Characterization of inside-out oriented H+-ATPases in cholate-pretreated renal brush-border membrane vesicles

Summary Exposure of porcine renal brush-border membrane vesicles to 1.2% cholate and subsequent detergent removal by dialysis reorients almost all N-ethylmaleimide (NEM)-sensitive ATPases from the vesicle inside to the outside. ATP addition to cholate-pretreated, but not to intact, vesicles causes H⁺ uptake as visualized by the pH indicator, acridine organge. The reoriented H⁺-pump is electrogenic because permeant extravesicular anions or intravesicular K⁺ plus valinomycin enhance H⁺ transport. ATP stimulates H⁺ uptake with an apparentK _m of 93 m. Support of H⁺ uptake andP _i liberation by ATP>GTPITP> UTP indicates a preference for ATP and utilization of other nucleotides at lower efficiency. ADP is a potent, competitive inhibitor of ATP-driven H⁺ uptake,(K _i , 24 m). Mg²⁺ and Mn^2– support ATP-driven H⁺ uptake, but Ca²⁺, Ba²⁺ and Zn²⁺ do not. Imm Zn²⁺ inhibits MgATP-driven H⁺ transport completely. NEM-sensitiveP _i liberation is stimulated by Mg²⁺ and Mg^2– and, unlike H⁺ uptake, also by Ca²⁺ suggesting Ca²⁺-dependent ATP hydrolysis unrelated to H⁺ transport. The inside-out oriented H⁺-pump is relatively insensitive toward oligomycin, azide, N,N-dicyclohexylcarbodiimide (DCCD) and vanadate, but efficiently inhibited by NEM (apparentK _i , 0.77 m), and 4-chloro-7-nitro-benzoxa-1,3-diazole (NBD-Cl; apparentK _i , 0.39 m). Taken together, the H⁺-ATPase of proximal tubular brush-border membranes exhibits characteristics very similar to those of vacuolar type (V-type) H⁺-ATPases. Hence,V-type H⁺-ATPases occur not only in intracellular organelles but also in specialized plasma membrane areas.     

pH-dependent Ca2+ binding to the F0 c-subunit affects proton translocation of the ATP synthase from Synechocystis 6803

It was shown before (Wooten, D. C., and Dilley, R. A. (1993) J. Bioenerg. Biomembr. 25, 557–567; Zakharov, S. D., Li, X., Red'ko, T. P., and Dilley, R. A. (1996) J. Bioenerg. Biomembr. 28, 483–493) that pH dependent reversible Ca²⁺ binding near the N- and C-terminal end of the 8 kDa subunit c modulates ATP synthesis driven by an applied pH jump in chloroplast and E. coli ATP synthase due to closing a proton gate proposed to exist in the F₀ H⁺ channel of the F₀F₁ ATP synthase. This mechanism has further been investigated with the use of membrane vesicles from mutants of the cyanobacterium Synechocystis 6803. Vesicles from a mutant with serine at position 37 in the hydrophilic loop of the c-subunit replaced by the charged glutamic acid (strain plc 37) has a higher H⁺/ATP ratio than the wild type and therefore shows ATP synthesis at low values of _H ⁺. The presence of 1 mM CaCl₂ during the preparation and storage of these vesicles blocked acid–base jump ATP formation when the pH of the acid side (inside) was between pH 5.6 and 7.1, even though the pH of the acid–base jump was thermodynamically in excess of the necessary energy to drive ATP formation at an external pH above 8.28. That is, in the absence of added CaCl₂, ATP formation did occur under those conditions. However, when the base stage pH was 7.16 and the acid stage below pH 5.2, ATP was formed when Ca²⁺ was present. This is consistent with Ca²⁺ being displaced by H⁺ ions from the F₀ on the inside of the thylakoid membrane at pH values below about 5.5. Vesicles from a mutant with the serine of position 3 replaced by a cysteine apparently already contain some bound Ca²⁺ to F₀. Addition of 1 mM EGTA during preparation and storage of those vesicles shifted the otherwise already low internal pH needed for onset of ATP synthesis to higher values when the external pH was above 8. With both strains it was shown that the Ca²⁺ binding effect on acid–base induced ATP synthesis occurs above an internal pH of about 5.5. These results were corroborated by ⁴⁵Ca²⁺- ligand blot assays on organic solvent soluble preparations containing the 8 kDa F₀ subunit c from the S-3-C mutant ATP synthase, which showed ⁴⁵Ca²⁺ binding as occurs with the pea chloroplast subunit III. The phosphorylation efficiency (P/2e), at strong light intensity, of Ca²⁺ and EGTA treated vesicles from both strains were almost equal showing that Ca²⁺ or EGTA have no other effect on the ATP synthase such as a change in the proton to ATP ratio. The results indicate that the Ca²⁺ binding to the F₀ H⁺ channel can block H⁺ flux through the channel at pH values above about 5.5, but below that pH protons apparently displace the bound Ca²⁺, opening the CF₀ H⁺ channel between the thylakoid lumen and H⁺ conductive channel.     

