Effect of hydroxyl radical on Na(+)-K(+)-ATPase activity of the brain microsomal membranes.

Sphingomyelin liposomes and brain microsomes were oxidized by exposure to hydrogen peroxide and ferrous ion. Lipid peroxidation were measured by the formation of thiobarbituric acid- reactive substances (TBAR). Hydroxyl radical was detected using the spin- trapping technique. Incubation of sphingomyelin liposomes with H2O2-Fe2+ resulted in an increase in the formation of TBAR. Na(+)-K(+)-ATPase activity was markedly inhibited and the SH group content decreased during incubation of microsomes in the presence of H2O2-Fe2+. Sodium ferulate effectively inhibited TBAR formation, protected Na(+)-K(+)-ATPase activity and prevented the oxidative modification of SH groups. Spin-trapping experiments showed that sodium ferulate effectively scavenged the hydroxyl radicals.     

Na(+)-K(+)-ATPase activity in alveolar epithelial cells increases with cyclic stretch

Na(+)-K(+)-ATPase pumps (Na(+) pumps) in the alveolar epithelium create a transepithelial Na(+) gradient crucial to keeping fluid from the pulmonary air space. We hypothesized that alveolar epithelial stretch stimulates Na(+) pump trafficking to the basolateral membrane (BLM) and, thereby, increases overall Na(+) pump activity. Alveolar type II cells were isolated from Sprague-Dawley rats and seeded onto elastic membranes coated with fibronectin or 5-day-conditioned extracellular matrix. After 2 days in culture, cells were uniformly stretched for 1 h in a custom-made device. Na(+) pump activity was subsequently assessed by ouabain-inhibitable uptake of (86)Rb(+), a K(+) tracer, and BLM Na(+) pump abundance was measured. In support of our hypothesis, cells increased Na(+) pump activity in a "dose-dependent" manner when stretched to 12, 25, or 37% change in surface area (DeltaSA), and cells stretched to 25% DeltaSA more than doubled Na(+) pump abundance in the BLM. Cells on 5-day matrix tolerated higher strain than cells on fibronectin before the onset of Na(+) pump upregulation. Treatment with Gd(3+), a stretch-activated channel blocker, amiloride, a Na(+) channel blocker, or both reduced but did not abolish stretch-induced effects. Sustained tonic stretch, unlike cyclic stretch, elicited no significant Na(+) pump response.     

大鼠单根近端肾小管Na+-K+-ATPase活性测定方法的改进

本研究旨在对Doucet等报道的定量检测大鼠单根近端肾小管Na^＋-K^＋-ATPase活性方法进行改进。取经过Ⅱ型胶原酶消化的大鼠肾脏皮质组织,在体视显微镜下手工分离单根近端肾小管,并测量其长度,经低渗和冻融处理后与[γ-^32P]ATP共同孵育,液闪法检测从[γ-^32P]ATP解离出的^32Pi,采用修正后的公式计算Na^＋-K^＋-ATPase活性。改良法与Doucet等的方法比较,测定单根近端肾小管Na^＋-K^＋-ATPase活性无显著性差异（P〉0．05）。改进后的方法节省试剂,操作简便、省时。     

Mechanical stretching of alveolar epithelial cells increases Na(+)-K(+)-ATPase activity.

Alveolar epithelial cells effect edema clearance by transporting Na(+) and liquid out of the air spaces. Active Na(+) transport by the basolaterally located Na(+)-K(+)-ATPase is an important contributor to lung edema clearance. Because alveoli undergo cyclic stretch in vivo, we investigated the role of cyclic stretch in the regulation of Na(+)-K(+)-ATPase activity in alveolar epithelial cells. Using the Flexercell Strain Unit, we exposed a cell line of murine lung epithelial cells (MLE-12) to cyclic stretch (30 cycles/min). After 15 min of stretch (10% mean strain), there was no change in Na(+)-K(+)-ATPase activity, as assessed by (86)Rb(+) uptake. By 30 min and after 60 min, Na(+)-K(+)-ATPase activity was significantly increased. When cells were treated with amiloride to block amiloride-sensitive Na(+) entry into cells or when cells were treated with gadolinium to block stretch-activated, nonselective cation channels, there was no stimulation of Na(+)-K(+)-ATPase activity by cyclic stretch. Conversely, cells exposed to Nystatin, which increases Na(+) entry into cells, demonstrated increased Na(+)-K(+)-ATPase activity. The changes in Na(+)-K(+)-ATPase activity were paralleled by increased Na(+)-K(+)-ATPase protein in the basolateral membrane of MLE-12 cells. Thus, in MLE-12 cells, short-term cyclic stretch stimulates Na(+)-K(+)-ATPase activity, most likely by increasing intracellular Na(+) and by recruitment of Na(+)-K(+)-ATPase subunits from intracellular pools to the basolateral membrane.     

