Activity Of Ouabain-sensitive Na Research Articles

Astrocyte membrane Na +, K +-ATPase and Mg 2+-ATPase as targets of organic solvent impact

Astrocytes serve to maintain the proper homeostatic environment for neurons. In these regulations, the astrocytic membrane ATPase plays an important role. In the present study, the astrocyte membrane ATPase as a target of organic solvent impact was investigated. The astrocytes were cultured in the modified Minimum Essential Medium (MEM), and the isolated cell membranes were exposed to solvents at different concentrations (3, 6 and 9 mM) for one hour. The activities of the membrane-bound Na + K +-ATPase and Mg 2+-ATPase were determined after exposure to aromatic, alicyclic and aliphatic hydrocarbons. Aromatic hydrocarbons decreased the enzyme activities dose-dependently according to their lipid solubilities. The slight enzyme-inhibiting effect of aliphatic and alicyclic hydrocarbons was not dependent on the lipophilicity of these solvents. Their molecular structure may have determined the solvent impact. In the present study, the activity of ouabain sensitive Na +, K +-ATPase in astrocytes was only 20-15% of the total ATPase activity, and in neurons 45–55%. The enzyme inhibition caused by organic solvents may disturb the homeostatic regulatory functions of astrocytes and thus have a toxic effect on the CNS.

Change in the Activity of Na1 , K+ -ATPase in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus, during Early Development: (sea urchin embryo/Na+ , K+ -ATPase/RNA synthesis/ectodermal cell/actinomycin D).

The activity of ouabain-sensitive Na+ , K+ -ATPase in sea urchin embryos at the morula and the swimming blastula stage was practically the same to that in unfertilized eggs. The activity increased during the period between the mesenchyme blastula and the late gastrula stages. In embryo-wall cell fraction, which contained presumptive ectodermal cells as well as those of other cell lineages at the pre-gastrula stage and ectodermal cells at the late gastrula stage, the Na+ , K+ -ATPase activity increased in this developmental period more largely than in another cell fraction, containing mesenchyme cells and archenteron cells. Cycloheximide did not only block the activity increase in this period but also caused evident decrease in the activity in embryos at all examined stages. The activity increase in this period was strongly blocked by the treatment with actinomycin D, starting before the mesenchyme blastula stage, and was not seriously inhibited by the treatment starting at the mesenchyme blastula stage. The treatment starting at the initiation of gastrulation only slightly blocked further increase in the activity. Probably, an accumulation of mRNA encoding Na+ , K+ -ATPase occurs mainly in ectodermal cells and is completed up to the early gastrula stage.

Myotube phospholipid synthesis and sarcolemmal ATPase activity in dystrophic (mdx) mouse muscle

Phospholipid incorporation of 32P by primary myotube cultures and the tissue activity of sarcolemmal Na+/K(+)-transporting ATPase were studied to determine whether the absence of dystrophin from dystrophic (mdx) muscle would affect membrane lipid synthesis and membrane function. The incorporation of 32P by phospholipid as a ratio with total protein was greater in cultured dystrophic cells compared with control cells. The mdx cells also incorporated more 32P than control cells into phosphatidylethanolamine, which is thought to increase prior to myoblast fusion, and less into phosphatidylserine, phosphatidylinositol, and lysophosphatidylcholine. There was no difference in total protein content or [3H]leucine or 32P incorporation into the aqueous fraction of dystrophic and control cells, although dystrophic cells incorporated less [35S]methionine into protein than controls. Isolated sarcolemma from mdx skeletal muscle tissue demonstrated a consistently greater specific activity of ouabain-sensitive Na+/K(+)-transporting ATPase than sarcolemmal preparations from control skeletal muscle. These observations suggest that cytoskeletal changes such as dystrophin deficiency may alter the differentiation of membrane composition and function.

The effect of culture density and proliferation rate on the expression of ouabain-sensitive [formula omitted] ATPase pumps in cultured human retinal pigment epithelium

The number and activity of ouabain-sensitive Na K ATPase pumps expressed by many cell types in vitro, including human retinal pigment epithelial cells (RPE), have been shown to decline with increasing culture density. Cell proliferation also declined as cultures became dense so it was unclear if pump number was modulated by cell proliferation or culture confluency. By exposing RPE cultures to various feeding regimens, using culture medium containing or lacking serum, it was possible to produce RPE cultures with a range of culture densities and growth rates. These were analyzed for proliferative activity by quantifying [ 3H]thymidine incorporation and for Na K ATPase pump number by measuring specific [ 3H]ouabain binding. The results suggest that pump number is modulated by culture density and, further, that the density-dependent regulation of pump number requires serum. Although density-dependent modulation of culture growth is also serum requiring, cell proliferation and pump number did not appear to be related; cultures of similar density which differed significantly in growth rate had similar numbers of pumps. The view that elevated numbers of pumps were not necessarily found in proliferating cells was further supported by qualitative examination of radioautographs of cells dually labeled with [ 3H]thymidine and [ 3H]ouabain. Cycling cells which had [ 3H]thymidine-labeled nuclei did not have notably higher labeling with [ 3H]ouabain. However, [ 3H]ouabain labeling, as an indicator of pump site number and distribution, did vary among cells in an RPE population and also within individual cells. This latter observation suggests that unpolarized RPE cells in sparse cultures may have regionally different requirements for ionic regulation.

