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1.
Na-K-ATPase activity of the rat heart was similar throughout the postnatal growth when measured from crude unpurified fraction. Instead in the cardiac sarcolemmal fraction, isolated by hypotonic shock LiBr-treatment method, the activity was over two times higher in 10-day old neonates than in adult rats. The conflicting results are partly explained by different effects of the isolation procedure on neonatal and adult tissues. Na concentration for half-maximal activity of the Na-K-ATPase was similar in neonates (7.0 mM) and adults (6.4 mM). Ca-ATPase activity was not affected by Na concentration (2-100 mM) in the two age-groups studied.  相似文献   

2.
1. Concentrations of 0.1-10 mM Ouabain do not affect oscillating contractions, when applied externally in physiological solutions. 2. Ouabain has no effect, when applied in solutions of increased sodium- (100 mM) and lowered potassium-concentration (0.1 or zero mM). 3. De novo generation of oscillating contractions in protoplasmic drops is not suppressed in Ouabain-solutions. 4. Biochemical studies of Na-K-ATPase did not show any Ouabain sensitive ATPase-activity. 5. Reaction of veins on high concentrations (300 mosm) is discussed as to be a physical effect. 6. It is concluded that no Na-K-ATPase is engaged in triggering oscillating contraction automaticity.  相似文献   

3.
Posterior gills (No. 7 and 8) of shore crabsCarcinus maenas were homogenized and fractionated by means of differential and density gradient centrifugation. Employment of marker enzymes Na-K-ATPase and carbonic anhydrase for plasma membranes and cytochrome oxidase for mitochondria showed that these structural elements were separated. Ultramicroscopic investigations of combined fractions confirmed the presence of the respective mitochondrial and vesicular plasma membrane structures. An ATPase which did not depend on the presence of sodium (20 mM) ions in the incubation medium but on the presence of potassium (20 mM) ions only was found in the mitochondrial fractions. The mitochondrial ATPase was tightly bound to cellular particulates and activated approximately threefold by bicarbonate (20 mM) ions. The activity of this ATPase was nearly completely inhibited by oligomycin (1 μg ml−1) and greatly inhibited by low levels (5 mM) of thiocyanate and calcium ions, the Ki for Ca2+ being ca 4 mM. The results obtained confirm literature data on high mitochondrial densities in crab gills and allow the assumption of significant rates of energy metabolism in these organs. Considering its properties the mitochondrial ATPase is clearly distinct from crab gill Na-K-ATPase and can be measured specifically in samples containing Na-K-ATPase. Mitochondrial ATPase is therefore considered a suitable and reliable marker enzyme for mitochondria.  相似文献   

4.
The dog tracheal epithelium actively secretes Cl and absorbs Na. The possible dependency of this electrolyte transport on a Mg-dependent, Na-K-activated adenosine triphosphatase (Na-K-ATPase, EC 3.6.1.3) was examined. The characteristics of this enzyme system were investigated using homogenates of tracheal epithelium. The electrical properties and ion fluxes of this epithelium were determined in tissues mounted in Ussing chambers. Addition of Na and K produced an approximate 50% activation of basal Mg-ATPase activity. The apparent Km values for ATP, Na, K, and Mg were 0.4, 12.7, 1.9, and 1.6 mM, respectively. The total specific ATPase activity was 8.1 +/- 0.4 and that of the Mg-ATPase 4.3 +/- 0.1 mumol Pi. mg protein -1.h-1. Addition of ouabain (1 muM) or omission of K from the submucosal bathing solution reduced potential difference (PD) and short-circuit current (SCC) significantly. Relatively low concentrations (0.1 mM or less) of ethacrynic acid, furosemide, or 2,4-dinitrophenol (2,4-DNP) depressed SCC and PD significantly, i.e., at concentrations that were without effect on the Na-K-ATPase activity. Ethacrynic acid inhibited Cl secretion, whereas 2,4-DNP lowered both Na and Cl transport. These data demonstrate that 1) the tracheal mucosa of dogs contains a Na-K-ATPase at relatively high specific activity, 2) this enzyme is likely contained in the basal aspect of this membrane, 3) it appears to be essential for maintenance of Cl secretion, and 4) Cl secretion can be reduced (by ethacrynic acid, furosemide, and 2,4-DNP) without Na-K-ATPase inhibition.  相似文献   

