesis, we examined the effect of exposure to high partial pressures of O2 or NO2 on the physical state and function of pulmonary endothelial cell plasma membranes. Both hyperoxia (95% O2 at 1 ATA) and NO2 exposure (5 ppm) caused early and significant decreases in fluidity in the hydrophobic interior of the plasma membrane lipid bilayer and subsequent depressions in plasma membrane-dependent transport of 5-hydroxytryptamine. Lipid domains at the surface of pulmonary endothelial cell plasma membranes are more susceptible to NO2-induced injury than to hyperoxic injury. Alterations in the fluidity of these more superficial domains are associated with derangements in surface dependent functions, such as receptor-ligand interaction. These results support our hypothesis and advance our understanding of how the chemical events of free radical injury associated with high O2 and NO2 tensions are translated into functional manifestations of O2 and NO2-induced cellular injury.     

Cyclic stretch translocates the alpha2-subunit of the Na pump to plasma membrane in skeletal muscle cells in vitro

The Na+-K+-ATPase and its regulation is important for maintaining membrane potential and transmembrane Na(+) gradient in all skeletal muscle cells and thus is essential for cell survival and function. In our previous study, cyclic stretch activated the Na pump in cultured skeletal muscle cells. Presently, we investigated whether this stimulation was the result of translocation of Na+-K+-ATPase from endosomes to the plasma membrane, and also evaluated the role of phosphatidylinositol 3-kinase (PI 3-kinase), the activation of which initiated vesicular trafficking and targeting of proteins to specific cell compartments. Skeletal muscle cells were stretched at 25% elongation continuous for 24h using the Flexercell Strain Unit. The plasma membrane and endosome fractions were isolated and Western blotted to localize the Na+-K+-ATPase alpha1- and alpha2-subunit protein. The results showed stretch increased Na+-K+-ATPase alpha1- and alpha2-subunit protein expression in plasma membrane fractions and decreased it in endosomes. The alpha2-subunit had a more dynamic response to mechanical stretch. PI 3-kinase inhibitors (LY294002) blocked the stretch-induced translocation of the Na+-K+-ATPase alpha2-subunit, while LY294002 had no effect on the transfer of alpha1-subunit. We concluded that cyclic stretch mainly stimulated the translocation of the alpha2-subunit of Na+-K+-ATPase from endosomes to the plasma membrane via a PI 3-kinase-dependent mechanism in cultured skeletal muscle cells in vitro, which in turn increased the activity of the Na pump.     

Gill Na+-K+-ATPase activity correlates with basolateral membrane lipid composition in seawater- but not freshwater-acclimated Arctic char (Salvelinus alpinus)

The successful migration of euryhaline teleost fish from freshwater to seawater requires the upregulation of gill Na+-K+-ATPase, an ion transport enzyme located in the basolateral membrane (BLM) of gill chloride cells. Following 39 days of seawater exposure, Arctic char had similar plasma sodium and chloride levels as individuals maintained in freshwater, indicating they had successfully acclimated to seawater. This acclimation was associated with an eightfold increase in gill Na+-K+-ATPase activity but only a threefold increase in gill Na+-K+-ATPase protein number, suggesting that other mechanisms may also modulate gill Na+-K+-ATPase activity. We therefore investigated the influence of membrane composition on Na+-K+-ATPase activity by examining the phospholipid, fatty acid, and cholesterol composition of the gill BLM from freshwater- and seawater-acclimated Arctic char. Mean gill BLM cholesterol content was significantly lower ( approximately 22%) in seawater-acclimated char. Gill Na+-K+-ATPase activity in individual seawater Arctic char was negatively correlated with BLM cholesterol content and positively correlated with %phosphatidylethanolamine and overall %18:2n6 (linoleic acid) content of the BLM, suggesting gill Na+-K+-ATPase activity of seawater-acclimated char may be modulated by the lipid composition of the BLM and may be especially sensitive to those parameters known to influence membrane fluidity. Na+-K+-ATPase activity of individual freshwater Arctic char was not correlated to any membrane lipid parameter measured, suggesting that different lipid-protein interactions may exist for char living in each environment.     

A 133Cs nuclear magnetic resonance study of endothelial Na(+)-K(+)-ATPase activity: can actin regulate its activity?

