Bicarbonate-stimulated ATPase in the renal proximal tubule luminal (brush border) memebrane.

Brush border membranes of the rabbit renal tubule have an ATPase which was stimulated 60% by 50 mm HCO₃^?. The K_a for HCO₃^? was 36 mm. Kinetic studies of the “HCO₃^?-ATPase” indicate that HCO₃^? had no effect on the K_m for ATP and ATP did not alter the K_a for HCO₃^?. Several anions, notably SO₃^2?, also accelerated the rate of dephosphorylation of ATP. The V for “SO₃^2?-ATPase” was fivefold greater than that for “HCO₃^?-ATPase.” The K_a for SO₃^2? was 0.78 mm. Other anions including Cl^? and phosphates, did not enhance ATPase activity. Thus, of the anions present in the glomerular filtrate in appreciable concentrations only HCO₃^? stimulated the luminal membrane enzyme. The anion-stimulated ATPase activity increased sharply from pH 6.1 to 7.1 and moderately with higher pH. The renal ATPase was not inhibited by SCN^? nor methyl sulfonyl chloride and was relatively insensitive to oligomycin and quercetin. Carbonyl cyanide p-trifluoromethoxy phenylhydrazone increased the basal rate of the membranal ATPase, suggesting that the ATPase activity is limited by transmembrane H⁺ flux. Carbonic anhydrase significantly increased the HCO₃^?-stimulated ATPase activity. This increment was blocked by Diamox. These findings provide evidence consistent with the hypothesis that the brush border membrane ATPase is involved in the extrusion of H⁺ from tubular cell to lumen and support suggested interrelationships between HCO₃^?-stimulated ATPase, H⁺ secretion, and bicarbonate transport in the kidney.     

Substance P inhibits bicarbonate secretion from guinea pig pancreatic ducts by modulating an anion exchanger

The stimulatory pathways controlling HCO₃^- secretion by the pancreatic ductal epithelium are well described. However, only a few data are available concerning inhibitory mechanisms, which may play an important role in the physiological control of the pancreas. The aim of this study was to investigate the cellular mechanism by which substance P (SP) inhibits pancreatic ductal HCO₃^- secretion. Small intra/interlobular ducts were isolated from the pancreas of guinea pigs. During overnight culture the ducts seal to form a closed sac. Transmembrane HCO₃^- fluxes were calculated from changes in intracellular pH (measured using the pH-sensitive dye BCECF) and the buffering capacity of the cells. We found that secretin can stimulate HCO₃^- secretion in guinea pig pancreatic ducts about fivefold and that this effect could be totally blocked by SP. The inhibitory effect of SP was relieved by spantide, an SP receptor antagonist. SP had no effect on the activity of basolateral Na⁺-HCO₃^- cotransporters and Na⁺/H⁺ exchangers. However, the peptide did inhibit a Cl^--dependent HCO₃^- efflux (secretory) mechanism, most probably the Cl^-/HCO₃ exchanger on the apical membrane of the duct cell. pancreas; Cl^-/HCO₃^- exchanger; tachykinin     

Stimulation of HCO3− secretion by the prostaglandin E2 analog enprostil: Studies in human stomach and rat duodenum

This study investigated the action of enprostil, a synthetic analog of PGE₂, on gastric HCO₃⁻ secretion in humans and on duodenal HCO₃⁻ secretion in the anesthetized rat. A previously validated 2-component model was used to calculate gastric HCO₃⁻ and H⁺ secretion in 10 human subjects. Compared to placebo, a single 70 μg oral dose of enprostil increased basal gastric HCO₃⁻ secretion from 1810 +- 340 to 3190 ± 890 μmol/hr (P < 0.05). In addition, enprostil reduced basal gastric H⁺ secretion from 5240 ± 1140 to 1680 ± 530 μmol/hr (P < 0.02). Enprostil also increased HCO₃⁻ and reduced H⁺ secretion during intravenous pentagastrin infusion. In the rat, duodenal HCO₃⁻ secretion was measured by direct titration in situ using perfused segments of duodenum just distal to the Brunner gland area dn devoid of pancreatic and biliary secretions. Addition of enprostil(10 μg/ml) to the duodenal bathing solution increased duodenal HOC₃⁻ secretion from 6.3 ± 1.3 to 15.1 ± 2.0 μmol/cm·hr (P < 0.01, n = 6). The stimulatory action of enprostil on duodenal HCO₃⁻ secretion at 10 μg/ml was comparable in magnitude and duration to that of 10 μg/ml natural PGE₂. In summary, the PGE₂ analog enprostil stimulated gastroduodenal HCO₃⁻ secretion, effects which may be beneficial in protection of the gastroduodenal mucosa against luminal acid.     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K⁺, Na⁺ and ATP on the gastric (H⁺ + K⁺)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min^?1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min^?1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min^?1. Additions of 5 mM K⁺ or Na⁺, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K⁺, but not on Na⁺. Alkaline pH increased the rate of dephosphorylation. K⁺ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K⁺ was inhibitory. Below pH 7.0 Na⁺ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na⁺ inhibited both activities. The effect of extravesicular pH on transport of H⁺ was investigated. At pH 6.5 the apparent K_m for ATP was 2.7 μM and increased little when K⁺ was added extravesicularly. At pH 7.5, millimolar concentrations of K⁺ increased the apparent K_m for ATP. Extravesicular K⁺ and Na⁺ inhibited the transport of H⁺. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H⁺-producing reactions as possible candidates as physiological regulators of (H⁺ + K⁺)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO₂ and the subsequent formation of H⁺ and HCO₃^?. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H⁺/ATP ratio was about 1 at pH 8.0. When CO₂ was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO₂. The results indicate a possible co-operation in the production of acid between the H⁺ + K⁺-ATPase and a carbonic anhydrase associated with the vesicular membrane.     