Erythrocyte Membrane Digoxin-Sensitive (Na+–K+)-ATPase of Non-Insulin Dependent Diabetic Humans

Erythrocyte plasma membranes of non-insulin dependent diabetic humans (NIDDM) and healthy humans were prepared by hypotonic lysis. The specific activity of (Na⁺–K⁺)-ATPase of NIDDM membranes, both in the absence and presence of digoxin were lower than the specific activity of normal enzymes (83.6 percent and 74.0 percent of the normal enzyme respectively). Addition of digoxin decreased the activity of this enzyme (38.0 percent in NIDDM and 30.0 percent in normal enzyme).Although the affinity of the pump for ATP was similar in both membranes of NIDDM and normal humans (K_m for ATP=19.9±0.24M ATP and 20.0±0.21 M ATP respectively), the V_max of NIDDM membranes was more than 20 percent lower than that of the normal enzyme. The specific activity of Mg²⁺-dependent Ca²⁺-pumping ATPase (Ca²⁺–Mg²⁺)-ATPase) of NIDDM membrane was lower than 80 percent of the specific activity of the normal enzymes. While the affinity of the pump for ATP was lower in the membranes of NIDDM (K_m for ATP=50.0±4.3 M ATP) in comparison to normal membranes (K_m for ATP=63.1±38M ATP), the V_max of NIDDM membranes was similar to the normal enzyme. Altogether, these findings suggest that both the (Na⁺–K⁺)-ATPase and Ca²⁺-pumping ATPase of NIDDM membranes are less functional than the enzymes in normal erythrocytes.     

H+/Ca2+ exchange in rabbit renal cortical endosomes

Summary We have examined the effect of second messengers on ATP-driven H⁺ transport in an H⁺ ATPase-bearing endosomal fraction isolated from rabbit renal cortex. cAMP (0.1mm) had no effect on H⁺ transport. Acridine orange fluorescence in the presence of 0.5mm Ca²⁺ (+1mm EGTA) was 19±6% of control. Inhibition of ATP-driven H⁺ transport by Ca²⁺ was concentration dependent; 0.25 and 0.5mm Ca²⁺ (+1mm EGTA) inhibited acridine orange fluorescence by 50 and 80%, respectively. Ca²⁺ also produced a concentration-dependent increase in the rate of pH-gradient dissipation. Ca²⁺ did not affect ATP hydrolysis. ATP-dependent Br^– uptake was virtually unchanged in the presence of 0.5mm Ca²⁺ (+1mm EGTA). These vesicles were also shown to transport Ca²⁺ in an ATP-dependent mode. Inositol 1, 4, 5-trisphosphate had no effect on ATP-dependent Ca²⁺ uptake. These results are consistent with the co-existence of an H⁺ ATPase and an H⁺/Ca²⁺ exchanger on these endosomes, the latter transport system using the H⁺ gradient to energize Ca²⁺ uptake. Attempts to demonstrate an H⁺/Ca²⁺ antiporter in the absence of ATP have been unsuccessful. Yet, when a pH gradient was established by preincubation with ATP and residual ATP was subsequently removed by hexokinase + glucose, stimulation of Ca²⁺ uptake could be demonstrated. A Ca²⁺-dependent increase in H⁺ permeability and an ATP-dependent Ca²⁺ uptake might have important implications for the regulation of vacuolar H⁺ ATPase activity as well as the homeostasis of cytosolic Ca²⁺ concentration.     

Interaction of polypeptides with the gastric (H+ + K+)ATPase: melittin,synthetic analogs,and a potential intracellular regulatory protein