Using (133)Cs+ NMR, we developed a technique to repetitively measure, in vivo, Na(+)-K(+)-ATPase activity in endothelial cells. The measurements were made without the use of an exogenous shift reagent, because of the large chemical shift of 1.36 +/- 0.13 ppm between intra- and extracellular Cs+. Intracellularly we obtained a spin lattice relaxation time (T1) of 2.0 +/- 0.3 s, and extracellular T1 was 7.9 +/- 0.4 s. Na(+)-K+ pump activity in endothelial cells was determined at 12 +/- 3 nmol Cs+ x min(-1) x (mg Prot)[-1] under control conditions. When intracellular ATP was depleted by the addition of 5 mM 2-deoxy-D-glucose (DOG) and NaCN to about 5% of control, the pump rate decreased by 33%. After 80 min of perfusion with 5 mM DOG and NaCN, reperfusion with control medium rapidly reestablished the endothelial membrane Cs+ gradient. Using (133)Cs+ NMR as a convenient tool, we further addressed the proposed role of actin as a regulator of Na(+)-K+ pump activity in intact cells. Two models of actin rearrangement were tested. DOG caused a rearrangement of F-actin and an increase in G-actin, with a simultaneous decrease in ATP concentration. Cytochalasin D, however, caused an F-actin rearrangement different from that observed for DOG and an increase in G-actin, and cellular ATP levels remained unchanged. In both models, the Na(+)-K(+)-pump activity remained unchanged, as measured with (133)Cs NMR. Our results demonstrate that (133)Cs NMR can be used to repetitively measure Na(+)-K(+)-ATPase activity in endothelial cells. No evidence for a regulatory role of actin on Na(+)-K(+)-ATPase was found.     

Isoproterenol improves ability of lung to clear edema in rats exposed to hyperoxia.

Exposure of adult rats to 100% O(2) results in lung injury and decreases active sodium transport and lung edema clearance. It has been reported that beta-adrenergic agonists increase lung edema clearance in normal rat lungs by upregulating alveolar epithelial Na(+)-K(+)-ATPase function. This study was designed to examine whether isoproterenol (Iso) affects lung edema clearance in rats exposed to 100% O(2) for 64 h. Active Na(+) transport and lung edema clearance decreased by approximately 44% in rats exposed to acute hyperoxia. Iso (10(-6) M) increased the ability of the lung to clear edema in room-air-breathing rats (from 0.50 +/- 0.02 to 0.99 +/- 0. 05 ml/h) and in rats exposed to 100% O(2) (from 0.28 +/- 0.03 to 0. 86 +/- 0.09 ml/h; P < 0.001). Disruption of intracellular microtubular transport of ion-transporting proteins by colchicine (0. 25 mg/100 g body wt) inhibited the stimulatory effects of Iso in hyperoxia-injured rat lungs, whereas the isomer beta-lumicolchicine, which does not affect microtubular transport, did not inhibit active Na(+) transport stimulated by Iso. Accordingly, Iso restored the lung's ability to clear edema after hyperoxic lung injury, probably by stimulation of the recruitment of ion-transporting proteins (Na(+)-K(+)-ATPase) from intracellular pools to the plasma membrane in rat alveolar epithelium.     

The Na+-K+-ATPase as self-adhesion molecule and hormone receptor

Thanks to the homeostasis of the internal milieu, metazoan cells can enormously simplify their housekeeping efforts and engage instead in differentiation and multiple forms of organization (tissues, organs, systems) that enable them to produce an astonishing diversity of mammals. The stability of the internal milieu despite drastic variations of the external environment (air, fresh or seawater, gastrointestinal fluids, glomerular filtrate, bile) is due to transporting epithelia that can adjust their specific permeability to H(2)O, H(+), Na(+), K(+), Ca(2+), and Cl(-) over several orders of magnitude and exchange substances with the outer milieu with exquisite precision. This exchange is due to the polarized expression of membrane proteins, among them Na(+)-K(+)-ATPase, an oligomeric enzyme that uses chemical energy from ATP molecules to translocate ions across the plasma membrane of epithelial cells. Na(+)-K(+)-ATPase presents two types of asymmetries: the arrangement of its subunits, and its expression in one pole of the epithelial cell ("polarity"). In most epithelia, polarity consists of the expression of Na(+)-K(+)-ATPase towards the intercellular space and arises in part from the interaction of the extracellular segment of the β-subunit with another β-subunit present in a Na(+)-K(+)-ATPase molecule expressed by a neighboring cell. In addition to enabling the Na(+)-K(+)-ATPase to transport ions and water vectorially, this position exposes its receptors to ouabain and analogous cardiotonic steroids, which are present in the internal milieu because these were secreted by endocrine cells.     