5.
Amines and amides were found to inhibit the process of stimulation of sugar transport in muscle tissue (N. A. Vinogradova et al., 1978). The present paper reports results of experiments on frog sartorius muscles acted upon by amines and other substances that inhibit induction of cultured cell proliferation. The stimulation of sugar transport induced by insulin, 2,4-dinitrophenol, or KCl was found to be inhibited by dansylcadaverine (1 mM), 5-methoxytryptamine (2 mM), or methylamine (100 mM). Such substances as chloroquine, bacitracin, or monensin exerted no effect. Besides, dansylcadaverine (1 mM), and 5-methoxytryptamine inhibited the stimulation of insulin induced glycogen synthesis. Dansylcadaverine was ineffective at concentrations lower than 0.5 mM. It is suggested that the inhibitory action of amines depends on their influence on the processes of membrane protein phosphorylation.  相似文献   

6.
The effects of light deprivation from birth to 40 days of age on the development of Na-K-ATPase and Mg-ATPase activity, enzymes significantly involved in cerebral energy exchange, ion transport, and synaptic function, were investigated in visual and non-visual brain areas of the rat. Although both enzymes generally showed a progressive depression with age in light-deprived rats, Na-K-ATPase was more depressed than Mg-ATPase, and significant effects were confined to the superior colliculi, visual cortex, frontal cortex and hypothalamus. A disparate developmental pattern was evidenced in Na-K-ATPase activity in the visual cortex, where it was higher than control values at day 10 but lower by day 40, and in the hypothalamus, where it was lower on days 10 and 25 but significantly higher on day 40. The depression of Mg-ATPase in the hypothalamus of light-deprived rats at all ages and the activation of Na-K-ATPase in this structure is interpreted to mean that discrete alterations may have occurred in neurosecretory functions of the hypothalamus, known to be responsive to light. Transferring dark-reared animals to normal light-cycle conditions at day 25 affected only Mg-ATPase in the visual cortex and Na-K-ATPase in the hypothalamus, both enzymes showing a significant increase by day 40 over values in continually light-deprived animals. These findings confirm that early light deprivation is associated with important biochemical and neuroendocrine changes that persist into adulthood.  相似文献   

7.
To evaluate a possible role of ornithine-delta-aminotransferase (EC 2.6.1.13; Orn-T) as a rate-limiting enzyme for the synthesis of transmitter glutamate and gamma-aminobutyric acid (GABA), respectively, its activity and kinetic properties were analyzed in cultured astrocytes as well as in neuronal cultures consisting mainly of glutamatergic neurons (cerebellar granule cells) or GABAergic neurons (cerebral cortex interneurons). For comparison the activity and kinetics of Orn-T were also assayed in mouse brain homogenates. The highest activity of Orn-T was found in astrocytes and in cerebral cortical neurons (5.3 +/- 0.5 and 5.3 +/- 0.4 nmol X mg-1 X min-1, respectively) whereas the activities of Orn-T in cerebellar granule cell cultures and in mouse brain were found to be about half of these values (3.1 +/- 0.3 and 2.8 +/- 0.1 nmol X min-1 X mg-1, respectively). From a kinetic study of Orn-T in the different preparations only a relatively low affinity for the enzyme with respect to ornithine was found in cerebellar granule cells, astrocytes, and whole brain [apparent Km values (at 0.5 mM alpha-ketoglutarate): 4.7 +/- 0.9, 4.3 +/- 2.2, and 6.8 +/- 2.2 mM, respectively] whereas the corresponding Km value for Orn-T in cerebral cortex interneurons was found to be significantly lower (apparent Km: 0.8 +/- 0.3 mM). The enzyme was not found to be inhibited by GABA (range 0.1 - 10 mM) in any of the preparations.  相似文献   

8.
The enzyme catalysing the polyamine-stimulated modification of Physarum ornithine decarboxylase in vivo was partially purified and its activity on purified ornithine decarboxylase was examined with respect to its specificity for various amines. Spermidine, spermine and several polyamine analogues strongly promoted this reaction in vitro (apparent Km in the 0.1--0.5 mM range), whereas putrescine (apparent Km 5.33 mM) and several related diamines were not nearly as effective. In agreement with this, sensitivity studies performed in vivo also suggested that cellular spermidine, and not putrescine, is critical in modulating ornithine decarboxylase activity by this post-translational control. Unlike putrescine, or other diamines, 1,3-diaminopropane demonstrated a functional similarity to the polyamines in stimulating this reaction. This study has demonstrated a method whereby non-physiological amines capable of depressing ornithine decarboxylase activity by this natural feedback mechanism can be readily identified for further evaluation of their potential use in the experimental and medical control of polyamine biosynthesis.  相似文献   