The 26 amino acid bee venom toxin, melittin, is an amphipathic helical polypeptide which inhibits the gastric (H⁺ + K⁺)ATPase. The site of interaction with the (H⁺ + K⁺)ATPase was shown to be the alpha subunit of the (H⁺ + K⁺)ATPase in studies using [¹²⁵I]azidosalicylyl melittin, a radioactive photoaffinity analog of melittin. A synthetic amphipathic polypeptide (Trp3) containing tryptophan, which exhibits a structure similar to that of melittin, also inhibited the gastric (H⁺ + K⁺)ATPase, and prevented labeling by [¹²⁵I]azidosalicylyl melittin. These findings suggested that melittin and the synthetic amphipathic helical polypeptide were bound to the same or overlapping site(s). In the present studies, novel tritiated photoaffinity analogs of Trp3 containing benzoylphenylalanine (in place of tryptophan) were used to photoaffinity label the (H⁺ + K⁺)ATPase. These studies help to establish that the (H⁺ + K⁺)ATPase contains a binding site for polypeptides which exhibit an amphipathic helical motif. The precise amino acid sequence of the polypeptide appears to be of secondary importance for interaction with the (H⁺ + K⁺)ATPase as long as the alpha helical motif is present. The benzoylphenylalanine containing polypeptides are ideal for mapping the binding site on the (H⁺ + K⁺)ATPase. Using an antibody which recognizes this amphipathic helical (melittin-like) motif, we have demonstrated that the gastric parietal cell contains a 67 kDa melittin-like protein. This protein was associated with the gastric parietal cell apical membrane in the stimulated (secreting) state, but not in the resting (non-secreting) state. The binding site for the gastric melittin-like protein appears to overlap with the melittin binding site on the alpha subunit of the (H⁺ + K⁺)ATPase. The potential physiological significance of the melittin binding site and the overlapping binding site for this newly identified endogenous melittin-like protein on the (H⁺ + K⁺)ATPase to regulated HCl secretion by the parietal cell is presently under investigation. Evidence is presented which demonstrates that melittin binds to other E₁-E₂ ion pumps, raising the possibility that there might exist similar intracellular proteins which interact with other ion pumps.     

Characterization of a plasma membrane H+-ATPase from the extremely acidophilic algaDunaliella acidophila

Summary Dunaliella acidophila is an unicellular green alga which grows optimally at pH 0–1 while maintaining neutral internal pH. A plasma membrane preparation of this algae has been purified on sucrose density gradients. The preparation exhibits vanadatesensitive ATPase activity of 2 mol P_i/mg protein/min, an activity 15 to 30-fold higher than that in the related neutrophilic speciesD. salina. The following properties suggest that the ATPase is an electrogenic plasma membrane H⁺ pump. (i) ATP induces proton uptake and generates a positive-inside membrane potential as demonstrated with optical probes. (ii) ATP hydrolysis and proton uptake are inhibited by vanadate, diethylstilbestrol, dicyclohexylcarbodiimide and erythrosine but not by molybdate, azide or nitrate. (iii) ATP hydrolysis and proton uptake are stimulated by fussicoccin in a pH-dependent manner as found for plants plasma membrane H⁺-ATPase. Unusual properties of this enzyme are: (i) theK _m for ATP is around 60 M, considerably lower than in other plasma membrane H⁺-ATPases, and (ii) the ATPase activity and proton uptake are stimulated three to fourfold by K⁺ and to a smaller extent by other monovalent cations. These results suggest thatD. acidophila possesses a vanadate-sensitive H⁺-ATPase with unusual features enabling it to maintain the large transmembrane pH gradient.     

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

Characterization of MgATP-Driven H+ uptake in to a microsomal vesicle franction from rat pancreatic acinar cells

Summary In microsomal vesicles, as isolated from exocrine pancreas cells, MgATP-driven H⁺ transport was evaluated by measuring H⁺-dependent accumulation of acridine orange (AO). Active H⁺ uptake showed an absolute requirement for ATP with simple Michaelis-Menten kinetics (K _m for ATP 0.43 mmol/liter) with a Hill coefficient of 0.99. H⁺ transport was maximal at an external pH of 6.7, generating an intravesicular pH of 4.8. MgATP-dependent H⁺ accumulatioin was abolished by protonophores. such as nigericin (10^–6 mol/liter) or CCCP (10^–5 mol/liter), and by inhibitors of nonmitochondria H⁺ ATPase, such as NEM or NBD-Cl, at a concentration of 10^–5 mol/liter. Inhibitors of both mitochondrial and nonmitochondrial H⁺ pumps, such as DCCD (10^–5 mol/liter) or Dio 9 (0.25 mg/ml), reduced microsomal H⁺ transport by about 90%. Vanadate (2×10^–3 mol/liter). a blocker of those ATPases, which form a phosphorylated intermediate, did not inhibit H⁺ transport. The stilbene derivative DIDS (10^–4 mol/liter), which inhibits anion transport systems, abolished H⁺ transport completely. MgATP-dependent H⁺ transport was found to be anion dependernt in the sequence Cl^–>Br^–>gluconate^–; in the presence of SO ₄ ^–2 . CH₃COO^– or No ₃ ^– , no H⁺ transport was observed. MgATP-dependent H⁺ accumulation was also cation dependent in the sequence K⁺>Li⁺>Na⁺=choline⁺, As shown by dissipation experiments in the presence of different ion gradients and ionophores, both a Cl^– and a K⁺ conductance, as well as a small H⁺ conductance. were found in the microsomal membranes. When membranes containing the H⁺ pump wer further purified by Percoll gradient centrifugatin (ninefold enrichment comparad to homogenate), no correlation with markers for endoplasmic reticulum., mitochondria, plasma membranes, zymogen graules or Golgi membranes was found.The present data indicate that the H⁺ pump located in microsomes from rat exocrine pancreas is a vacuolar-or V-type H⁺ ATPase and has most similarities to that described in endoplasmic reticulum. Golgi apparatus or endosomes.     