Cytochemical localization of Na+-K+-ATPase in rat type II pneumocytes

The distribution of sodium-potassium-activated adenosinetriphosphatase (Na+-K+-ATPase) in the alveolar portion of rat lungs was examined by indirect immunofluorescence with the use of a mouse monoclonal anti-rat Na+-K+-ATPase and by ultrastructural cytochemistry using p-nitrophenylphosphate as substrate. The reaction was inhibitable by 10 mM ouabain or by the omission of K+ from the reaction mixture. Cysteine or levamisole was used to inhibit alkaline phosphatase activity. By immunofluorescence, staining was confined to cuboidal cells in alveolar spaces. These were tentatively identified as type II pneumocytes. By ultrastructural cytochemistry reaction product was present on the cytoplasmic side of the basolateral membranes of type II pneumocytes. No reaction product was observed in type I pneumocytes or in endothelium. These results indicate that type II pneumocytes contain more Na+-K+-ATPase, an enzyme important in vectorial electrolyte transport, than type I pneumocytes or endothelial cells. More sensitive methods, however, are required to determine the amounts and distribution of this enzyme in type I pneumocytes and pulmonary vascular endothelial cells.     

Regulation of membrane lipid bilayer structure during salinity adaptation: a study with the gill epithelial cell membranes of Oreochromis niloticus

A significant variation in the membrane fluidity (as assessed by DPH-fluorescence polarisation) and membrane lipid bilayer composition is noticed in the subcellular membranes of the gill epithelial cells of Oreochromis niloticus due to exposure of the fish to 1% saline water for 1 month. Also, a 70% enhanced activity of Na(+)-K(+)-ATPase in plasma membranes and a 2.5-fold increase of glucose-6-phosphate dehydrogenase in microsomal membranes are recorded in the treated fish. The changed membrane structure and fluidity along with the changed enzymatic activity of Na(+)-K(+)-ATPase help the influx the Na(+) rather than the efflux of K(+) through the gill epithelial cells during salinity adaptation.     

栀子提取物ZG对副流感病毒1型感染后宿主细胞膜的影响

为了探讨栀子提取物ZG抗病毒作用的生物学机制,观察了栀子提取物ZG对副流感病毒1型(PIV-1)感染后宿主细胞膜电位、膜Na -K -ATP酶活性和膜流动性的影响。以氯化乙酰胆碱为阳性对照,采用荧光探针Di-BAC4(3)标记Hep-2细胞膜电位,借助流式细胞仪检测膜电位;定磷法,分光光度计检测Na -K -ATP酶活性;荧光探针NBD-C6-HPC标记细胞膜磷脂,以荧光漂白恢复法和激光扫描共聚焦显微镜检测膜流动性。结果显示:PIV-1感染后宿主细胞膜电位下降,处于超极化状态;膜Na -K -ATP酶活性显著增加,膜流动性显著降低。栀子提取物ZG作用后,对宿主细胞膜的超极化状态没有明显影响;对膜Na -K -ATP酶活性没有明显影响;而对膜流动性则有明显的恢复作用。阳性对照药乙酰胆碱能明显改善病毒感染后膜电位的超极化状态。PIV-1感染后膜电位、Na -K -ATP酶活性和膜流动性等细胞膜能态和功能的改变,可能为病毒感染的生物学机制之一;栀子提取物ZG可能是通过改善细胞膜流动性,维持细胞膜的正常功能来发挥抗病毒感染的作用,而与膜电位和膜Na -K -ATP酶活性等能态来源的环节可能无关。     

Relationships between membrane lipids and ion transport in red blood cells of Dahl rats

Distinct changes of membrane lipid content could contribute to the abnormalities of ion transport that take part in the development of salt hypertension in Dahl rats. The relationships between lipid content and particular ion transport systems were studied in red blood cells (RBC) of Dahl rats kept on low- and high-salt diets for 5 weeks since weaning. Dahl salt-sensitive (SS/Jr) rats on high-salt diet had increased blood pressure, levels of plasma triacylglycerols and total plasma cholesterol compared to salt-resistant (SR/Jr) rats. Furthermore, RBC of SS/Jr rats differed from SR/Jr ones by increased content of total membrane phospholipids, but membrane cholesterol was not changed significantly. SS/Jr rats had higher RBC intracellular Na+ (Na(i)+) content and enhanced bumetanide-sensitive Rb+ uptake. RBC membrane content of cholesterol and phospholipids correlated positively with RBC Na(i)+ content, with the activity of Na+-K+ pump and Na+-K+-2Cl- cotransport and also with Rb+ leak. The content of phosphatidylserines plus phosphatidylinositols was positively associated with RBC Na(i)+ content, with the activity of Na+-K+ pump and Na+-K+-2Cl- cotransport and with Rb+ leak. The content of sphingomyelins was positively related to Na+-K+-2Cl- cotransport activity and negatively to ouabain-sensitive Rb+-K+ exchange. We can conclude that observed relationships between ion transport and the membrane content of cholesterol and/or sphingomyelins, which are known to regulate membrane fluidity, might participate in the pathogenesis of salt hypertension in Dahl rats.     

