An improved approach for determination of Na(+)-K(+)-ATPase activity in single proximal renal tubule of rat reported by Doucet and his colleagues has been suggested. The single proximal renal tubules were isolated by hand under stereomicroscope from collagenase II-treated renal cortical tissue in rats. The length of every single renal tubule segment obtained was measured. The single proximal renal tubules were treated with a hypoosmotic solution and with freeze-thaw successively, before incubation with [gamma-(32)P]-ATP. (32)Pi hydrolyzed from [gamma-(32)P]ATP by Na(+)-K(+)-ATPase in the single proximal renal tubules was assayed by liquid scintillation counting. The activity of Na(+)-K(+)-ATPase in the single proximal renal tubules was calculated by applying a modified formula. There was no significant difference in the measurement result of Na(+)-K(+)-ATPase activity between the method of Doucet et al. and the improved one, but the latter has advantage of less time, less reagents and being easy to operate.
Adenosine Triphosphate ; metabolism ; Animals ; Kidney Tubules, Proximal ; enzymology ; Male ; Microscopy ; Rats ; Rats, Wistar ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Time Factors

AIMTo study the effect of Na+, K(+)-ATPase inhibition by ouabain on growth and death of vascular endothelial cells ECV304 and involved mechanisms.

METHODSGrowth inhibition of ouabain on ECV304 cells was analyzed using MTT assay. The feature of cell death was studied by Hoechst 33342/PI staining, transmission electron microscopy and DNA agarose gel electrophoresis in ECV304 cells treated with ouabain. The mRNA expression of Na+, K(+)-ATPase alpha1, beta1-subunit was examined by reverse transcription PCR (RT-PCR).

RESULTSOuabain inhibited the growth of ECV304 cells in a dose and time-dependent manner. 10 micromol/L ouabain treated for 24 hours could stimulate the necrosis of ECV304 cells; When treated with 0.1 micromol/L ouabain for 24-48 hours, the cells showed obviously defluxion, the loss of cell-cell contacts, nuclear chromatin condensation, chromatin margination and DNA fragmentation. Na+, K(+)-ATPase alpha1-subunit mRNA expression was significantly up-regulated in ECV304 cells treated with ouabain while the beta1-subunit expression conversely showed a significant decrease.

CONCLUSIONOuabain could up-regulate Na+, K(+)-ATPase alpha1-Subunit expression and reduce beta1-Subunit expression which mediated signal transduction and decreased cell-cell adhesions and induced ECV304 cells death.

Cell Death ; drug effects ; Cell Line ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Ouabain ; pharmacology ; Sodium-Potassium-Exchanging ATPase ; metabolism

OBJECTIVETo explore the mechanism of cytotoxic effect of 2, 5-hexanedione (2, 5-HD) on motor neuron.

METHODSVsc4.1 (a cell line from motor neuron) was incubated with a series concentration of 2, 5-HD. The cell viability, Ca(2+) Mg(2+) ATPase and Na(+)K(+) ATPase were detected. Laser scanning confocal microscope (LSCM) was used for detecting intracellular calcium level. The average calcium level in VSC4.1 was measured by flow cytometry.

RESULTSThe cell viability was decreased when Vsc4.1 cells were treated with 2, 5-HD at the dosage of 2.5, 5.0, 7.5, 10, 15 and 20 mmol/L for 24 hours. Compared with the control group the activity of Ca(2+) Mg(2+) ATPase was decreased to 70.02%, 77.44% and 47.47% respectively; the activity of Na(+)K(+) ATPase was decreased to 82.07%, 72.45% and 50.71%. The difference was significant. Intracellular free calcium of VSC4.1 cell was increased rapidly within 10 s and then recovered within 40 seconds when it was exposed to 33.5 mmol/L 2, 5-HD. An increase in intracellular calcium was observed when the VSC4.1 was treated with 33.5 mmol/L 2, 5-HD. The peak of intracellular calcium level occurred ten minutes later.

CONCLUSIONThe disturbance of calcium homeostasis may be involved in the mechanisms of neurotoxicity of 2, 5-HD.

Animals ; Ca(2+) Mg(2+)-ATPase ; metabolism ; Calcium ; metabolism ; Cell Line ; Dose-Response Relationship, Drug ; Hexanones ; toxicity ; Motor Neurons ; drug effects ; metabolism ; Rats ; Sodium-Potassium-Exchanging ATPase ; metabolism