Electrogenic calcium transport in plasma membrane of rat pancreatic acinar cells

Summary ATP-dependent⁴⁵Ca²⁺ uptake was investigated in purified plasma membranes from rat pancreatic acinar cells. Plasma membranes were purified by four subsequent precipitations with MgCl₂ and characterized by marker enzyme distribution. When compared to the total homogenate, typical marker enzymes for the plasma membrane, (Na⁺,K⁺)-ATPase, basal adenylate cyclase and CCK-OP-stimulated adenylate cyclase were enriched by 43-fold, 44-fold, and 45-fold, respectively. The marker for the rough endoplasmic reticulum was decreased by fourfold compared to the total homogenate. Comparing plasma membranes with rough endoplasmic reticulum, Ca²⁺ uptake was maximal with 10 and 2 mol/liter free Ca²⁺, and half-maximal with 0.9 and 0.5 mol/liter free Ca²⁺. It was maximal at 3 and 0.2 mmol/liter free Mg²⁺ concentration, at an ATP concentration of 5 and 1 mmol/liter, respectively, and at pH 7 for both preparations. When Mg²⁺ was replaced by Mn²⁺ or Zn²⁺ ATP-dependent Ca²⁺ uptake was 63 and 11%, respectively, in plasma membranes; in rough endoplasmic reticulum only Mn²⁺ could replace Mg²⁺ for Ca²⁺ uptake by 20%. Other divalent cations such as Ba²⁺ and Sr²⁺ could not replace Mg²⁺ in Ca²⁺ uptake. Ca²⁺ uptake into plasma membranes was not enhanced by oxalate in contrast to Ca²⁺ uptake in rough endoplasmic reticulum which was stimulated by 7.3-fold. Both plasma membranes and rough endoplasmic reticulum showed cation and anion dependencies of Ca²⁺ uptake. The sequence was K⁺>Rb⁺>Na⁺>Li⁺>choline⁺ in plasma membranes and Rb⁺K⁺Na⁺>Li⁺>choline⁺ for rough endoplasmic reticulum. The anion sequence was Cl^–Br^–I^–>SCN^–>NO ₃ ^– >isethionate^– >cyclamate^–>gluconate^–>SO ₄ ^2– glutarate^– and Cl^–>Br>gluconate>SO ₄ ^2– >NO ₃ ^– >I^–>cyclamate^–SCN^–, respectively. Ca²⁺ uptake into plasma membranes appeared to be electrogenic since it was stimulated by an inside-negative K⁺ and SCN diffusion potential and inhibited by an inside-positive diffusion potential. Ca²⁺ uptake into rough endoplasmic reticulum was not affected by diffusion potentials. We assume that the Ca²⁺ transport mechanism in plasma membranes as characterized in this study represents the extrusion system for Ca²⁺ from the cell that might be involved in the regulation of the cytosolic Ca²⁺ level.     

In vitro analysis of ion channels in periaxolemmal-myelin and white matter clathrin coated vesicles: Modulation by calcium and GTPγS

This study reports the analysis of K⁺ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K⁺ channels were observed with amplitudes of 25–30, 45–55, and 80–100 pS, all of which exhibited mean open-times of 1–2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K⁺ channel activity was regulated by Ca²⁺. Little or no change in activity was observed when Ca²⁺ was added to thecis side of the bilayer. Addition of 10 M total Ca²⁺ also resulted in little change in K⁺ channel activity. However, at 80 M total Ca²⁺ all K⁺ channel activity was suppressed along with the activation of a 100 pS Cl^– channel. The K⁺ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTPS to thetrans side of the bilayer resulted in a restriction of activity to the 45–50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K⁺ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the subunits of G₀, G_s, and G_i and the low molecular weight G protein,ras. As with periaxolemmal-myelin treated with GTPS, the vesicle membrane exhibited only the 50 pS channel. The channel was active at all holding potentials and had open times of 1–6 msec. Addition of GTPS to the bilayer fused with vesicle membrane appeared to suppress this channel activity at low voltages yet induced a hyperactive state at holding potentials of 45 mV or greater. The vesicle 50 pS K⁺ channel was also activated by the 6-methyl-dihydropyron-2-one (20 M).Abbreviations CNPase 2–3 cyclic nucleotide phosphohydrolase - EDTA ethylenediamine N,N,N,N-tetraacetic acid - G-protein GTP(guanosine triphosphate) binding protein - GTPS guanosine 5-O-(3-thiotriphosphate) - MAG myelin associated glycoprotein - Na⁺ K⁺ ATPase, Na⁺ and K⁺ stimulated adenosine triphosphatase - PLP myelin proteolipid protein Special issue dedicated to Dr. Majorie B. Lees.     

