Influence of monensin on cation influx and glycolysis of Eimeria tenella sporozoites in vitro

Extracellular Eimeria tenella sporozoites exposed to 1.0 microgram/ml monensin at 40 C had an accelerated rate of sodium influx as well as an increased rubidium uptake that was inhibited by the cardiac glycoside, ouabain. These results suggested the presence of a functional (Na+-K+)-ATPase and its stimulation by monensin. Under the same conditions, sporozoite ATP concentrations declined, lactate production increased and the rate of amylopectin utilization was enhanced. Exposure to monensin also appeared to stimulate the rate of sporozoite glycolysis. The results of this study demonstrated that the cidal effect of monensin on extracellular sporozoites was caused by the capability of the ionophore to act as a transmembrane sodium carrier.     

In vitro activity of the human neutrophil cathepsin G on Eimeria tenella sporozoites

The role of human neutrophil cathepsin G (Cat G) on Eimeria tenella sporozoites was studied in vitro. Sporozoites were incubated for 2 hr at 37 C in PO4 buffer, 0.9% NaCl (PBS), pH 7.6 in the presence of Cat G (50 micrograms/ml), diisopropyl fluorophosphate-inhibited Cat G (DFP-Cat G) (50 micrograms/ml) or PBS alone, prior to being inoculated into embryonated eggs. As judged by oocyst production on day 7 postinoculation, embryo mortality and the hemorrhage scores, both Cat G and DFP-Cat G demonstrated anticoccidial activity; greater activity was obtained with the DFP-Cat G. Sporozoites were exposed also to increasing concentrations of native and trypsin-digested DFP-Cat G (0-100 micrograms/ml) under the same conditions. Significant protection (37% and 49% for native and digested DFP-Cat G, respectively) was obtained with a low concentration (5 mu/ml), and higher concentrations resulted in 70% and 84% protection, respectively. The primary bactericidal domain of Cat G, the HPQYNQR peptide, at 3 concentrations (25, 50, and 100 micrograms/ml), reduced the oocyst production by 46%, 16%, and 15%, respectively. The anticoccidial activity of Cat G may involve a peptide fragment different from the antimicrobial domain of the enzyme.     

Glucose supplements increase human muscle in vitro Na+-K+-ATPase activity during prolonged exercise

Regulation of maximal Na(+)-K(+)-ATPase activity in vastus lateralis muscle was investigated in response to prolonged exercise with (G) and without (NG) oral glucose supplements. Fifteen untrained volunteers (14 males and 1 female) with a peak aerobic power (Vo(2)(peak)) of 44.8 +/- 1.9 ml.kg(-1).min(-1); mean +/- SE cycled at approximately 57% Vo(2)(peak) to fatigue during both NG (artificial sweeteners) and G (6.13 +/- 0.09% glucose) in randomized order. Consumption of beverage began at 30 min and continued every 15 min until fatigue. Time to fatigue was increased (P < 0.05) in G compared with NG (137 +/- 7 vs. 115 +/- 6 min). Maximal Na(+)-K(+)-ATPase activity (V(max)) as measured by the 3-O-methylfluorescein phosphatase assay (nmol.mg(-1).h(-1)) was not different between conditions prior to exercise (85.2 +/- 3.3 or 86.0 +/- 3.9), at 30 min (91.4 +/- 4.7 vs. 91.9 +/- 4.1) and at fatigue (92.8 +/- 4.3 vs. 100 +/- 5.0) but was higher (P < 0.05) in G at 90 min (86.7 +/- 4.2 vs. 109 +/- 4.1). Na(+)-K(+)-ATPase content (beta(max)) measured by the vanadate facilitated [(3)H]ouabain-binding technique (pmol/g wet wt) although elevated (P < 0.05) by exercise (0<30, 90, and fatigue) was not different between NG and G. At 60 and 90 min of exercise, blood glucose was higher (P < 0.05) in G compared with NG. The G condition also resulted in higher (P < 0.05) serum insulin at similar time points to glucose and lower (P < 0.05) plasma epinephrine and norepinephrine at 90 min of exercise and at fatigue. These results suggest that G results in an increase in V(max) by mechanisms that are unclear.     