Effect of oxygen and endotoxin on lactate dehydrogenase release, 5-hydroxytryptamine uptake, and antioxidant enzyme activities in endothelial cells

We compared the effects of 95% O2 (hyperoxia) alone, endotoxin (20 ng/ml) alone, and 95% O2 plus endotoxin on the release of lactate dehydrogenase (LDH), uptake of 5-hydroxytryptamine (5-HT), and antioxidant enzyme activities in porcine pulmonary arterial and aortic endothelial cells in monolayer culture. Hyperoxia increased LDH release and decreased 5-HT in both endothelial cell types. Hyperoxia also caused a decrease in catalase (CAT) activity and an increase in total superoxide dismutase (SOD) and glutathione reductase (GSH-Red) activities in both cell types. Endotoxin alone had no effect on LDH release, 5-HT uptake, or antioxidant enzyme activities. However, endotoxin prevented the hyperoxic increase in LDH release and the hyperoxic decrease in 5-HT uptake. Endotoxin plus 95% O2 had no consistent effect on the antioxidant enzyme profile in pulmonary artery or aortic endothelial cells. These results indicate that (1) hyperoxia injures both pulmonary artery and aortic endothelial cells in culture and causes changes in the antioxidant enzyme profile that are similar in the two cell types; (2) hyperoxia-induced decreases in CAT activity and increases in SOD activity may be responsible for increased sensitivity of endothelial cells to O2 toxicity; and (3) endotoxin protects against hyperoxic injury to endothelial cells in vitro, but increases in antioxidant enzyme activities are not the mechanism for this protection.     

Characterization of sodium transport in Acholeplasma laidlawii B cells and in lipid vesicles containing purified A. laidlawii (Na+-Mg2+)-ATPase by using nuclear magnetic resonance spectroscopy and 22Na tracer techniques.

The active transport of sodium ions in live Acholeplasma laidlawii B cells and in lipid vesicles containing the (Na+-Mg2+)-ATPase from the plasma membrane of this microorganism was studied by 23Na nuclear magnetic resonance spectroscopic and 22Na tracer techniques, respectively. In live A. laidlawii B cells, the transport of sodium was an active process in which metabolic energy was harnessed for the extrusion of sodium ions against a concentration gradient. The process was inhibited by low temperatures and by the formation of gel state lipid in the plasma membrane of this organism. In reconstituted proteoliposomes containing the purified (Na+-Mg2+)-ATPase, the hydrolysis of ATP was accompanied by the transport of sodium ions into the lipid vesicles, and the transport process was impaired by reagents known to inhibit ATPase activity. At the normal growth temperature (37 degrees C), this transport process required a maximum of 1 mol of ATP per mol of sodium ion transported. Together, these results provide direct experimental evidence that the (Na+-Mg2+)-ATPase of the Acholeplasma laidlawii B membrane is the cation pump which maintains the low levels of intracellular sodium characteristic of this microorganism.     

Sodium pump hyperpolarization-relaxation in rat caudal artery

Electrogenic ion transport contributes vitally to the Em in vascular muscle and thus is an important influence on contraction and relaxation. Agents that act on membrane ion transport will cause depolarization or hyperpolarization of sufficient magnitude to cause contraction or relaxation, respectively. In the caudal artery of the rat, the principal ion involved appears to be Na+. The transport process appears to be the Na+, K+-ATPase, which is ouabain sensitive, rather than other possible candidates such as the Na+-Ca2+ countertransport mechanism. The hyperpolarization and parallel relaxation found in caudal artery on return to K+ provide unequivocal evidence for an electrogenic Na+ pump. In contrast, the lack of a contraction on transition to O Na+ suggests that the caudal artery does not show an Na+-K+ countertransport system. Although other ion transport systems might be established later for caudal artery and other kinds of vascular muscle, it now appears that the electrogenic Na+ pump is the main ion transport system controlling contraction through a continuous contribution to Em.     