Role of MMP-2 in inhibiting Na+ dependent Ca2+ uptake by H2O2 in microsomes isolated from pulmonary smooth muscle

Treatment of microsomes (preferentially enriched with endoplasmic reticulum) isolated from bovine pulmonary artery smooth muscle tissue with H₂O₂ (1 mM) markedly stimulated matrix metalloproteinase activity and also inhibited Na⁺ dependent Ca²⁺ uptake. Electron micrograph revealed that H₂O₂ (1 mM) does not cause any damage to the microsomes. MMP-2 and TIMP-2 were determined to be the ambient protease and corresponding antiprotease of the microsomes. Pretreatment with vitamin E (1 mM) and TIMP-2 (50 g/ml) reversed the effect produced by H₂O₂ (1 mM) on Na⁺ dependent Ca²⁺ uptake in the microsomes. However, H₂O₂ (1 mM) caused changes in MMP-2 activity and Na⁺ dependent Ca²⁺ uptake were not reversed upon pretreatment of the microsomes with a low concentration of 5 g/ml of TIMP-2 which otherwise reversed MMP-2 (1 g/ml) mediated increase in ¹⁴C-gelatin degradation and inhibition of Na⁺ dependent Ca²⁺ uptake. Combined treatment of the microsomes with a low dose of MMP-2 (0.5 g/ml) and H₂O₂ (0.5 mM) inhibited Na⁺ dependent Ca²⁺ uptake in the microsomes compared to the respective low dose of either of them. Direct treatment of TIMP-2 (5 g/ml) with H₂O₂ (1 mM) abolished the inhibitory effect of the inhibitor on ¹⁴C-gelatinolytic activity elicited by 1 g/ml of MMP-2. Thus, one of the mechanisms by which H₂O₂ activates MMP-2 could be due to inactivation of TIMP-2 by the oxidant. The resulting activation of MMP-2 subsequently inhibits Na⁺ dependent Ca²⁺ uptake in the microsomes. (Mol Cell Biochem 270: 79–87, 2005)     

Relation of ATPases in rat renal brush-border membranes to ATP-driven H+ secretion

Summary In the presence of inhibitors for mitochondrial H⁺-ATPase, (Na⁺+K⁺)- and Ca²⁺-ATPases, and alkaline phosphatase, sealed brush-border membrane vesicles hydrolyse externally added ATP demonstrating the existence of ATPases at the outside of the membrane (ecto-ATPases). These ATPases accept several nucleotides, are stimulated by Ca²⁺ and Mg²⁺, and are inhibited by N,N-dicyclohexylcarbodiimide (DCCD), but not by N-ethylmaleimide (NEM). They occur in both brushborder and basolateral membranes. Opening of brush-border membrane vesicles with Triton X-100 exposes ATPases located at the inside (cytosolic side) of the membrane. These detergent-exposed ATPases prefer ATP, are activated by Mg²⁺ and Mn²⁺, but not by Ca²⁺, and are inhibited by DCCD as well as by NEM. They are present in brush-border, but not in basolateral membranes. As measured by an intravesicularly trapped pH indicator, ATP-loaded brush-border membrane vesicles extrude protons by a DCCD- and NEM-sensitive pump. ATP-driven H⁺ secretion is electrogenic and requires either exit of a permeant anion (Cl^–) or entry of a cation, e.g., Na⁺ via electrogenic Na⁺/d-glucose and Na⁺/l-phenylalanine uptake. In the presence of Na⁺, ATP-driven H⁺ efflux is stimulated by blocking the Na⁺/H⁺ exchanger with amiloride. These data prove the coexistence of Na⁺-coupled substrate transporters, Na⁺/H⁺ exchanger, and an ATP-driven H⁺ pump in brush-border membrane vesicles. Similar location and inhibitor sensitivity reveal the identity of ATP-driven H⁺ pumps with (a part of) the DCCD- and NEM-sensitive ATPases at the cytosolic side of the brush-border membrane.     