番茄碱对人红细胞膜Na+-K+-ATPase活性影响的研究

以低渗法从新鲜健康人红细胞中制得膜Na^ -K^ -ATPase,研究了番茄碱(tomatine)X~人红细胞膜Na^ -K^ -ATPase活性的影响。实验结果表明,反应体系中的tomatine浓度低于1mmol／L时,对不依赖钙调蛋白(CAM)激活的Na^ -K^ -ATPase(称之为基本酶活)影响不大,浓度达1mmol／L时,该酶活性仍保持在95％左右;而在此浓度范围内,tomatine对依赖CaM的Na^ -K^ -ATPase有明显的抑制作用,其IC50值为0．16mmol／L．说明tomatine对膜酶Na^ -K^ -ATPase活性的影响可能是通过阻断CaM激活的途径而起作用,从而为进一步研究番茄碱的作用机制奠定了基础。     

Penetration of Eimeria tenella sporozoites under different oxygen concentrations in vitro.

Sporozoites of Eimeria tenella (Wisconsin strain) were inoculated onto monolayers of normal chicken kidney fibroblasts and cultured in RPMI-1640 supplemented with 5% fetal bovine serum, sodium bicarbonate, and gentamicin under either aerobic, 5% CO2/95% air, or anaerobic conditions. Penetration of fibroblasts by sporozoites under CO2 or anaerobic conditions at 2 and 24 hr postinoculation was 3-4 times greater than that in the aerobic atmosphere. Effect of reduced oxygen concentrations, i.e., 20.0, 12.5, and 5.0% oxygen, was also investigated in an N2-O2-CO2 incubator. Under 5.0 and 12.5% oxygen at 2 and 24 hr postinoculation, the number of sporozoites that penetrated was about 4 and 2 times greater, respectively, than under 20.0% O2. These results indicate that lower oxygen concentrations provide for greater penetration by E. tenella sporozoites in cultured cells.     

Depressed Na+-K+-ATPase activity in skeletal muscle at fatigue is correlated with increased Na+-K+-ATPase mRNA expression following intense exercise

We investigated whether depressed muscle Na(+)-K(+)-ATPase activity with exercise reflected a loss of Na(+)-K(+)-ATPase units, the time course of its recovery postexercise, and whether this depressed activity was related to increased Na(+)-K(+)-ATPase isoform gene expression. Fifteen subjects performed fatiguing, knee extensor exercise at approximately 40% maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue, 3 h, and 24 h postexercise and analyzed for maximal Na(+)-K(+)-ATPase activity via 3-O-methylfluorescein phosphatase (3-O-MFPase) activity, Na(+)-K(+)-ATPase content via [(3)H]ouabain binding sites, and Na(+)-K(+)-ATPase alpha(1)-, alpha(2)-, alpha(3)-, beta(1)-, beta(2)- and beta(3)-isoform mRNA expression by real-time RT-PCR. Exercise [352 (SD 267) s] did not affect [(3)H]ouabain binding sites but decreased 3-O-MFPase activity by 10.7 (SD 8)% (P < 0.05), which had recovered by 3 h postexercise, without further change at 24 h. Exercise elevated alpha(1)-isoform mRNA by 1.5-fold at fatigue (P < 0.05). This increase was inversely correlated with the percent change in 3-O-MFPase activity from rest to fatigue (%Delta3-O-MFPase(rest-fatigue)) (r = -0.60, P < 0.05). The average postexercise (fatigue, 3 h, 24 h) alpha(1)-isoform mRNA was increased 1.4-fold (P < 0.05) and approached a significant inverse correlation with %Delta3-O-MFPase(rest-fatigue) (r = -0.56, P = 0.08). Exercise elevated alpha(2)-isoform mRNA at fatigue 2.5-fold (P < 0.05), which was inversely correlated with %Delta3-O-MFPase(rest-fatigue) (r = -0.60, P = 0.05). The average postexercise alpha(2)-isoform mRNA was increased 2.2-fold (P < 0.05) and was inversely correlated with the %Delta3-O-MFPase(rest-fatigue) (r = -0.68, P < 0.05). Nonsignificant correlations were found between %Delta3-O-MFPase(rest-fatigue) and other isoforms. Thus acute exercise transiently decreased Na(+)-K(+)-ATPase activity, which was correlated with increased Na(+)-K(+)-ATPase gene expression. This suggests a possible signal-transduction role for depressed muscle Na(+)-K(+)-ATPase activity with exercise.     

