Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The electron-microscopic localization of ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity of the Na - K-ATPase complex was studied in the exorbital lacrimal gland of the untreated rat with the use of a newly developed one-step lead-citrate method (Mayahara and Ogawa 1980; Mayahara et al. 1980). In the rat lacrimal gland fixed for 15 min in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde, an electron-dense reaction product was observed on the plasma membrane of the basal infoldings and the lateral interdigitations of the ductal cells. The most intense reaction product - and thus the major site of the Na - K-ATPase activity - was evident on the basolateral membranes of the cells of the large interlobular ducts; a weak reaction was seen on the basolateral, extensively folded plasma membranes of the small intercalated ducts; no reaction product was observed on the plasma membranes of the acinar cells. Addition of 1) 10 mM ouabain, 2) p-chloromercuri-phenyl-sulfonic acid (PCMB-S), 3) elimination of K-ions from the incubation medium, or 4) preheating abolished completely the K-NPPase reaction. The activity was also substrate-dependent. Mg-ATPase-activity was observed not only in the basolateral membranes of all ductal cells but also in the basal part of the acinar cells and on the walls of blood vessels. This reaction was neither inhibited by ouabain nor activated by K-ions. The precipitate of the Mg-ATPase-activity was localized at the extracellular side of the plasma membrane, whereas the K-NPPase-reaction product was restricted to the cytoplasmic side of the plasmalemma. In contrast, non-specific alkaline-phosphatase (ALPase) activity was missing in cells of the large interlobular ducts, but obvious on the apical plasmalemma of cells lining the small intercalated ducts. With respect to its localization and reactivity pattern the activity of the K-NPPase (member of the Na - K-ATase complex) differs markedly from the Mg-ATPase- and ALPase-activity.
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PMID:Ultracytochemical localization of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity in the lacrimal gland of the rat. 614 Oct 8

With the goal of isolating and identifying plasma membrane vesicle populations from epithelial cells of the rat exorbital lacrimal gland, we have designed an analytical fractionation of homogenates of the gland parenchyma. This fractionation utilizes separation procedures based on three independent physical properties of subcellular particles: sedimentation coefficient, density, and density after interaction of membrane cholesterol with digitonin. A commonly accepted marker for basal-lateral membranes, Na-K-ATPase, is associated with at least two physically distinct membrane populations. One population can be identified as basal-lateral membrane fragments on the basis of its fractional and specific contents of Na-K-ATPase; it accounts for 50% of the total Na-K-ATPase activity, enriched 29-fold with respect to the initial homogenate. With these values we calculate that the sample of basal membranes has been purified 60-fold with respect to the initial homogenate. The remaining Na-K-ATPase activity appears to be associated, at three- to fivefold lower specific activities, with intracellular membrane populations. We speculate that these populations have been derived from the Golgi complex.
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PMID:Basal-lateral and intracellular membrane populations of rat exorbital lacrimal gland. 630 62

We performed analytical fractionation studies with the goal of isolating basal-lateral and apical membrane vesicles from epithelial cells of rat exorbital lacrimal gland. A density region designated window II contained elements of three physically and biochemically distinct membrane populations. These were resolved by countercurrent distribution in an aqueous polymer two-phase system. One population contained 50% of the NADPH-cytochrome c reductase activity recovered from window II and appeared to be of intracellular origin. A second population contained 70% of the recovered Na-K-ATPase, maximally enriched 42-fold with respect to the initial homogenate. This population was the major locus of alanine transport activity, roughly 85% of which was mediated by systems believed to be characteristic of epithelial cell basal-lateral membranes. It also contained portions of the alkaline phosphatase, galactosyltransferase, acid phosphatase, and NADPH-cytochrome c reductase activities. A third population accounted for 33% of the recovered alkaline phosphatase and was a secondary locus of Na-alanine transport activity, 45% of which could be attributed to systems believed to be characteristic of epithelial cell apical membranes.
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PMID:Resolution of apical and basal-lateral membrane populations from rat exorbital gland. 631 24