9.
The ouabain sensitive and K+-dependent p-nitrophenyl-phosphatase was inhibited by polyamines. The order of effectiveness was spermine spermidine putrescine = cadaverine. The half maximum inhibition concentration of spermine was approximately 0.03 mM and 0.8 mM in the presence of 0.5 mM and 3.0 mM KCl in the reaction mixtures, respectively. Basic amino acids and hydroxylamine inhibited slightly. Other amines such as glycine and histamine were without effect. Spermine did not inhibit other membrane bound phosphatases, such as glucose-6-phosphatase, 5′-nucleotidase, alkaline phosphatase and ouabain insensitive p-nitrophenylphosphatase activity at pH 7.5  相似文献   

10.
Several amines were shown to inhibit growth stimulation in quiescent confluent and sparse cultures of Swiss 3T3 and 3T6 cells by changing for a fresh medium containing 10-20% serum. Proliferation was inhibited by dansylcadaverine (0.1 mM), chloroquine (0.05 mM), 5-methoxytryptamine (0.1 mM), cystamine (0.1 mM), dimethylurea (100 mM), methylurea (100 mM), and in some experiments--by urea (100 mM). The inhibitory activity was not associated with a direct influence of amines on DNA synthesis or thymidine phosphorylation. The findings suggest that amines may influence the process of clustering of growth factor-receptor complexes, or the fusion of plasma membrane and intracellular vesicles containing some components required for cell proliferation.  相似文献   

11.
The effects of endothelin on cellular Ca2+ mobilization were examined in cultured rat vascular smooth muscle cells (VSMC). Endothelin (10(-8)M) induced a rapid transient increase of [Ca2+]i from 77 +/- 3 to 104 +/- 5 nM (p less than .05) in VSMC. Preincubation (60 min) with endothelin (2 x 10(-6)M) increased basal [Ca2+]i from 77 +/- 3 to 105 +/- 8 nM (p less than .05). Preincubation with endothelin also enhanced vasopressin (10(-7)M)-stimulated peak levels of [Ca2+]i (528 +/- 20 nM vs 969 +/- 21 nM, p less than .01). Endothelin (10(-7)M) induced an intracellular alkalinization (7.18 +/- 0.03 vs 7.37 +/- 0.04, p less than .01) which was blocked by pretreatment with amiloride. The biphasic effects of endothelin on [Ca2+]i were similar to those of an endogenous inhibitor of Na-K-ATPase that we examined in a previous study. Therefore, we examined the effects of endothelin on Na-K-ATPase in an enzyme preparation from hog cerebral cortex. At high concentrations, endothelin (10(-5)M) inhibited Na-K-ATPase in vitro. Thus, endothelin may exert its vasoconstrictor effects at least in part via alterations of cellular Ca2+ mobilization in VSMC. While the rapid transient increase of [Ca2+]i appears to reflect intracellular Ca2+ mobilization, the sustained effect on [Ca2+]i may be related to an increase of intracellular sodium mediated by inhibition of Na-K-ATPase and/or more likely by stimulation of the Na+/H+-antiport.  相似文献   

12.
The ion gradients generated by the Na-K-ATPase play a critical role in epithelia by driving transepithelial transport of various solutes. The efficiency of this Na-K-ATPase-driven vectorial transport depends on the integrity of epithelial junctions that maintain polar distribution of membrane transporters, including the basolateral sodium pump, and restrict paracellular diffusion of solutes. The review summarizes the data showing that, in addition to pumping ions, the Na-K-ATPase located at the sites of cell-cell junction acts as a cell adhesion molecule by interacting with the Na-K-ATPase of the adjacent cell in the intercellular space accompanied by anchoring to the cytoskeleton in the cytoplasm. The review also discusses the experimental evidence on the importance of a specific amino acid region in the extracellular domain of the Na-K-ATPase β(1) subunit for the Na-K-ATPase trans-dimerization and intercellular adhesion. Furthermore, a possible role of N-glycans linked to the Na-K-ATPase β(1) subunit in regulation of epithelial junctions by modulating β(1)-β(1) interactions is discussed.  相似文献   