Density of sodium channels in insect synaptic nerve endings

Specific binding of [11-3H]saxitoxin (STX) and activity of ouabain sensitive adenosine-triphosphatase (Na+, K+-ATPase) were determined in neuronal membrane fractions using a subcellular preparation from the central nervous system of the cockroach Periplaneta americana. The nerve ending fractions (synaptosomes) contained 90–95% of the total specific activity of ouabain sensitive Na+, K+-ATPase, and 60–70% of specific STX binding of the crude nerve homogenate. Sodium influx induced by veratridine in synaptosomes was inhibited by saxitoxin at a half-maximal concentration of 4 nM, and kinetics were consistent with reversible binding of one molecule of saxitoxin to each sodium channel receptor site, with an equilibrium dissociation constant (KD) of approx. 3 nM. The density of saturable binding sites was 2 pmol/mg protein which was estimated to correspond to about 95 binding sites per μm2 of synaptic membrane. The results of transport and binding data show that insect synaptosomes possess the capability to conduct inward sodium currents at least comparable to those found in other neuronal membranes, and thus provide a physiologically viable preparation to assess the effect of cation fluxes on the synaptic transmitter release process.

Depression of membrane-bound Na+-K+-ATPase activity induced by free radicals and by ischemia of kidney

A partially purified, membrane-bound Na+-K+-ATPase fraction, prepared from the outer medulla of porcine kidney, was incubated in the presence of 0.1 mM FeCl3, 1 mM ADP, and 0.1-100 mM H2O2 for either 15 or 30 min at 37 degrees C. The activity of ouabain-sensitive Na+-K+-ATPase was reduced proportionally to the concentration of H2O2 and the duration of incubation. There were decreases in SH contents and turnover rates of the Na+-K+-ATPase preparation, while malondialdehyde (MDA) and conjugated dienes were generated from the membrane lipids in the course of the incubation. The concentrations of ethanolamine (E) plasmalogen and of arachidonic acid in the E glycerophospholipid molecules were reduced by the free radical reaction. Similarly, a reduction in Na+-K+-ATPase activity and the formation of MDA and conjugated dienes, together with a decrease in E glycerophospholipids, were observed when the membrane fraction was exposed to ultraviolet irradiation (254 nm) for 30 min at 4 degrees C. Administration of 10 mM dithiothreitol alleviated the reductions in enzyme activity, in turnover rate, and in SH content without suppressing MDA formation. Addition of 2 mM butylated hydroxytoluene to the incubation mixture prevented the lipid peroxidation without totally normalizing the enzyme activity in the H2O2 experiment, whereas this antioxidant restored the ATPase activity to normal in the ultraviolet experiment. Microsomal fractions, prepared from the outer medulla of canine kidney after 1 h of unilateral ischemia and 1 h of reperfusion, showed a decreased Na+-K+-ATPase activity, a reduced amount of SH groups, and an increased MDA.(ABSTRACT TRUNCATED AT 250 WORDS)

Early postnatal development of transport functions in the rabbit choroid plexus.

The development of transport functions in the rabbit choroid plexus was followed postnatally up to 2 months after birth. The activity of ouabain-sensitive Na+, K+-ATPase in newborn rabbit choroid plexus composes about one-fourth (lateral and third ventricle) to one-half (fourth ventricle) of the activity in the adult animal, and it increases markedly within the first 3 weeks of early life. A similar profile of postnatal changes is observed for the capacity to take up and accumulate the organic base choline, which is about three to five times higher for the adult rabbit than for the newborn animal. This coincides with the maturation of the epithelial cells as well as with the development of the sympathetic nerve supply in the choroid plexus. The results suggest that energy-dependent translocation systems influenced by local sympathetic nerves in the choroid plexus, at the interface between blood and CSF, have a functional role shortly after birth.

Sodium—Potassium Pump Activity in Mineralocorticoid- and Glucocorticoid-Induced Hypertension

1. The activity of ouabain-sensitive Na+,K+-dependent ATPase and ouabain-sensitive ATPase was measured in the microsomal fractions of kidney, liver and heart tissue of Sprague-Dawley rats treated with DOCA (10 mg day−1 kg−1 for 6 days, systolic blood pressure 143 ± 13 mmHg, n = 9) or 6 α-methylprednisolone (100 mg day−1 kg−1 for 6 days, systolic blood pressure 140 ± 19 mmHg, n = 9) and of a control group (systolic blood pressure 124 ± 12 mmHg, n = 9). 2. In the kidney, the ouabain-sensitive Na+,K+-ATPase activity (μmol of phosphate h−1 mg−1 of protein) was increased in the DOCA-treated (33.0 ± 6.9) and in the prednisolone-treated groups (30.8 ± 6.9) compared with that in the control group (26.4 ± 3.4) (P < 0.05). In the liver, the ouabain-sensitive Na+,K+-ATPase activity was elevated in the prednisolone-treated animals only (4.8 ± 1.3 vs 3.1 ± 0.8 in the controls, P < 0.025). The ouabain-sensitive Na+,K+-ATPase activity in heart tissue was similar in all three groups. The ouabain-insensitive ATPase activity was not altered by DOCA or prednisolone in the tissues studied. 3. In a separate study, the activity of the ouabain-sensitive Na+,K+-ATPase in erythrocyte ghosts was found to be elevated in 10 patients with Cushing's syndrome (0.91 ± 0.35 μmol of phosphate h−1 mg−1 of protein) compared with five patients with primary aldosteronism and compared with 12 normotensive control subjects (0.38 ± 0.08) (P < 0.005). 4. These data demonstrate an increased Na-K pump activity in the kidney in mineralocorticoid-induced hypertension. Glucocorticoids in addition activate the Na-K pump in a variety of other tissues. This could partially explain the redistribution of volume from the intracellular to the extracellular space.