Changes in Na+-K+-ATPase activity influence cell attachment to fibronectin

Most vital cellular functions aredependent on a fine-tuned regulation of intracellular ion homeostasis.Here we have demonstrated, using COS cells that were untransfected ortransfected with wild-type rat ouabain-resistantNa⁺-K⁺-ATPase, that partial inhibition ofNa⁺-K⁺-ATPase has a dramatic influence oncell attachment to fibronectin. Ouabain dose-dependently decreasedattachment in untransfected cells and in cells expressing wild-typeNa⁺-K⁺-ATPase, but not in cells expressingouabain-insensitive Na⁺-K⁺-ATPase, whereasinhibition of Na⁺-K⁺-ATPase by loweringextracellular K⁺ concentration decreased attachment in allthree cell types. Thirty percent inhibition ofNa⁺-K⁺-ATPase significantly attenuatedattachment. Na⁺-K⁺-ATPase inhibition caused asustained increase in the intracellular Ca²⁺ concentrationthat obscured Ca²⁺ transients observed in untreated cellsduring attachment. Inhibitors of Ca²⁺ transporterssignificantly decreased attachment, but inhibition ofNa⁺/H⁺ exchanger did not. Ouabain reduced focaladhesion kinase autophosphorylation but had no effect on cell surfaceintegrin expression. These results suggest that the level ofNa⁺-K⁺-ATPase activity strongly influences cellattachment, possibly by an effect on intracellular Ca²⁺.

     

Cigarette smoking impairs Na+-K+-ATPase activity in the human coronary microcirculation

The extracellular K(+) concentration ([K(+)](o)) has been proposed to link cardiac metabolism with coronary perfusion and arrhythmogenesis, particularly during ischemia. Several animal studies have also supported K(+) as an EDHF that activates Na(+)-K(+)-ATPase and/or inwardly rectifying K(+) (K(ir)) channels. Therefore, we examined the vascular reactivity of human coronary arterioles (HCAs) to small elevations in [K(+)](o), the influence of risk factors for coronary disease, and the role of K(+) as an EDHF. Changes in the internal diameter of HCAs were recorded with videomicroscopy. Most vessels dilated to increases in [K(+)](o) with a maximal dilation of 55 ± 6% primarily at 12.5-20.0 mM KCl (n = 38, average: 16 ± 1 mM). Ouabain, a Na(+)-K(+)-ATPase inhibitor, alone reduced the dilation, and the addition of Ba(2+), a K(ir) channel blocker, abolished the remaining dilation, whereas neither endothelial denudation nor Ba(2+) alone reduced the dilation. Multivariate analysis revealed that cigarette smoking was the only risk factor associated with impaired dilation to K(+). Ouabain significantly reduced the vasodilation in HCAs from subjects without cigarette smoking but not in those with smoking. Cigarette smoking downregulated the expression of the Na(+)-K(+)-ATPase catalytic α(1)-subunit but not Kir2.1 in the vessels. Ouabain abolished the dilation in endothelium-denuded vessels to a same extent to that with the combination of ouabain and Ba(2+) in endothelium-intact vessels, whereas neither ouabain nor ouabain plus Ba(2+) reduced EDHF-mediated dilations to bradykinin and ADP. A rise in [K(+)](o) dilates HCAs primarily via the activation of Na(+)-K(+)-ATPase in vascular smooth muscle cells with a considerable contribution of K(ir) channels in the endothelium, indicating that [K(+)](o) may modify coronary microvascular resistance in humans. Na(+)-K(+)-ATPase activity is impaired in subjects who smoke, possibly contributing to dysregulation of the coronary microcirculation, excess ischemia, and arrhythmogenesis in those subjects. K(+) does not likely serve as an EDHF in the human coronary arteriolar dilation to bradykinin and ADP.     

Eimeria tenella: immunogenicity of arrested sporozoites in chickens

Groups of chickens were medicated with the anticoccidial drug, decoquinate, and starting 1 day after this medication they were given daily inoculations of either 1 X 10(4) (Experiment 1) or 1 X 10(5) (Experiment 2) oocysts of a decoquinate-sensitive strain of Eimeria tenella. This assured the presence of large numbers of drug-inhibited sporozoites in the cecal tissues. The immunity arising from the presence of these inhibited sporozoites was assessed by challenging the medicated chickens with a 2.5 X 10(5) oocysts of a decoquinate-resistant strain of E. tenella. The response to challenge was assessed by weight gain, the severity of cecal lesions, hematocrits, and cecal oocyst numbers. The inhibited sporozoites promoted little (if any) immunity judged by clinical signs of disease. However, judged by body weight changes after challenge, the presence of inhibited sporozoites provided substantial protection against the body-weight-depressing effects of the challenge dose. These findings emphasize the importance of stage-specific antigen expression in Eimeria spp. infections and support the notion that immunogenicity is associated with tropic stages of the parasite.     

Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase

Although inhibition of the sarcolemmal (SL) Na(+)-K(+)-ATPase is known to cause an increase in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) by stimulating the SL Na(+)/Ca(2+) exchanger (NCX), the involvement of other SL sites in inducing this increase in [Ca(2+)](i) is not fully understood. Isolated rat cardiomyocytes were treated with or without different agents that modify Ca(2+) movements by affecting various SL sites and were then exposed to ouabain. Ouabain was observed to increase the basal levels of both [Ca(2+)](i) and intracellular Na(+) concentration ([Na(+)](i)) as well as to augment the KCl-induced increases in both [Ca(2+)](i) and [Na(+)](i) in a concentration-dependent manner. The ouabain-induced changes in [Na(+)](i) and [Ca(2+)](i) were attenuated by treatment with inhibitors of SL Na(+)/H(+) exchanger and SL Na(+) channels. Both the ouabain-induced increase in basal [Ca(2+)](i) and augmentation of the KCl response were markedly decreased when cardiomyocytes were exposed to 0-10 mM Na(+). Inhibitors of SL NCX depressed but decreasing extracellular Na(+) from 105-35 mM augmented the ouabain-induced increase in basal [Ca(2+)](i) and the KCl response. Not only was the increase in [Ca(2+)](i) by ouabain dependent on the extracellular Ca(2+) concentration, but it was also attenuated by inhibitors of SL L-type Ca(2+) channels and store-operated Ca(2+) channels (SOC). Unlike the SL L-type Ca(2+)-channel blocker, the blockers of SL Na(+) channel and SL SOC, when used in combination with SL NCX inhibitor, showed additive effects in reducing the ouabain-induced increase in basal [Ca(2+)](i). These results support the view that in addition to SL NCX, SL L-type Ca(2+) channels and SL SOC may be involved in raising [Ca(2+)](i) on inhibition of the SL Na(+)-K(+)-ATPase by ouabain. Furthermore, both SL Na(+)/H(+) exchanger and Na(+) channels play a critical role in the ouabain-induced Ca(2+) increase in cardiomyocytes.     

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.     

Studies on (Na+-K+)-activated ATPase XXXIV. Phosphatidylserine not essential for (Na+-K+)-ATPase activity

An (Na⁺-K⁺)-ATPase preparation, consisting of NaI-treated microsomes from cattle brain, was incubated with a phosphatidylserine decarboxylase preparation from Escherichia coli. This led to a reduction in the phosphatidylserine content from 10.1 % to less than 0.1%, accompanied by an equimolar formation of phosphatidylethanolamine. Since the (Na⁺-K⁺)-ATPase activity was not reduced, it can be concluded that phosphatidylserine is not essential for the Na⁺-K⁺)-ATPase activity.     

Effects of 20-HETE on Na+ transport and Na+ -K+ -ATPase activity in the thick ascending loop of Henle

Previous studies have indicated that 20-hydroxyeicosatetraenoic acid (20-HETE) inhibits Na+ transport in the medullary thick ascending loop of Henle (mTALH), but the mechanisms involved remain uncertain. The present study compared the effects of 20-HETE with those of ouabain and furosemide on intracellular Na+ concentration ([Na+]i), Na+ -K+ -ATPase activity, and 86Rb+ uptake, an index of Na+ transport, in mTALH isolated from rats. Ouabain (2 mM) increased, whereas furosemide (100 microM) decreased, [Na+]i in the mTALH of rats. Ouabain and furosemide inhibited 86Rb+ uptake by 91 and 30%, respectively. 20-HETE (1 microM) had a similar effect as ouabain and increased [Na+]i from 19 +/- 1 to 30 +/- 1 mM. 20-HETE reduced Na+ -K+ -ATPase activity by 30% and 86Rb+ uptake by 37%, but it had no effect on 86Rb+ uptake or [Na+]i in the mTALH of rats pretreated with ouabain. 20-HETE inhibited 86Rb+ uptake by 12% and increased [Na+]i by 19 mM in mTALH pretreated with furosemide. These findings indicate that 20-HETE secondarily inhibits Na+ transport in the mTALH of the rat, at least, in part by inhibiting the Na+ -K+ -ATPase activity and raising [Na+]i.