The present study was designed to investigate the effect of administration of adrenomedullin (ADM) into subfornical organ (SFO) on renal tubular Na(+),K(+)-ATPase activity in rats. Rats under anesthesia were injected with ADM 0.1 mL (20 ng/mL) via an implanted cannula into SFO (n=6). Plasma ADM and serum endogenous digitalis-like factor (EDLF) levels were assayed with radioimmunoassay, and urine samples were collected via a canoula intubated in bladder. Urinary sodium concentration was assayed with flame spectrophotometry. Single proximal renal tubule segments were obtained by hand under stereomicroscope and its Na(+),K(+)-ATPase activity was measured by liquid scintillation counting. In addition, single proximal renal tubule segments from normal rats (n=6) were incubated with serum from animals administered with ADM into SFO, and then the Na(+),K(+)-ATPase activity was determined. The results showed that both urinary volume and sodium excretion amounted to the peak value at 30 min after ADM administration, and sustained a significant high level at 60 min (P<0.01). At 30 min after ADM administration, there was a significant increase in serum EDLF and a decrease in Na(+),K(+)-ATPase activity of proximal tubule (P<0.01, respectively), but not in plasma ADM level. Na(+),K(+)-ATPase activity was decreased significantly in single proximal renal tubule segments from normal rats incubated with serum from rats administered with ADM into SFO (P<0.01). These results suggest that the diuretic and natriuretic responses following administration of ADM into SFO are associated with the inhibition of renal tubule Na(+),K(+)-ATPase activity. The inhibition of renal tubule Na(+),K(+)-ATPase activity is related to the increase in the serum level of EDLF.
Adrenomedullin ; pharmacology ; Animals ; Kidney Tubules, Proximal ; drug effects ; enzymology ; Rats ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Subfornical Organ

Animals ; Enzyme Inhibitors ; pharmacology ; Hepatitis ; immunology ; Immune Sera ; pharmacology ; Mice ; Sodium-Potassium-Exchanging ATPase ; antagonists & inhibitors ; metabolism

OBJECTIVETo explore the effects of low temperature on the functions of learning and memory and activities of ATPase in brain tissue of mice.

METHODS120 mice were randomly divided into 3 groups of A, B, and C with different cold exposure time. After low temperature test, learning and memory ability and activities of ATPase in brain tissue of the mice were measured.

RESULTSCompared with corresponding control group, in the test of learning ability, the total electric shock period [(41.00 +/- 12.06), (45.90 +/- 13.61) min], the total electric times (85.00 +/- 15.81, 89.00 +/- 17.29), and the error reaction times (33.60 +/- 10.69, 39.00 +/- 11.63) were increased in group A and group C significantly (P < 0.01, P < 0.05), but the rate of right reaction (53.60% +/- 11.23%, 54.59% +/- 6.14%) were decreased in group A and group C significantly (P < 0.01, P < 0.05). In the test of memory ability, the total electric shock period [(19.00 +/- 4.62), (51.70 +/- 15.19) min] in group A and group C were increased significantly (P < 0.05, P < 0.01), the rate of right reaction (86.17% +/- 6.34%, 65.92% +/- 8.17%) in group A and group C were decreased significantly (P < 0.05). In test of activities of ATPase in brain tissue, the activities of Na(+)-K(+)-ATPase and activities of Ca(2+)-Mg(2+)-ATPase in brain tissue in group A and group C were decreased significantly (P < 0.05, P < 0.01).

CONCLUSIONLow temperature could decrease the functions of learning and memory and the activities of Na+-K+-ATPase and Ca(2+)-Mg(2+)-ATPase in brain tissue of mice.

Animals ; Brain ; enzymology ; Ca(2+) Mg(2+)-ATPase ; metabolism ; Cold Temperature ; adverse effects ; Environmental Exposure ; adverse effects ; Female ; Male ; Maze Learning ; Memory ; Mice ; Sodium-Potassium-Exchanging ATPase ; metabolism

The presence of serum in a culture medium makes it impossible to identify whether changed cellular functions are directly caused by a manipulation itself or mediated by a component in serum. Madin Darby canine kidney cells can survive in a serum-free medium for about 48 h. We took this advantage to examine whether low K(+)-induced up-regulation of Na,K-ATPase requires serum. We found that serum was essential for low K(+) to induce an increase in Na,K-ATPase binding sites as quantified by ouabain factor binding assays. In an attempt to identify which component was critical, we screened EGF, IGF1, PGE1 and transferrin to identify which one can replace serum. We discovered that transferrin was the single most important factor that mimicked about 80% to 90% of the effect of serum. Transferrin potentiated the effect of low K(+) on the Na,K-ATPase binding sites in a time- and dose-dependent manner. Furthermore, transferrin was also required for low K(+)-induced increase in alpha(1)-promoter activity, alpha(1)- and beta(1)-subunit protein abundance of the Na,K-ATPase. In the presence of transferrin, low K(+) enhanced cellular uptake of iron approximately by 70%. Inhibition of intracellular iron activity by deferoxamine (30 micromol/L) abrogated the effect of low K(+). We conclude that stimulation of the Na,K-ATPase by low K(+) is critically dependent on transferrin. The effect of transferrin is mediated by increased iron transport.
Animals ; Cell Line ; Culture Media, Serum-Free ; Dogs ; Kidney ; cytology ; Potassium ; metabolism ; pharmacology ; Sodium-Potassium-Exchanging ATPase ; genetics ; metabolism ; Transferrin ; metabolism ; pharmacology

OBJECTIVETo study the mutual interaction of vestibular afferent nervous system and vestibular efferent nervous system in vestibular compensation.

