Distribution of bicarbonate-stimulated ATPase in rat intestinal epithelium

This study reports on the distribution of bicarbonate-stimulated ATPase in rat intestinal epithelial cells. Brush-border membranes and basolateral membranes were separated from each other and from mitochondrial and other intracellular membranes by differential and density gradient centrifugation. Bicarbonate-sensitive ATPase activity followed the mitochondrial marker succinic dehydrogenase closely throughout all the centrifugation steps. The low HCO3--ATPase activity in purified brush-border and basolateral plasma membranes could be accounted for quantitatively by the small mitochondrial contamination. Consequently, there are no grounds for postulating that this enzyme has a direct role in H+ or HCO3- transport across the rat small intestine.     

Investigation of adenosinetriphosphatase activity of rat liver and thymus cell nuclei]

The activity of ATPase was studied in highly purified rat liver and thymus cell nuclei, HCO3-, CO3(2-) and SO3(2-) stimulated nuclear ATPase in 1.5--2 times. HSO3- did not affect the enzyme activity, and NO3-, J-, ClO4-,F- and SCN- inhibited it. Bicarbonate increased V and decreased Ka for ATP. SCN- inhibited HCO3--ATPase activity non-competitively with respect to HCO3-. Mg2+-ATPase activity did not depend on pH, and HCO3-component of the activity was decreased under alkaline pH. Mg2+, Mn2+ and Co2+ increased the initial ATPase activity and helped its stimulation with HCO3-. Ba2+, Ni2+ and Zn2+ inhibited the ATPase activity, and Ca2+ did not affect it, Nuclear ATPase is sensitive to 2,4-dinitrophenol and DNAase. It is suggested that cell nuclei have their own H+-ATPase differing for some characteristics from mitochondrial H+-ATPase.     

The localization of the anion-sensitive ATPase activity in corneal endothelium

The localization of the anion-sensitive ATPase (EC 3.6.1.3) of bovine corneal endothelium has been investigated. Homogenates were fractionated by differential and density gradient centrifugation, into fractions enriched in plasma membranes and mitochondria. (Na+ + K+)-ATPase (EC 3.6.1.3) and cytochrome oxidase (EC 1.9.3.1) were used as marker enzymes for these two cell components, and glucose-6-phosphatase (EC 3.1.3.5) was used to identify endoplasmic reticulum. 5'-Nucleotidase (EC 3.1.3.5) was also measured but was found not to be exclusively associated with any one cell component. The activity of the anion-sensitive ATPase (HCO3--ATPase) was measured in suspensions that were frozen and thawed before assay in order to expose latent enzyme activity. The fraction containing the greatest amount of (Na+ + K+)-ATPase (35%) contained only 6% of the cytochrome oxidase and HCO3--ATPase. Conversely, the mitochondrial fraction, containing 40% of the cytochrome oxidase, contained about 40% of the HCO3--ATPase, but only 7% of the (Na+ + K+)-ATPase. The recoveries and relative degree of purification of the cytochrome oxidase and HCO3--ATPase were also nearly identical in the other fractions examined. It was concluded that the anion-sensitive ATPase activity of the corneal endothelium is located solely in the mitochondria and not in the plasma membrane. Consequently, any role that the enzymes may have in the transport of bicarbonate across this tissue, which had been suggested in earlier studies, must be an indirect one.     

Requirement of Zn to demonstrate HCO3-stimulated ATPase activity of rat small intestinal brush border

The existence of a membrane-bound HCO3-stimulated ATPase in intestinal mucosa is controversial. A crude brush border fraction of rat small intestinal homogenates contained HCO3-ATPase activity which was inhibited by preincubation with 3 mM EDTA. Alkaline phosphatase activity of this preparation was also inhibited in a parallel, time-dependent fashion by preincubation with EDTA. When 5 mM ZnSO4 accompanied 3 mM EDTA in the preincubation mix, preservation of both enzyme activities occurred, demonstrating a requirement of Zn for the activity of both these phosphatases. These studies support the earlier contention that HCO3-ATPase and alkaline phosphatase activities may be different properties of the same enzyme, and raise the possibility that the ATPase could play a role in intestinal ion transport. The failure to identify a membrane-bound HCO3-ATPase by other workers could be due to the exposure of EDTA which occurred in their tissue preparation.     