Relationship between Ca2+-transport and ATP hydrolytic activities in guinea-pig pancreatic acinar plasma membranes

Partially purified plasma membrane fractions were prepared from guinea-pig pancreatic acini. These membrane preparations were found to contain an ATP-dependent Ca²⁺-transporter as well as a heterogenous ATP-hydrolytic activity. The Ca²⁺-transporter showed high affinity for Ca²⁺ (K_Ca ²⁺ = 0.04 ± 0.01 M), an apparent requirement for Mg²⁺ and high substrate specificity. The major component of ATPase activity could be stimulated by either Ca²⁺ or Mg²⁺ but showed a low affinity for these cations. At low concentrations, Mg²⁺ appeared to inhibit the Ca²⁺-dependent ATPase activity expressed by these membranes. However, in the presence of high Mg²⁺ concentration (0.5–1 mM), a high affinity Ca²⁺-dependent ATPase activity was observed (K_Ca ²⁺ = 0.08 ± 0.02 M). The hydrolytic activity showed little specificity towards ATP. Neither the Ca²⁺-transport nor high affinity Ca²⁺-ATPase activity were stimulated by calmodulin. The results demonstrate, in addition to a low affinity Ca²⁺ (or Mg⁺)-ATPase activity, the presence of both a high affinity Ca²⁺-pump and high affinity Ca²⁺-dependent ATPase. However, the high affinity Ca²⁺-ATPase activity does not appear to be the biochemical expression of the Ca²⁺-pump.Abbreviations Ca²⁺-ATPase calcium-activated, magnesium-dependent adenosine triphosphatase - CaM calmodulin - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate - EDTA ethylene-diaminetetraacetate - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate - NADPH reduced form of nicotinamide adenine dinucleotide phosphate     

Expression and regulation of insulin-like growth factor binding protein-1 in the rat uterus throughout estrous cycle

The role of Ca²⁺-stimulated adenosine 5-triphosphatase (Ca²⁺-ATPase) in Ca²⁺ sequestering of rat liver nuclei was investigated. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺–Mg²⁺)-ATPase activity. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Nuclear Ca²⁺-ATPase activity increased linearly in the range of 10–40 M Ca²⁺ addition. With those concentrations, Ca²⁺ was completely taken up by the nuclei dependently on ATP (2 mM). Nuclear Ca²⁺-ATPase activity was decreased significantly by the presence of arachidonic acid (25 and 50 M), nicotinamide-adenine dinucleotide (NAD⁺; 2 mM) and zinc sulfate (2.5 and 5.0 M). These reagents caused a significant decrease in the nuclear Ca²⁺ uptake and a corresponding elevation in Ca²⁺ release from the nuclei. Moreover, calmodulin (10 g/ml) increased significantly nuclear Ca²⁺-ATPase activity, and this increase was not seen in the presence of trifluoperazine (10 M), an antogonist of calmodulin. The present findings suggest that Ca²⁺-ATPase plays a role in Ca²⁺ sequestering by rat liver nuclei, and that calmodulin is an activator. Moreover, the inhibition of Ca²⁺-ATPase may evoke Ca²⁺ release from the Ca²⁺-loaded nuclei.     

Thermal stability of soluble mitochondrial H+-ATPase

ATPase melting has been studied by circular dichroism and differential scanning microcalorimetry. Decomposition of the -helix of H⁺-ATPase (in which about 80% of the peptide groups of the enzyme are involved) following thermal treatment is shown to proceed gradually, beginning with room temperature. Effect of nucleotides upon melting is detected in the range of 20–40 C. Above 40 C, the pattern of thermal decomposition of the three-dimensional structure of H⁺-ATPase is independent of the nature of nucleotides present. Highly stable -helical sites have been found in the enzyme molecule. Possible mechanism of formation of such sites is discussed, and the results obtained are compared with data on thermal stability of ATPase from thermophilic bacteria. Structural changes in the molecule following thermal treatment are compared with ATPase activity changes under similar experimental conditions.     

Catecholamine- and volume-dependent ion fluxes in carp (Cyprinus carpio) red blood cells

In carp erythrocytes, noradrenaline (10^-6 mol·l^-1) induces a 30- to 40-fold activation of Na⁺/H⁺ exchange (the ethylisopropylamiloride-inhibited component of the ²²Na influx) and a fourfold stimulation of the Na⁺, K⁺ pump (ouabain-inhibited component of ⁸⁶Rb influx). In both cases the effect of noradrenaline is blocked by propranolol but not phentolamine and is imitated by forskolin. An activator of protein kinase C (-phorbol 12-myristate, 13-acetate) increases Na⁺/H⁺ exchange by 10 times and decreases the Na⁺, K⁺ pump activity by 20–30 percent. In the presence of ethylisopropylamiloride the increment of the Na⁺, K⁺ pump activity induced by noradrenaline is reduced by 35–45 percent, indicating the existence of a Na⁺/H⁺ exchange-independent mechanism of the Na⁺, K⁺ pump regulation by -adrenergic catecholamines. Hypertonic shrinkage of carp erythrocytes results in a 40- to 80-fold activation of Na⁺/H⁺ exchange, whereas hypotonic swelling induces an increase in the rate of ⁸⁶Rb⁺ efflux which is inhibited by furosemide by about 30–40 percent. The rate of pH₀ recovery in response to acidification or alkalinization in rat erythrocytes is approximately 15 times as fast as in carp erythrocytes. Unlike in rat erythrocytes, valinomycin does not cause an alkalinization of incubation medium in carp erythrocytes indicating the absence of conductive pathway in the operation of anion transporter protein. A scheme is suggested which describes the interrelation of Na⁺/H⁺ exchange, Na⁺, K⁺ pump and a non-identified system providing for K⁺ efflux in cell swelling, regulation of cell volume and cytoplasmic pH in fish erythrocytes under conditions of deep hypoxia and high activity.Abbreviations cAMP cyclic adenosine monophosphate - CCCP carbonylcyamide m-chlorophenylhydrazone - DMSO dimethylsulphoxide - EIPA ethylisopropylamiloride - NA noradrenaline - PMA -phorbol 12-myristate, 13-acetate - RVD regulatory volume decrease - RVI regulatory volume increase     