1. The rate of acetylcholine (ACh)-induced fluid secretion was measured from the main secretory duct of rabbit lacrimal glands perfused in vivo with Krebs Henseleit bicarbonate solutions. 2. Perfusion with ouabain (10(-5) M) decreased the rate of lacrimal gland fluid secretion to 23% of the control value. 3. Perfusion with furosemide (10(-4) and 10(-3) M), which has been shown to inhibit the coupled transport of Na+ and Cl-, reversibly decreased the rate of secretion to 43 and 33% of the control value respectively. 4. (Na+ + K+)-activated ATPase was localized in slices of rabbit lacrimal gland using autoradiography with [3H]ouabain. 5. A high density of [3H]ouabain binding sites was present on ductal cells, whereas a very low density was found on acinar cells. For both types of cells the [3H]ouabain binding sites were located on the basolateral plasma membranes. 6. It is concluded that ACh-induced secretion of electrolytes and water is dependent upon (Na+ + K+)-activated ATPase. In addition, coupled transport of Na+ and Cl- appears to be involved in secretion. 7. Basolateral location of the (Na+ + K+)-activated ATPase implies that it plays an indirect role in electrolyte and water secretion. A possible role may be to energize a secondary active transport of Cl- that is mediated by a NaCl cotransport system.
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PMID:Lacrimal gland electrolyte and water secretion in the rabbit: localization and role of (Na+ + K+)-activated ATPase. 646 55

Acetylcholine (ACh)-evoked flow from the main excretory duct as well as ACh-induced secretory and resting membrane potentials from cells of the rabbit lacrimal gland were recorded during perfusion with inhibitors. During perfusion with 2,4-dinitrophenol (DNP), ouabain, or ethacrynic acid, ACh-induced flow was 5%, 20%, and 8% of control, respectively; ACh-induced hyperpolarizing secretory potential was 64%, 50%, and 63% of control, respectively; and the resting membrane potential was 79%, 64%, and 73% of control, respectively. Only during perfusion with ethacrynic acid was there a significant increase in the number of cells that did not respond to ACh with a change in potential. We have concluded that (1) ACh-induced secretion is highly dependent on oxidative metabolism and Na-K ATPase; (2) ACh-induced hyperpolarization is dependent on changes in ionic permeabilities, Na-K ATPase, and to lesser extent oxidative metabolism; and (3) the resting membrane potential is much less dependent on oxidative metabolism and Na-K ATPase activity.
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PMID:Lacrimal gland flow and potentials during dinitrophenol, ouabain, and ethacrynic acid perfusion. 721 69

The tumour-promoting agent thapsigargin has been shown to inhibit the microsomal Ca(2+)-ATPase and cause Ca2+ mobilization in a variety of cell types including exocrine acinar cells [Bird, Obie and Putney (1992) J. Biol. Chem. 267, 18382-18386]. When applied to acutely isolated lacrimal acinar cells, thapsigargin caused a slow biphasic activation of both the Ca(2+)-dependent K+ and Cl- currents measured using the whole-cell patch-clamp technique. If the only action of thapsigargin is to inhibit sequestration into Ca2+ pools, then Ca2+ mobilization following exposure to thapsigargin indicates that there is a significant 'leak' of Ca2+ into the cytoplasm, which is normally countered by Ca(2+)-ATPase activity. In the present study, we introduced the Ins(1,4,5)P3 receptor antagonist heparin (200 micrograms/ml) into lacrimal acinar cells via the patch-clamp pipette. Following a 5 min preincubation in the presence of heparin, neither acetylcholine (1 microM) nor thapsigargin (1 microM) caused any significant increase in either Ca(2+)-dependent current. Caffeine has been shown to suppress basal Ins(1,4,5)P3 levels in exocrine acinar cells [Toescu, O'Neill, Petersen and Eisner (1992) J. Biol. Chem. 267, 23467-23470]. Preincubation with caffeine (10 mM) also inhibited the response to subsequent exposure to thapsigargin. These data suggest that, in acutely isolated lacrimal cells, the source of the Ca2+ leak which gives rise to Ca2+ mobilization following inhibition of Ca2+ re-uptake by thapsigargin is Ca2+ release, from Ins(1,4,5)P3-dependent Ca2+ pools, caused by resting Ins(1,4,5)P3 levels.
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PMID:Thapsigargin-induced Ca2+ mobilization in acutely isolated mouse lacrimal acinar cells is dependent on a basal level of Ins(1,4,5)P3 and is inhibited by heparin. 816 57