13.
Gastric K+-stimulated p-nitrophenylphosphatase cytochemistry   总被引:1,自引:0,他引:1  
K Fujimoto  K S Ogawa  K Ogawa 《Histochemistry》1986,84(4-6):600-608
A cytochemical study of gastric K+-stimulated p-nitrophenylphosphatase (K-NPPase) activity, corresponding to a K+-stimulated phosphoprotein phosphatase of H-K-ATPase system, has been made by a new cytochemical method. Sections of fixed guinea pig gastric mucosa in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde, were incubated with the incubation medium (1.0 M glycine-0.1 M KOH buffer, pH 9.0, 2.5 ml; 1.1 M KCl, 0.5 ml; 10 mM lead citrate dissolved in 50 mM KOH, 4 ml; levamisole, 6.0 mg; dimethyl sulfoxide, 2.0 ml; 0.1 M p-nitrophenylphosphate (Mg-salt), 1.0 ml; ouabain, 73.0 mg) for 30 min at room temperature. Under a light microscope the specific gastric K-NPPase reaction was distributed only in the parietal cells of the fundic glands. The electron microscopic cytochemistry showed that the gastric K-NPPase activity was localized on the membrane lining the apical surfaces, secretory canaliculi and tubulovesicles. On the other hand, ouabain-sensitive K-NPPase activity (Na-K-ATPase) was demonstrated to localize only in the basolateral membrane of parietal cells with Mayahara's method. These findings support the interrelationships between the apical surface membrane, secretory canalicular membrane and tubulovesicles, and the functional differentiation of the membrane between the secretory membrane and basolateral membrane.  相似文献   

14.
Mechanical ventilation with high tidal volumes (HV(T)) impairs lung liquid clearance (LLC) and downregulates alveolar epithelial Na-K-ATPase. We have previously reported that the Na-K-ATPase alpha(2)-subunit contributes to LLC in normal rat lungs. Here we tested whether overexpression of Na-K-ATPase alpha(2)-subunit in the alveolar epithelium would increase clearance in a HV(T) model of lung injury. We infected rat lungs with a replication-incompetent adenovirus that expresses Na-K-ATPase alpha(2)-subunit gene (Adalpha(2)) 7 days before HV(T) mechanical ventilation. HV(T) ventilation decreased LLC by approximately 50% in untreated, sham, and Adnull-infected rats. Overexpression of Na-K-ATPase alpha(2)-subunit prevented the decrease in clearance caused by HV(T) and was associated with significant increases in Na-K-ATPase alpha(2) protein abundance and activity in peripheral lung basolateral membrane fractions. Ouabain at 10(-5) M, a concentration that inhibits the alpha(2) but not the Na-K-ATPase alpha(1), decreased LLC in Adalpha(2)-infected rats to the same level as sham and Adnull-infected lungs, suggesting that the increased clearance in Adalpha(2) lungs was due to Na-K-ATPase alpha(2) expression and activity. In summary, we provide evidence that augmentation of the Na-K-ATPase alpha(2)-subunit, via gene transfer, may accelerate LLC in the injured lung.  相似文献   

15.
Escherichia coli RNA polymerase was assayed with 4 mM Mg2+ and 1 mM Mn2+ using native DNA, heat-denatured DNA, histone-nucleate and isolated rat liver nuclei as the template source. With purified DNA and either or both divalent metal ions, 0.1--5 mM amine stimulated enzyme activity. Spermidine resulted in the greatest stimulation (1.7-fold at 5 mM); whereas, spermine or methylglyoxal bis(guanylhydrazone) first stimulated, then above 3 mM inhibited, the reaction. The addition of unfractionated histone to purified DNA inhibited the reaction by 90%. The subsequent addition of amines resulted in a slight stimulation in incorporation (1.5-fold) in the range of 1--3 mM amine. Alternatively, when enzyme was combined with DNA before histone, only a 20% inhibition was observed and this could be completely prevented by 3 mM spermidine. The addition of amines to isolated nuclei resulted in marked alterations in ultrastructure and Mg2+ content; however, relatively small effects on RNA polymerase activity were observed. With the E. coli enzyme, 0.1--1.0 mM amine stimulated RNA synthesis (1.5-fold) whereas, none of the amines stimulated endogeneous activity in the absence or presence of 300 mM (NH4)2SO4.  相似文献   

16.
Purification and fluorometric assay of proteinase A from yeast   总被引:2,自引:0,他引:2  
A kinetic assay system which provides reliable measurements of Na-K-ATPase activity on 0.2 to 0.5-mm segments of renal proximal convoluted tubules isolated from collagenase-digested renal cortical slices is described. The use of collagenase digestion provides higher values for Na-K-ATPase, possibly by making the enzyme more accessible to the reaction system. The advantages of a kinetic vs an endpoint assay include the ability to use the same tubule as its own reference for the determination of total, ouabain-sensitive, and ouabain-insensitive ATPase activity. In addition, it allows dose-response studies on the effect of inhibitors on ATPase activity in the same tubule segment.  相似文献   