METHODSBuild up animal model of vestibular compensation by destroying single side vestibule of wistar rat. In the study the rats were divided into 3 groups: Group A 16 normal rats; Group B 15 rats, after 7 days of left vestibular damage; Group C 7 rats 3 months after left vestibular damage; and Group D 7 rats, after vestibular compensation. Electromyography of the rats was recorded and the expression of calcitonin gene relative peptide (CGRP), choline acetyltransferase (AChT) and Na-K-ATPase were investigated in efferent vestibular nervous system.

RESULTSElectric potential activity of muscles of injury side decreased while that of the opposite side increased. In animals of vestibular compensation electric potential of bilateral musculus longus capitis at quiescent stage recovered symmetrically. CGRP positive cells of efferent vestibular nervous system increased bilaterally, and their activity enhanced, especially obvious at the acute stage. AChT positive cells of injury side of efferent vestibular nervous system decreased, but reaction degree of two sides enhanced. Reaction degree of the opposite side enhanced obviously at the stage of vestibular compensation. Expression of Na-K-ATPase mRNA of the same side was lower, but vestibular signal of the opposite side enhanced, clinically head and neck inclined obliquely by means of medial fasciculus of tractus vestibulospinalis. Months later, vestibular signal of the same side enhanced, and that of the opposite side enhanced also, clinical symptoms improved slightly. At the vestibular compensation stage, expression of Na-K-ATPase mRNA of the same side enhanced, and it was same as that of the opposite side or much higher, clinically it reached vestibular compensation.

CONCLUSIONComprehensive effect of the above results maybe as follows: Efferent vestibular nervous system inhibited afferent signal of the opposite vestibule, and it modulated excitement of vestibular center of the same side, and it worked in the complicated mechanisms of vestibular compensation. CGRP may have facilitation function to the vestibular afferent signal of injury side. While Ach improved vestibule compensation by means of inhibition of vestibule excitement of the healthy side.

Afferent Pathways ; metabolism ; Animals ; Efferent Pathways ; metabolism ; Male ; Rats ; Rats, Wistar ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Vestibular Nerve ; metabolism ; Vestibule, Labyrinth ; metabolism

Animals ; Brain ; metabolism ; Female ; Ginkgolides ; pharmacology ; Hypoxia ; metabolism ; Lactic Acid ; metabolism ; Male ; Malondialdehyde ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sodium-Potassium-Exchanging ATPase ; metabolism

OBJECTIVETo investigate the effect of phenylephrine (an α-adrenergic agonist) on alveolar fluid clearance (AFC) in ventilator-induced lung injury and the possible mechanism involved.

METHODSA total of 170 male Wistar rats were randomly allocated into 17 groups (n=10) using random number tables. Short-term (40 minutes) mechanical ventilation with high tidal volume (HVT) was performed to induce lung injury, impair active Na+ transport and lung liquid clearance in the rats. Unventilated rats served as controls. To demonstrate the effect of phenylephrine on AFC, phenylephrine at different concentrations (1×10(-5), 1×10(-6), 1×10(-7), 1×10(-8), and 1×10(-9) mol/L) was injected into the alveolar space of the HVT ventilated rats. To identify the influence of adrenergic antagonists, Na(+) channel, and microtubular system on the effect of phenylephrine, phenylephrine at 1×10(-5) mol/L combined with prazosin (an α1-adrenergic antagonist, 1×10(-4) mol/L), yohimbine (an α2-adrenergic antagonist, 1×10(-4) mol/L), atenolol (a β1- adrenergic antagonist, 1×10(-5) mol/L), ICI-118551 (an β2-adrenergic antagonist, 1×10(-5) mol/L), amiloride (a Na+ channel blocker, 5×10(-4) mol/L), ouabain (a Na(+)/K(+)-ATPase blocker, 5×10(-4) mol/L), colchicine (a microtubular disrupting agent, 0.25 mg/100 g body weight), or β-lumicolchicine (an isomer of colchicine, 0.25 mg/100 g body weight) were perfused into the alveolar space of the rats ventilated with HVT for 40 minutes. AFC and total lung water content were measured.

RESULTSBasal AFC in control rats was (17.47±2.56)%/hour, which decreased to (9.64± 1.32)%/hour in HVT ventilated rats (P=0.003). The perfusion of phenylephrine at 1×10(-8), 1×10(-7), 1×10(-6), and 1×10(-5) mol/L significantly increased the AFC in HVT ventilated rats (all P<0.05). This effect of phenylephrine on AFC was suppressed by prazosin, atenolol, and ICI-118551 in HVT ventilated rats by 53%, 31%, and 37%, respectively (all P<0.05). The AFC-stimulating effect of phenylephrine was lowered by 33% and 42% with amiloride and ouabain, respectively (both P<0.05). Colchicine significantly inhibited the effect of phenylephrine (P=0.031).

CONCLUSIONPhenylephrine could increase the AFC in HVT-ventilated rats and accelerate the absorption of pulmonary edema.

Animals ; Male ; Phenylephrine ; therapeutic use ; Pulmonary Alveoli ; metabolism ; Rats ; Rats, Wistar ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Ventilator-Induced Lung Injury ; drug therapy ; metabolism ; pathology