Characterization of two different Ca2+ uptake and IP3-sensitive Ca2+ release mechanisms in microsomal Ca2+ pools of rat pancreatic acinar cells

We have examined the effect of the Ca²⁺ (Mg²⁺)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent ⁴⁵Ca²⁺ uptake into IP₃-sensitive Ca²⁺ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40–50% of total Ca²⁺ uptake was inhibited by TG up to 10 nm (apparent K_i4.2 nm, Ca²⁺ pool I). An additional increase of inhibition up to 85–90% of total Ca²⁺ uptake could be achieved at 15 to 20 nm of TG (apparent K_i12.1 nm, Ca²⁺ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent K_i10 m). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca²⁺ uptake. About 30–40% of total Ca²⁺ uptake was inhibited by 100 m of vanadate (apparent K_i18 m, Ca²⁺ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent K_i300 m) or by TG up to 10 nm (Ca²⁺ pool I). The amount of IP₃-induced Ca²⁺ release was constant at 25% over a wide range of Ca²⁺ filling. About 10–20% remained unreleasable by IP₃. Reduction of IP₃ releasable Ca²⁺ in the presence of inhibitors showed similar dose-response curves as Ca²⁺ uptake (apparent K_i 3.0 nm for IP₃-induced Ca²⁺ release as compared to 4.2 nm for Ca²⁺ uptake at TG up to 10 nm) indicating that the highly TG-sensitive Ca²⁺ pump fills the IP₃-sensitive Ca²⁺ pool I. At TG concentrations >10 nm which blocked Ca²⁺ pool II the apparent K_i values were 11.3 and 12.1 nm, respectively. For inhibition by vanadate up to 100 m the apparent K_i values were 18 m for Ca²⁺ uptake and 7 m for Ca²⁺ release (Ca²⁺ pool II). At vanadate concentrations up to 1 mm the apparent K_i values were 300 and 200 m, respectively (Ca²⁺ pool I). Both Ca²⁺ pools I and II also showed different sensitivities to IP₃. Dose-response curves for IP₃ in the absence of inhibitors (control) showed an apparent K_m value for IP₃ at 0.6 m. In the presence of TG (inhibition of Ca²⁺ pool I) the curve was shifted to the left with an apparent K_m for IP₃ at 0.08 m. In the presence of vanadate (inhibition of Ca²⁺ pool II), the apparent K_m for IP₃ was 2.1 m. These data allow the conclusion that there are at least three different Ca²⁺ uptake mechanisms present in pancreatic acinar cells: TG- and IP₃ insensitive but highly vanadate-sensitive Ca²⁺ uptake occurs into membrane vesicles derived from plasma membranes. Two Ca²⁺ pools with different TG-, vanadate- and IP₃-sensitivities are most likely located in the endoplasmic reticulum at different cell sites, which could have functional implications for hormonal stimulation of pancreatic acinar cells.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 246. The authors wish to thank Dr. KlausDieter Preuß for valuable discussions and Mrs. Gabriele Mörschbächer for excellent secretarial help.     

The Nootropic Drug Vinpocetine Inhibits Veratridine-Induced [Ca2+]i Increase in Rat Hippocampal CA1 Pyramidal Cells