17.
Thyroid hormone (T3) increases Na-K-ATPase activity in rat adult alveolar type II cells via a PI3K-dependent pathway. In these cells, dopamine and beta-adrenergic agonists can stimulate Na-K-ATPase activity through either PI3K or MAPK pathways. We assessed the role of the MAPK pathway in the stimulation of Na-K-ATPase by T3. In the adult rat alveolar type II-like cell line MP48, T3 enhanced MAPK/ERK1/2 activity in a dose-dependent manner. Increased ERK1/2 phosphorylation was observed within 5 min, peaked at 20 min, and then decreased. Two MEK1/2 inhibitors, U0126 and PD-98059, each abolished the T3-induced increase in the quantity of Na-K-ATPase alpha(1)-subunit plasma membrane protein and Na-K-ATPase activity. T3 also increased the phosphorylation of MAPK/p38; however, SB-203580, a specific inhibitor of MAPK/p38 activity, did not prevent the T3-induced Na-K-ATPase activity. SP-600125, a specific inhibitor of the MAPK/JNK pathway, also did not block the T3-induced Na-K-ATPase activity. Phorbol 12-myristate 13-acetate (PMA) significantly increased ERK1/2 phosphorylation and Na-K-ATPase activity. The PMA-induced Na-K-ATPase activity was inhibited by U0126. These data indicate that activation of MAPK-ERK1/2 was required for the T3-induced increase in Na-K-ATPase activity in addition to the requirement for the PI3K pathway.  相似文献   

18.
A novel in situ kidney perfusion technique is described in Sprague-Dawley rats. The procedure involves retrograde perfusion from the renal veins via the kidneys, and then through the renal arteries and dorsal aorta. Ouabain (15 mM) in perfusate increased Na retention by 92%, decreased K retention by 53% and produced no effect on Cl retention. Ethacrynic acid (1 mM) in perfusate decreased Na retention by 52%, increased K retention by 105% and decreased Cl retention by 27%. Furosemide (1.5 mM) in perfusate decreased Na retention by 52%, increased K retention by 47% and decreased Cl retention by 56%. The Na-K-ATPase pump localized at the peritubular side of the proximal tubule cell is ouabain sensitive and Mg dependent. An Na-K pump responsible for Na influx and K effux exists at the luminal side of the proximal tubule cell and is ethacrynic acid and furosemide sensitive.  相似文献   

19.
Angiotensin (ANG) II via ANG II type 1 receptors (AT1R) activates renal sodium transporters including Na-K-ATPase and regulates sodium homeostasis and blood pressure. It is reported that at a high concentration, ANG II either inhibits or fails to stimulate Na-K-ATPase. However, the mechanisms for these phenomena are not clear. Here, we identified the signaling molecules involved in regulation of renal proximal tubular Na-K-ATPase at high ANG II concentrations. Proximal tubules from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were incubated with low concentrations of ANG II (pM), which activated Na-K-ATPase in both the groups; however, the stimulation was more robust in SHR. A high concentration of ANG II (μM) failed to stimulate Na-K-ATPase in WKY rats. However, in SHR ANG II (μM) continued to stimulate Na-K-ATPase, which was sensitive to the AT1R antagonist candesartan. In the presence of N(G)-nitro-l-arginine methyl ester (l-NAME), a nitric oxide (NO) synthase (NOS) inhibitor, ANG II (μM) caused stimulation of Na-K-ATPase in proximal tubules of WKY rats while having no further stimulatory effect in SHR. ANG II (μM), via AT1R, increased proximal tubular NO levels in WKY rats but not in SHR. In SHR, NOS was uncoupled as incubation of proximal tubules with ANG II and l-arginine, a NOS substrate, caused superoxide generation only in SHR and not in WKY rats. The superoxide production in SHR was sensitive to l-NAME. There was exaggerated proximal tubular AT1R-G protein coupling and NAD(P)H oxidase activation in response to ANG II (μM) in proximal tubules of SHR compared with WKY rats. In SHR, inhibition of NADPH oxidase restored NOS coupling and ANG II-induced NO accumulation. In conclusion, at a high concentration ANG II (μM) activates renal NO signaling, which prevents stimulation of Na-K-ATPase in WKY rats. However, in SHR ANG II (μM) overstimulates NADPH oxidase, which impairs the NO system and leads to continued Na-K-ATPase activation.  相似文献   

20.
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