Effect of aldosterone antagonists on aldosterone-induced activation of Mg2+ -HCO3- -ATPase and carbonic anhydrase in rat intestinal mucosa

In previous studies, Mg2+ -dependent, HCO3- -activated ATPase in the brush border and carbonic anhydrase in the cytoplasm of rat duodenal and jejunal mucosa decreased after adrenalectomy. Both enzyme activities increased to near normal levels 4 h after i.p. injection of aldosterone (40 micrograms/kg). These results suggest the possibility that both enzymes in the small intestinal mucosa may be mediators of the action of aldosterone. In the present studies, therefore, the effects of actinomycin D (500 micrograms/kg, i.p.), spironolactone (50 mg/kg, s.c.) and potassium canrenoate (50 mg/kg, s.c.) on aldosterone-induced activation of both enzymes in the upper small intestinal mucosa from adrenalectomized rats were examined to clarify the mechanism of action of aldosterone in enzyme levels. Actinomycin D inhibited carbonic anhydrase activity in small intestinal mucosa from normal rats 4 h after i.p. injection but had no effect on ATPase activity, while two other drugs had no effect on either enzyme activity in normal rats up to 4 h later. Pretreatment with these 3 drugs 1 h before aldosterone administration (40 micrograms/kg, i.p.) to adrenalectomized rats blocked the aldosterone-induced activation of ATPase and carbonic anhydrase in the upper small intestine. On the other hand, adrenalectomy and administration of aldosterone and its antagonists, alone or in combination, had no effect on kidney enzyme activities. These results confirm that Mg2+ -HCO3- -ATPase and carbonic anhydrase are mediators of the action of aldosterone in the upper small intestinal mucosa.     

Studies on the localization and properties of rat duodenal HCO3--ATPase with special relation to alkaline phosphatase

Brush-border membrane fractions were isolated from rat duodenum. Purity and integrity of the fraction was confirmed by electron microscopy, enzymic analysis and demonstration of Na+-dependent glucose uptake. The membranes were enriched 15-fold in alkaline phosphatase and alpha-glucosidase and 6-fold in HCO3--ATPase activities. Assays of latent activity indicated that these enzymes were predominantly localised to the external aspect of the microvillus membrane. The enzymes were solubilised and subjected to analysis by gel filtration, ion exchange and phenylboronate chromatography. No separation of alkaline phosphatase and HCO3--ATPase was obtained and it is suggested that they reflect the same enzyme activity. The apparent activation by HCO3- was investigated, and was found to be due to shifts in the pH dependency of the activity due to changes in ionic strength.     

Phosphorylation of Cl-, HCO3--stimulated Mg2+-ATPase of plasma membranes of carp (Cyprinus carpio L.) brain sensitive to GABA(A)-ergic ligands

Phosphorylation of the sensitive to GABA(A)-ergic ligands Cl-, HCO3--stimulated Mg2+-ATPase of the plasma membranes from fish brain by [gamma-32P]ATP was investigated in the presence of Mg2+. It was established, that formation of the phosphoprotein at 0-1 degrees C is dependent on time incubation and concentration of Mg2+ in the incubation medium. Hydroxylamine (50 mM) and pH (10) completely inhibited formation of phosphorylated intermediate. Ions of Cl- (10 mM)+HCO3- (2 mM) and also GABA (1-100 microM) dephosphorylated the enzyme. The dephosphorylating effect of GABA on the membrane samples did not appear in the presence of bicuculline. o-Vanadate (10 microM) eliminates the dephosphorylating effect of anions and GABA on the phosphoprotein. It was established by SDS-PAAG electrophoresis and autoradiographia that investigated phosphorylation and GABA(A)-induced dephosphorylation is performed by the protein with molecular weight aproximately 56 kDa. Such molecular weight has a subunit which forms oligomer composition of the sensitive to GABA(A)-ergic ligands Cl-, HCO3--ATPase from fish brain. The obtained data demonstrated that Cl, HCO3- ATPase from fish brain can be directly phosphorylated by [gamma-32P]ATP in the presence of Mg2+ and forms the phosphorylation intermediate.     