The alkaloid derivative vinpocetine (14-ethoxycarbonyl-(3,16-ethyl)-14,15-eburnamine; Cavinton) has a well known beneficial effect on brain function in hypoxic and ischemic conditions. While it increases CNS blood flow and improves cellular metabolism, relatively little is known about vinpocetine's underlying molecular mechanisms on the single cell level. Since apoptotic and necrotic cell damage is always preceded by an increase in [Ca²⁺]_i, this study investigated the effect of vinpocetine on [Ca²⁺]_i increases in acute brain slices. Sodium influx is an early event in the biochemical cascade that takes place during ischemia. The alkaloid veratridine can activate this Na⁺ influx, causing depolarization and increasing [Ca²⁺]_i in the cells. Therefore, it can be used to simulate an ischemic attack in brain cells. Using a cooled CCD camera-based ratio imaging system and cell loading with fura 2/AM, the effect of vinpocetine on [Ca²⁺]_i changes in single pyramidal neurons in the vulnerable CA1 region of rat hippocampal slices was investigated. Preperfusion and continuous administration of vinpocetine (10 M) significantly inhibited the elevation in [Ca²⁺]_i induced by veratridine (10 M). When the drug was administered after veratridine, it could accelerate the recovery of cellular calcium levels. Piracetam, another nootropic used in clinical practice, could attenuate the elevation of [Ca²⁺]_i only at a high, 1 mM, concentration. We have concluded that vinpocetine, at a pharmacologically relevant concentration, can decrease pathologically high [Ca²⁺]_i levels in individual rat hippocampal CA1 pyramidal neurons; this effect might contribute to the neuroprotective property of the drug.     

Kinetic properties of the ATP-dependent Ca2+ pump and the Na+/Ca2+ exchange system in basolateral membranes from rat kidney cortex

Summary Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be LROIO=431. High-affinity Ca²⁺-ATPase, ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange have been studied with special emphasis on the relative transport capacities of the two Ca²⁺ transport systems. The kinetic parameters of Ca²⁺-ATPase activity in digitonin-treated membranes are:K _m =0.11 m Ca²⁺ andV _max=81±4 nmol P_i/min·mg protein at 37°C. ATP-dependent Ca²⁺ transport amounts to 4.3±0.2 and 7.4±0.3 nmol Ca²⁺/min·mg protein at 25 and 37°C, respectively, with an affinity for Ca²⁺ of 0.13 and 0.07 m at 25 and 37°C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca²⁺ transport, a stoichiometry of 0.7 mol Ca²⁺ transported per mol ATP is found for the Ca²⁺-ATPase. In the presence of 75mm Na⁺ in the incubation medium ATP-dependent Ca²⁺ uptake is inhibited 22%. When Na⁺ is present at 5mm an extra Ca²⁺ accumulation is observed which amounts to 15% of the ATP-dependent Ca²⁺ transport rate. This extra Ca²⁺ accumulation induced by low Na⁺ is fully inhibited by preincubation of the vesicles with 1mm ouabain, which indicates that (Na⁺–K⁺)-ATPase generates a Na⁺ gradient favorable for Ca²⁺ accumulation via the Na⁺/Ca²⁺ exchanger. In the absence of ATP, a Na⁺ gradient-dependent Ca²⁺ uptake is measured which rate amounts to 5% of the ATP-dependent Ca²⁺ transport capacity. The Na⁺ gradient-dependent Ca²⁺ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na⁺/Ca²⁺ exchange system for Ca²⁺ is between 0.1 and 0.2 m Ca²⁺, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca²⁺ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca²⁺-ATPase system exceeds the capacity of the Na⁺/Ca²⁺ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca²⁺ homeostasis of rat kidney cortex cells.     

Calcium efflux from platelet vesicles of the dense tubular system. Analysis of the possible contribution of the Ca2+ pump

The ATP dependent Ca²⁺ uptake of platelet vesicles was inhibited by the two hydrophobic drugs trifluoperazine (TFP) and propranolol (PROP). Inhibition was significantly lowered when Pi was used instead of oxalate as a precipitant agent. When the ATPase ligands substrate (Mg²⁺ and Pi) were absent of the efflux medium, a slow release of Ca²⁺ which did not couple with ATP synthesis (passive Ca²⁺ efflux) was observed. Both, TFP and PROP enhanced the passive Ca²⁺ efflux. This enhanced efflux was partially inhibited only when Mg²⁺ and Pi were added together to the efflux reaction media, but it was not affected by spermidine, ruthenium red or thapsigargin (TG). The Ca²⁺ ionophores A23187 and ionomycin, also enhanced passive Ca²⁺ efflux. However, in this case, Ca²⁺ efflux was inhibited just by inclusion of Mg²⁺ to the medium. Ca²⁺ efflux promoted by Triton X-100 was not affected by either Mg²⁺ or Pi, included together or separately into the efflux medium. The ATP Pi measured in the presence of Triton X-100 and millimolar Ca²⁺ concentrations was inhibited by both TFP and PROP, but not by Ca²⁺ ionophores up to 4 M. The data suggest that the observed enhancement of passive Ca²⁺ efflux promoted by TFP and PROP could be attributed to a direct effect of these drugs over the platelet Ca²⁺ pump isoforms (Sarco Endoplasmic Reticulum Calcium ATPase, SERCA_2b and SERCA₃) themselves, as it was reported for the sarcoplasmic reticulum Ca²⁺ ATPase (SERCA₁).