Reduced activity of muscle Na(+)-K(+)-ATPase after prolonged running in rats.

The purpose of this study was to investigate the hypothesis that Na(+)-K(+)-ATPase activity is reduced in muscle of different fiber composition after a single session of aerobic exercise in rats. In one experiment, untrained female Sprague-Dawley rats (weight 275 +/- 21 g; means +/- SE; n = 30) were run (Run) on a treadmill at 21 m/min and 8% grade until fatigue, or to a maximum of 2 h, which served as control (Con), or performed an additional 45 min of low-intensity exercise at 10 m/min (Run+). In a second experiment, utilizing rats of similar characteristics (weight 258 +/- 18 g; n = 32), Run was followed by passive recovery (Rec). Directly after exercise, rats were anesthetized, and tissue was extracted from Soleus (Sol), red vastus lateralis (RV), white vastus lateralis (WV), and extensor digitorum longus (EDL) and frozen for later analysis. 3-O-methylfluorescein phosphatase activity (3-O-MFPase) was determined as an indicator of Na(+)-K(+)-ATPase activity, and glycogen depletion identified recruitment of each muscle during exercise. 3-O-MFPase was decreased (P < 0.05) at Run+ by an average of 12% from Con in all muscles (P < 0.05). No difference was found between Con and Run. Glycogen was lower (P < 0.05) by 65, 57, 44, and 33% (Sol, EDL, RV, and WV, respectively) at Run, and there was no further depletion during the continued low-intensity exercise period. No differences in Na(+)-K(+)-ATPase activity was observed between Con and Rec. The results of this study indicate that inactivation of Na(+)-K(+)-ATPase can be induced by aerobic exercise in a volume-dependent manner and that the inactivation that occurs is not specific to muscles of different fiber-type composition. Inactivation of Na(+)-K(+)-ATPase suggests intrinsic structural modifications by mechanisms that are unclear.     

Effect of prostaglandin F2 alpha on Na+-K+-ATPase activity in luteal membranes

Slices of rat corpora lutea (CL) incubated with prostaglandin F2 alpha (PGF2 alpha) in Krebs-Hensenleit (K-H) Ringer solution showed a decrease in Na+-K+-ATPase activity after 60 min of incubation. However, PGF2 alpha in vitro did not alter Na+-K+-ATPase activity of isolated luteal membrane fractions. Following PGF2 alpha-induced in vivo luteal regression, reduction of Vmax and elevation of the activation energy above transition temperature of the lipid phase of the membrane occurred without changes in Km, optimum pH and transition temperature. These results suggest that reduction of Na+-K+-ATPase activity after PGF2 alpha treatment may be due to reduction in the number of enzyme molecules or to masking of the active site of the enzyme without any change in enzyme characteristics. In addition, a change in membrane-bound enzyme activity may be an early step in PGF2 alpha-induced luteolysis.     

Effect of increased calcium intake on cardiac and vascular Na(+)-K(+)-ATPase activity in oral contraceptive-treated female Sprague-Dawley rats

The present study aimed at investigating the influence of increased dietary calcium on Na(+)-K(+)-ATPase activity in heart and aorta of female Sprague-Dawley rats treated with oral contraceptive (OC) steroids. Rats were grouped as control (CR), OC-treated and OC+calcium-treated. OC-treated and OC+calcium-treated received a combination of OC steriods (ethinyloestradiol and norgestrel; ig). OC+calcium-treated rats were fed with 2.5% calcium diet, while OC-treated and CR groups were fed on 0.9% calcium diet. The activity of Na(+)-K(+)-ATPase in heart and aorta was significantly lower in OC-treated rats than those in the other groups. OC treatment caused significant increase in plasma glucose and significant decrease in plasma K+ as compared to control group. Decrease in Na(+)-K(+)-ATPase activity and plasma K+ was abrogated by increased calcium intake, while increase in plasma glucose was not normalized by calcium supplementation. Plasma levels of Na+, lipid peroxidation index and ascorbic acid were comparable among the three groups. These results showed that OC treatment could lead to impaired activity of cardiac and vascular Na(+)-K(+)-ATPase, possibly due to reduced plasma K+ level and these effects could be abolished by high calcium diet.