Properties of the V-type ATPase from the excretory system of the usherhopper,Poekilocerus bufonius

The bafilomycin A(1) and N-ethylmaleimide (NEM)-sensitive (V-type) ATPase was partially purified from the apical membrane-rich fractions of excretory system (Malpighian tubules and hind gut) of P. bufonius. Enzymatic activity was inhibited by bafilomycin A(1) (IC(50) = 1.3 nM) and NEM (IC(50) = 10.1 microM). The V-type ATPase activity is confined to the apical membrane fraction, while the activity of Na(+)/K(+) -ATPase forms the major part of the basal membrane fraction. The optimal pH required for maximal activity of V-type ATPase was pH 7.5. The effect of 30 mM of various salts on ATPase activity was investigated. NaCl and KCl caused increases of 175% and 184%, respectively. Other chloride salts also caused an increase in activity in the following ascending order: RbCl, LiCI, choline Cl, NaCI, KCl and tris-HCl. The activity of V-type ATPase was stimulated by a variety of different anions and cations, and HCO(3)(-) was found to be the most potent cationic activator of ATPase activity. The present results show that the properties of V-type ATPase of P. bufonius are similar to those reported for other insect tissues.     

Cl- -HCO3- dependent ATPase in gills of freshwater and seawater adapted eels (Anguilla anguilla L.)

The gills of both seawater and freshwater adapted eels have an ATPase activity which is stimulated by anions in the presence of Mg2+. Plasma membranes were distinguished from mitochondrial membranes with specific enzyme markers, the membrane fractions separated on a discontinuous sucrose gradient, and the ATPase activity of the plasma membranes studied. Activation by the anions of Cl- or HCO3- followed Michaelis-Menten kinetics and was competitively inhibited by SCN-. The Cl- and HCO3- activation characteristics were determined: no differences between the plasma membrane ATPase activities of freshwater and seawater-adapted fishes were observed. Maximal activity measurements after solubilization of the enzymes by Triton X 100 confirmed these findings. The function of a membrane anion-dependent ATPase in the brachial epithelium of euryhaline fish is discussed.     

Na~ ,K~ 和ABA对盐胁迫大麦根液泡膜ATPase活性的影响

许多植物液泡膜ATPaso活性与植物抗盐性有关(Bremberger等1988,Matsumoto和Chung 1988,Gabarino和Dupont 1988)。当植物生长在高浓度NaCl环境中时,液泡膜上Na~ /K~ 交换对维持细胞质中高K~ /Na~ 起重要作用(Jeschke     

Unique regulation of anion/HCO3- exchangers by constitutive nitric oxide in rabbit small intestine

In the rabbit small intestine, there are three functionally different brush-border membrane (BBM) anion/HCO3- exchangers: 1) Cl/HCO3- exchange on the BBM of villus cells responsible for coupled NaCl absorption; 2) Cl/HCO3- exchange on the BBM of crypt cells possibly involved in HCO3- secretion; and 3) short-chain fatty acid (SCFA)/HCO3- exchange on the BBM of villus cells, which facilitates SCFA absorption. Although constitutive nitric oxide (cNO) has been postulated to alter many gastrointestinal tract functions, how cNO may specifically alter these three transporters is unknown. Inhibition of cNO synthase with NG-nitro-L-arginine methyl ester (L-NAME) 1) did not affect villus cell BBM Cl/HCO3 change, 2) stimulated crypt cell BBM Cl/HCO3- exchange, and 3) inhibited villus cell BBM SCFA/HCO3- exchange. D-NAME, an inactive analog of L-NAME, and L-N6-(1-iminoethyl)lysine, a more selective inhibitor of inducible NO, did not affect these transport processes. Kinetic studies demonstrated that 1) the mechanism of inhibition of crypt cell BBM Cl/HCO3- exchange is secondary to a decrease in the maximal rate of uptake of Cl, without an alteration in the affinity of the transporter for Cl, and 2) the mechanism of stimulation of villus cell BBM SCFA/HCO3- exchange is secondary to an increase in the affinity of the transporter for SCFA without an alteration in the maximal rate of uptake of SCFA. These results indicate that cNO uniquely regulates the three BBM anion/HCO3- transporters in the rabbit small intestine.     