Dopamine increases lung liquid clearance during mechanical ventilation

Short-term mechanical ventilation with high tidal volume (HVT) causes mild to moderate lung injury and impairs active Na+ transport and lung liquid clearance in rats. Dopamine (DA) enhances active Na+ transport in normal rat lungs by increasing Na+-K+-ATPase activity in the alveolar epithelium. We examined whether DA would increase alveolar fluid reabsorption in rats ventilated with HVT for 40 min compared with those ventilated with low tidal volume (LVT) and with nonventilated rats. Similar to previous reports, HVT ventilation decreased alveolar fluid reabsorption by ~50% (P < 0.001). DA increased alveolar fluid reabsorption in nonventilated control rats (by ~60%), LVT ventilated rats (by approximately 55%), and HVT ventilated rats (by ~200%). In parallel studies, DA increased Na+-K+-ATPase activity in cultured rat alveolar epithelial type II cells (ATII). Depolymerization of cellular microtubules by colchicine inhibited the effect of DA on HVT ventilated rats as well as on Na+-K+-ATPase activity in ATII cells. Neither DA nor colchicine affected the short-term Na+-K+-ATPase alpha1- and beta1-subunit mRNA steady-state levels or total alpha1- and beta1-subunit protein abundance in ATII cells. Thus we reason that DA improved alveolar fluid reabsorption in rats ventilated with HVT by upregulating the Na+-K+-ATPase function in alveolar epithelial cells.     

Human nongastric H+-K+-ATPase: transport properties of ATP1al1 assembled with different beta-subunits

To investigate whether nongastric H+-K+-ATPases transport Na+ in exchange for K+ and whether different beta-isoforms influence their transport properties, we compared the functional properties of the catalytic subunit of human nongastric H+-K+-ATPase, ATP1al1 (AL1), and of the Na+-K+-ATPase alpha1-subunit (alpha1) expressed in Xenopus oocytes, with different beta-subunits. Our results show that betaHK and beta1-NK can produce functional AL1/beta complexes at the oocyte cell surface that, in contrast to alpha1/beta1 NK and alpha1/betaHK complexes, exhibit a similar apparent K+ affinity. Similar to Na+-K+-ATPase, AL1/beta complexes are able to decrease intracellular Na+ concentrations in Na+-loaded oocytes, and their K+ transport depends on intra- and extracellular Na+ concentrations. Finally, controlled trypsinolysis reveals that beta-isoforms influence the protease sensitivity of AL1 and alpha1 and that AL1/beta complexes, similar to the Na+-K+-ATPase, can undergo distinct K+-Na+- and ouabain-dependent conformational changes. These results provide new evidence that the human nongastric H+-K+-ATPase interacts with and transports Na+ in exchange for K+ and that beta-isoforms have a distinct effect on the overall structural integrity of AL1 but influence its transport properties less than those of the Na+-K+-ATPase alpha-subunit.     

Phosphorylation of the red blood cell membrane during the active transport of C++

The phosphorylation of red blood cell membrane fragments (RBCMF) during Ca++ transport was investigated. When red cell membrane fragments are incubated with [gamma-32P]ATP under the experimental condition which minimizes the phosphorylation of Na+-K+-ATPase, RBCMF are labeled in the presence of Mg++ without Ca++. When Ca++ is added, the labeling decreases due to dephosphorylation of RBCMF. The initial reaction of phosphorylation is reversed in the presence of excess ADP. The treatment of RBCMF with n-ethylmaleimide (NEM) does not interfere with the initial phosphorylation reaction, but blocks the dephosphorylation in the presence of Ca++. These data suggest that the enzymatic sequence of the Ca++ transport mechanism may be very similar to that of the Na+ transport mechanism.     

Continuous enteral nutrition attenuates pulmonary edema in rats exposed to 100% oxygen.

Adult rats exposed to hyperoxia develop anorexia, weight loss, and a lung injury characterized by pulmonary edema and decreased lung liquid clearance. We hypothesized that maintenance of nutrition during hyperoxia could attenuate hyperoxia-induced pulmonary edema. To test this hypothesis, we enterally fed adult male Sprague-Dawley rats via gastrostomy tubes and exposed them to oxygen (inspired O(2) fraction >0.95) for 64 h. In contrast to controls, enterally fed hyperoxic animals did not lose weight and had smaller pleural effusions and wet-to-dry weight ratios (a measure of lung edema) that were not different from room air controls. Enterally fed rats exposed to hyperoxia had increased levels of mRNA for the Na(+)-K(+)-ATPase alpha(1)- and beta(1)-subunits and glutathione peroxidase. These findings suggest that maintenance of nutrition during an oxidative lung injury reduces lung edema, perhaps by allowing for continued expression and function of protective proteins such as the Na(+)-K(+)-ATPase.