Electrogenic transport of protons driven by the plasma membrane ATPase in membrane vesicles from radish : biochemical characterization

Mg:ATP-dependent H⁺ pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Δ ψ). ΔpH was measured as the decrease of absorbance of Acridine orange and Δ ψ as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Δ pH and Δ ψ generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Δ pH generation is not inhibited by NO₃⁻. These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H⁺-ATPases. Both intravesicular acidification and Δ ψ generation are influenced by anions: Δ pH increases and Δ ψ decreases following the sequence SO₄²⁻, Cl⁻, Br⁻, NO₃⁻. ATP-dependent H⁺ pumping strictly requires Mg²⁺. It is very specific for ATP (apparent K_m 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Δ pH generation is inhibited by CuSO₄ and diethylstilbestrol as well as vanadate. Δ pH generation is specificially stimulated by K⁺ (+ 80%) and to a lesser extent by Na⁺ and choline (+28% and +14%, respectively). The characteristics of H⁺ pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials.     

Evolution of the sodium pump

Eukaryotic plasma membranes (PMs) are energized by electrogenic P-type ATPases that generate either Na⁺ or H⁺ motive forces to drive Na⁺ and H⁺ dependent transport processes, respectively. For this purpose, animal rely on Na⁺/K⁺-ATPases whereas fungi and plants employ PM H⁺-ATPases. Prokaryotes, on the other hand, depend on H⁺ or Na⁺-motive electron transport complexes to energize their cell membranes. This raises the question as to why and when electrogenic Na⁺ and H⁺ pumps evolved? Here it is shown that prokaryotic Na⁺/K⁺-ATPases have near perfect conservation of binding sites involved in coordination of three Na⁺ and two K⁺ ions. Such pumps are rare in Eubacteria but are common in methanogenic Archaea where they often are found together with P-type putative PM H⁺-ATPases. With some exceptions, Na⁺/K⁺-ATPases and PM H⁺-ATPases are found everywhere in the eukaryotic tree of life, but never together in animals, fungi and land plants. It is hypothesized that Na⁺/K⁺-ATPases and PM H⁺-ATPases evolved in methanogenic Archaea to support the bioenergetics of these ancestral organisms, which can utilize both H⁺ and Na⁺ as energy currencies. Both pumps must have been simultaneously present in the first eukaryotic cell, but during diversification of the major eukaryotic kingdoms, and at the time animals diverged from fungi, animals kept Na⁺/K⁺-ATPases but lost PM H⁺-ATPases. At the same evolutionary branch point, fungi did loose Na⁺/K⁺-ATPases, and their role was taken over by PM H⁺-ATPases. An independent but similar scenery emerged during terrestrialization of plants: they lost Na⁺/K⁺-ATPases but kept PM H⁺-ATPases.     

Transport Properties of the Tomato Fruit Tonoplast : I. Identification and Characterization of an Anion-Sensitive H-ATPase

An anion-sensitive H⁺-translocating ATPase was identified in membrane vesicles isolated from mature green tomato (Lycopersicon esculentum) fruit. The H⁺-ATPase was associated with a low density membrane population having a peak density of 1.11 grams per cubic centimeter, and its activity was inhibited by NO₃⁻, N,N′-dicyclohexylcarbodiimide and diethylstilbestrol but not by vanadate, azide, molybdate, or oligomycin. This H⁺-ATPase has an unusual pH dependence indicating both a slightly acidic and a near neutral peak of activity. Chloride was found to be a potent stimulator of ATPase activity. The K_m for the H⁺-ATPase was approximately 0.8 millimolar ATP. The characteristics of this H⁺-ATPase are very similar to those described for a number of plant cell tonoplast H⁺-ATPases suggesting that the activity identified in tomato fruit membranes is tonoplast-associated. This report demonstrates the feasibility of isolating tonoplast vesicles from acidic fruit tissues for studies of transport activities associated with fruit development and maturation.     