Effects of suppression of eggshell calcification and of 1,25(OH)2D3 on Mg2+, Ca2+ and Mg2+HCO-3 ATPase, alkaline phosphatase, carbonic anhydrase and CaBP levels--II. The laying hen intestine

Activity of a HCO-3 stimulated Mg2+ dependent ATPase is demonstrated in mitochondrial fractions of the avian duodenum. Suppression of eggshell calcification resulted in a slight reduction in Mg2+, Ca2+ and Mg2+HCO-3 ATPase activities. Duodenal carbonic anhydrase activity was lower in birds laying soft-shelled eggs than in birds laying normal eggs. Alkaline phosphatase and calcium binding protein levels both decreased along the length of the small intestine, but the effect was more pronounced for alkaline phosphatase. Suppression of eggshell calcification and treatment of shell-less laying hens with 1,25(OH)2D3 influenced alkaline phosphatase activity only in the duodenal mucosa. Suppression of eggshell calcification reduced CaBP levels in all sections of the intestine. Treatment with 1,25(OH)2D3 restored CaBP levels. Regulation of intestinal CaBP levels by 1,25(OH)2D3 would therefore, seem to be controlled more directly by calcium requirements associated with eggshell calcification than by gonadal hormones.     

Extraction of mitochondrial protein-lipid complexes into organic solvents: an approach to study the interaction between the ATPase and the mitochondrial ATPase-inhibitor protein

Protein-lipid complexes were transferred directly from mitochondria and submitochondrial particles into hexane and ether. The protein-lipid residue left after solvent removal from these extracts was used to form liposomes which display low-temperature-resistant ATPase activity. Centrifugation experiments indicate that the ATPase activity is associated to the vesicles. Most of the F1-ATPases appear to be accessible to the external water phase of the liposomes. The ATPase activity of these particles was insensitive to dicyclohexylcarbodiimide and oligomycin. Incubation of these vesicles at room temperature activated (4--10-fold) the ATPase through a process that is partially sensitive to phenylmethylsulfonyl fluoride. The results with purified ATPase-inhibitor protein and (F1--ATPase)-inhibitor complex indicate that the activation process in the liposomes is due to the abolition of the inhibitory action of the inhibitor protein bound to a large fraction of the extracted ATPases. Liposomes prepared from hexane extracts obtained from submitochondrial particles having different levels of ATPase activity displayed an activation ratio which correlated with the number of ATPases that are inhibited by the inhibitor protein in the submitochondrial particles. The extraction of mitochondrial ATPase and its incorporation into liposomes followed by activity measurements may be used to judge the number of ATPases that in a given preparation contain the inhibitor protein in its inhibiting site.     