The lacrimal glands of males and females of various species differ with respect to several morphological, biochemical and functional characteristics. The purpose of this study was to determine the effect of sexual maturation on Na+,K(+)-ATPase, muscarinic cholinergic receptors and beta-adrenergic receptors, which are closely related to the secretion of electrolytes and fluid by the gland, and on other membrane-associated enzymes, specifically galactosyltransferase and alkaline and acid phosphatase. Soluble and total membrane fractions were obtained from lacrimal glands of prepubertal (1.0 kg), pubertal (2 kg), and mature (4 kg) of New Zealand white rabbits of both sexes. Prepubertal and pubertal rabbits exhibited no sex differences in the total amount of lacrimal gland protein or in any of the enzymes or receptors, with the exception of galactosyltransferase and alkaline phosphatase. Galactosyltransferase had higher total and specific activities in prepubertal and pubertal males, and alkaline phosphatase had higher specific activity in prepubertal males. As animals matured, total protein and activities of the enzymes increased, and several quantitative differences between males and females became apparent. Samples from mature females contained significantly less DNA and membrane and total protein. Specific activities of Na+,K(+)-ATPase, cholinergic receptors, galactosyltransferase, and acid and alkaline phosphatase were 40% to 80% greater (p < 0.05) in mature females. Total and specific activity for beta-adrenergic receptors, on the other hand, were higher in the male rabbits. These findings suggest that sex hormones play a role in regulating the levels of expression of a number of enzymes and receptors, including several which are clearly involved in lacrimal secretory functions.
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PMID:Sex-dependent parameters related to electrolyte, water and glycoprotein secretion in rabbit lacrimal glands. 826 91

The subcellular distribution of Na,K-ATPase in rat lacrimal gland acinar cells was surveyed by subcellar fractionation followed by determination of two Na,K-ATPase catalytic activities, K(+)-dependent p-nitrophenylphosphate hydrolysis and ouabain-sensitive ATP hydrolysis, by Western blotting of isolated membrane fractions, and by analysis of lacrimal tissue with immunocytochemical methods at the light and EM levels. Both catalytic activities distributed in parallel after centrifugation on sorbitol gradients. They were associated with membrane samples that appeared to be derived from: (1) acinar cell basal-lateral membranes; (2) the Golgi complex; and (3) endocytic compartments. beta 1-subunit immunoreactivity closely paralleled catalytic activity. The alpha 1-subunit immunoreactivity distribution suggested the presence of alpha 1-subunits in alpha beta complexes and excess alpha 1 subunits which were not assembled with beta subunits and not catalytically active. In the putative basal-lateral membrane and endocytic samples, alpha 1-reactivity was associated primarily with a 100-kDa band, while in the Golgi samples it was associated primarily with 40 and 60-kDa bands. beta 1-reactivity was also heterogeneous, with reactivity in basal-lateral membrane and putative endocytic samples associated with a broad band of 50-54 kDa, and reactivity in Golgi samples associated with discrete bands of 50, 52, and 54 kDa. Staining with anti-holoenzyme and anti-alpha 1-subunit antibodies yielded strong indirect immunofluorescence signals both in plasma membrane and in intracellular regions of acinar cells. beta 1-like immunoreactivity was concentrated in cytoplasmic regions of acinar cells. Immunogold electron microscopy revealed positive staining with anti-holoenzyme in Golgi membranes of acinar cells and in basal-lateral membranes of duct cells. These data support the hypothesis that lacrimal acinar cells contain substantial cytoplasmic pools of Na,K-ATPase and that there is a location-dependent heterogeneity which is not detected by immunocytochemical methods.
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PMID:Surface and intracellular pools of Na,K-ATPase catalytic and immuno-activities in rat exorbital lacrimal gland. 828 26

Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear.
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PMID:Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+,K(+)-ATPase in rabbit lacrimal acini. 839 79

Na-K-ATPase is associated with a variety of membrane populations in lacrimal acinar cells. Acinus-like structures formed by rabbit acinar cells in primary culture were incubated with horseradish peroxidase (HRP) to label basolateral and endosomal membranes and then analyzed by electron microscopy cytochemistry with the 3-3'-diaminobenzidine reaction or by fractionation and measurement of marker catalytic activities or immunoreactivities. HRP adsorbed to basolateral membranes at 4 degrees C. Fractionation showed it associated with low-density membranes enriched in acid phosphatase and TGN38 but containing only minor amounts of Na-K-ATPase. Cells internalized HRP to cytoplasmic vesicles, Golgi structures, and lysosomes at 37 degrees C. The major endosomal compartment revealed by fractionation coincided with major peaks of Na-K-ATPase and Rab6 and secondary peaks of galactosyltransferase and gamma-adaptin. Carbachol (10 microM) increased lysosomal and Golgi labeling. Thus most of the Na-K-ATPase is located in the basolateral membrane-oriented endosomal system, concentrated in a compartment possibly related to the trans-Golgi network. Constitutive and stimulation-accelerated traffic to and from this compartment may serve several exocrine cell functions.
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PMID:Na-K-ATPase in lacrimal gland acinar cell endosomal system: correcting a case of mistaken identity. 894 53


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