Ion-transporting ATPases and matrix mineralization in cultured osteoblastlike cells

Cultures of osteoblastlike cells obtained from the endosteal surfaces of rabbit long bones formed and mineralized an extracellular matrix when they were supplied daily with medium containing fresh ascorbate. No matrix formed without this supplementation. The matrix mineralized whether or not beta-glycerophosphate, a substrate of alkaline phosphatase, was added to the medium. The ion-transporting ATPase activities of untreated, ascorbate-treated, and ascorbate plus beta-glycerophosphate-treated cells were measured. Ascorbate-treated and ascorbate plus beta-glycerophosphate-treated cells had similar enzyme activities. The activities of the Ca2+-ATPase; Ca2+,Mg2+-ATPase; and alkaline phosphatase in treated cells were elevated over the activities in untreated cells. Na+,K+-ATPase activity was lower in treated than in untreated cells. HCO3--ATPase activity was not changed by treatment. Alkaline phosphatase activity was 20 times higher in freshly isolated osteoblastlike cells than in cells grown to confluence in primary culture. In addition, subculturing further reduced the activity of this osteoblast-marker enzyme. The activities of the ion-transporting ATPases and alkaline phosphatase in second passage cells were similar to the activities of these enzymes in fresh, noncalcifying tissues. Nevertheless, second passage cells retain the ability to mineralize an extracellular matrix, and their ion-transporting ATPase and alkaline phosphatase activities are altered when the cells mineralize a matrix.     

Purification and characterization of a membrane-associated ATPase from Natronococcus occultus, a haloalkaliphilic archaeon

Isolated membranes of the extreme haloalkaliphilic archaeon Natronococcus occultus were able to hydrolyze ATP via an ATPase, which required the presence of Mg(2+), high concentrations of NaCl, and a pH value of 9. The native molecular mass of the purified ATPase was 130 kDa and was composed of 74- and 61-kDa subunits. Enzyme activity was specific for the hydrolysis of ATP with slight activity towards GTP, CTP, and ITP. The enzyme required NaCl for maximal activity but Na(2)SO(4) and (NH(4))(2)SO(4) could substitute. The enzyme showed no activity if Na(2)SO(3) or sodium citrate was substituted for NaCl. The ATPase from N. occultus was inhibited by NBD-Cl, NaN(3), and ouabain, and was sensitive to nitrate, vanadate, DCCD, and bafilomycin A(1). It was not inhibited by NEM in contrast to other previously characterized halophile ATPases. The ATPase had a K(M) of 0.5 mM and appeared to be non-competitively inhibited by NaN(3) with a K(I) of 3.1 mM.     

Mechanism of regulation of rabbit intestinal villus cell brush border membrane Na/H exchange by nitric oxide

In the mammalian small intestine, coupled NaCl absorption occurs via the dual operation of Na/H and Cl/HCO(3) exchange on the villus cell brush border membrane (BBM). Although constitutive nitric oxide (cNO) has been demonstrated to alter gastrointestinal tract functions, how cNO may specifically alter these two transporters to regulate coupled NaCl absorption is unknown. In villus cells, inhibition of cNO synthase (cNOS) with l-N(G)-nitroarginine methylester (l-NAME) stimulated Na/H exchange whereas Cl/HCO(3) exchange was unaffected. In villus cell BBM vesicles (BBMV) prepared from rabbits treated with l-NAME, Na/H exchange was also stimulated. d-NAME, an inactive analog of l-NAME, and N(6)-(1-imonoethyl)-l-lysine dihydrochloride, a more selective inhibitor of inducible NO synthase, did not affect Na/H exchange. Kinetic studies demonstrated that the mechanism of stimulation is secondary to an increase in the maximal rate of uptake of Na, without an alteration in the affinity of the transporter for Na. Northern blot studies demonstrated an increase in the message for the BBM Na/H exchanger NHE3, and Western blot studies showed that the immunoreactive protein levels of NHE3 was increased when cNOS was inhibited. Thus these results indicate that cNO under nominal physiological states most likely maintains an inhibitory tone on small intestinal coupled NaCl absorption by specifically inhibiting BBM Na/H expression.     

Similarities and differences between the tonoplast-type and the mitochondrial H+-ATPases of oat roots

The native tonoplast and the mitochondrial H+-ATPase from oat roots were compared to determine whether the two enzymes have similar mechanisms. H+ pumping in low-density microsomal vesicles reflected activity from the tonoplast-type ATPase, as ATPase activity and ATP-dependent H+ pumping (quinacrine fluorescence quenching) showed similar sensitivities to inhibition by N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, 4,4'-diisothiocyano-2,2'-stilbene disulfonate, nitrate, quercetin, or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. The tonoplast-type ATPase was stimulated by C1-,Br- greater than HCO3- whereas the mitochondrial ATPase was stimulated by HCO3- much greater than C1-,Br-. Both enzymes hydrolyzed ATP preferentially and were inhibited competitively by AMP or ADP. Apart from resistance to azide, the tonoplast-type ATPase was strikingly similar in its inhibitor sensitivities to the mitochondrial ATPase. The insensitivity to vanadate of both enzymes suggests the reaction mechanisms do not involve a covalent phosphoenzyme. Inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide and protection by ATP suggests tyrosine and cysteine residues are in the catalytic site of the tonoplast ATPase. The mitochondrial ATPase was 100 times more sensitive to N,N'-dicyclohexyl-carbodiimide inhibition than the tonoplast H+-ATPase. These results suggest the tonoplast and the mitochondrial H+-ATPases share common steps in their catalytic and vectorial reaction mechanisms, yet sufficient differences exist to indicate they are two distinct ATPases.     

Restitution of the bullfrog gastric mucosa is dependent on a DIDS-inhibitable pathway not related to HCO3- ion transport

This study was conducted to determine the contribution of ion transport to restitution after injury in the gastric mucosa. For this, intact sheets of stomach from the bullfrog, Rana catesbeiana, were mounted in Ussing chambers. Restitution was evaluated in the presence or absence of ion transport inhibitors amiloride, DIDS, and bumetanide to block Na(+)/H(+) exchange, Cl(-)/HCO(3)(-) exchange and Na(+)/HCO(3)(-) co-transport, and Na(+)-K(+)-2Cl(-) cotransport, respectively. Ion substitution experiments with Na(+)-free, Cl(-)-free, and HCO(3)(-)-free solutions were also performed. Injury to the mucosa was produced with 1 M NaCl, and restitution was evaluated by recovery of transepithelial resistance (TER), mannitol flux, and morphology. Amiloride, bumetanide, Cl(-)-free, or HCO(3)(-)-free solutions did not affect restitution. In Na(+)-free solutions, recovery of TER and mannitol flux did not occur because surface cells did not attach to the underlying basement membrane. In contrast, all aspects of restitution were inhibited by DIDS, a compound that inhibits Na(+)-dependent HCO(3)(-) transport. Because HCO(3)(-)-free solutions did not inhibit restitution, it was concluded that DIDS must block a yet undefined pathway not involved in HCO(3)(-) ion transport but essential for cell migration after injury and restitution in the gastric mucosa.     

Ouabain-insensitive Na+ stimulation of an Mg-2+ -dependent ATPase in kidney tissue.

1. Freshly prepared microsomal fractions of the outermost cortex of guinea pig kidney show an Mg-2+-dependent ATPase activity which is partially inhibited by 100 mM NaCl, LiCl, KCl, RbCl, CsCl, NH4Cl or choline chloride. 2. If the microsomal preparation is aged by storage at 4 degrees C for 10-15 days, the Mg-2+-dependent activity shows stimulation by Na-+ and Li-+ but not by K-+, Rb-+, Cs-+, NH4-+ or choline. 3. Stimulation is similar with sodium salts of Cl-minus, HCO3-minus, CH3COO-minus, BR-minus, SO4-2-minus or methylsulphonate. 4. Stimulation is insensitive to 1 mM and 10 mM ouabain. 5. Stimulation is unaltered by the presence of 0.5 mM ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetracetic acid. 6. Stimulation is 100% inhibited by 2 mM ethacrynic acid, a concentration which inhibits only 30% of the Mg-2+-dependent ATPase and 50% of the (Na-++K-+)-stimulated ATPase. 7. Some of these characteristics coincide with those of an ouabain-resistant, K-+-independent, ethacrynic acid-sensitive mode of Na-+ extrusion out of guinea pig kidney cortex cells.