Relations between K+ uptake and photosynthetic uptake of inorganic carbon by aquatic plants

The uptake of K⁺ by the leafy shoots of four submersed higher aquatic plants (Elodea canadensis, Ranunculus aquatilis, R. trichophyllus, and Callitriche hamulata) with different HCO₃ ^- affinity was measured in successive 2-h periods under the conditions of high or low photosynthetic rates (i.e. at pH 7.5 or 10). At pH 7.5 the uptake of K⁺ by species with the higher HCO₃ ^- affinity (E. canadensis, R. trichophyllus) was significantly faster than that by species with a lower HCO₃ ^- affinity (R. aquatilis, C. hamulata). In the former group of species, the K⁺ uptake rate at pH 7.5 was 1.7 - 3.5 times higher than at pH 10. At pH 10, the soft-water species, R. aquatilis, had the lowest net photosynthetic rate (PN) of the three HCO₃ ^- users but, in contrast to the relative hard-water species, R. trichophyllus, showed a small K⁺ efflux (47 nmol kg^-1 s^-1). Thus, K⁺ uptake by shoots was not strictly correlated with PN. A significant K⁺ efflux (73 - 86 nmol kg^-1 s^-1) occurred from all HCO₃ ^- users in darkness. The relatively low K⁺ uptake by the strict CO₂ user, C. hamulata, was quite independent of PN and light or darkness. It may be suggested that uptake of K⁺ by shoots of submersed plants depends on their HCO₃ ^- affinity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Morphogenesis of “duct-like” structures in three-dimensional cultures of human cancerous pancreatic duct cells (Capan-1)

Summary Pancreatic duct cells secrete water and ions, bicarbonate in particular. The study of these secretion processes is hindered by the unavailability of human pancreatic tissue. In this study, pancreatic human cells of the Capan-1 cell line were employed to investigate secretion in vitro. These cells are of ductal origin because in standard culture they polarize spontaneously forming domes in the culture dishes, indicating the existence of transepithelial exchange of water and electrolytes. In culture in suspension, Capan-1 cells form hollow spheroids bounded by a cell monolayer in a radial organization. These three-dimensional structures could be maintained in culture for more than 140 days. In young cultures, the cells of these spheroids grew rapidly (mitotic index=9.2% on Day 2). Their cytologic features were analyzed by immunocytochemical, cytoenzymatic methods, and by electron microscopy. We showed that they are : a) polarized with an apical pole facing the culture medium; b) organized in a monolayer; c) bound by tight junctions and desmosomes; d) characterized by a particular distribution of enzyme systems known to play a role in ion exchanges, with placental-type alkaline phosphatases and carbonic anhydrases IV on their apical membranes and Ca²⁺-ATPases on their basolateral membranes. Crystalline structures were detected histochemically in the closed cavities and in the intercellular spaces of the spheroids. X-ray emission spectroscopy and electron diffraction showed that they consisted of calcium phosphate in an apatite structure. They were assumed to derive from a raised concentration of Ca²⁺ and phosphate ions under the impermeable monolayer of the spheroids. In addition, numerous cells secreted M1 gastric-type mucins, and acquired the ability to produce colonic-type M3 mucins. These hollow spheroids swelled during the culture period. Taken together these results suggest that the Capan-1 cells organized in these hollow spheroids exchange ions. Their three-dimensional structure resembles that of human pancreatic ducts, and they may therefore represent a useful model system for investigation of Cl⁻ and HCO₃ ⁻ ion exchange processes in the human pancreas.     

Dicyclohexylcarbodiimide and vanadate sensitive ATPase of lung lamellar bodies

Lung surfactant is synthesized in lung epithelial type II cells and stored in the lamellar bodies prior to its secretion onto the alveolar surface. The lamellar bodies, like other secretory organelles, maintain an ATP-dependent pH gradient that is sensitive to inhibitors of H⁺-ATPase. This report shows that the ATPase activity of lamellar bodies is enriched in a fraction prepared from lamellar bodies that were disrupted after isolation. The apparent V_max for this enzyme was 150 nmol ATP hydrolyzed per min per mg protein and apparent K_m for ATP was approximately 50 μM. The enzyme activity was sensitive to N-ethylmaleimide (NEM), dicyclohexylcarbodiimide (DCCD) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) (all inhibitors of vacuolar-type H⁺-ATPase) and vanadate (inhibitor of phosphoenzyme-type ATPase). Besides, the activity could also be inhibited with diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and Ca²⁺. Two proteins (of approximately 45 kDa and 17 kDa) of this fraction showed acid-stable phosphorylation with ATP. The labeling of proteins with ATP (-γ-³²P) could be chased with unlabelled ATP, suggesting that phosphorylation and dephosphorylation of these proteins is associated with the ATPase activity. Our results on inhibition characteristics of the enzyme activity suggest that besides a vacuolar type H⁺-ATPase, the lamellar bodies also contain a phosphoenzyme type ATPase that is sensitive to inhibitors of vacuolar type H⁺-ATPase.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: II. H/ATP Stoichiometry of an Anion-Sensitive H-ATPase

The H⁺/ATP stoichiometry was determined for an anion-sensitive H⁺-ATPase in membrane vesicles believed to be derived from tonoplast. Initial rates of proton influx were measured by monitoring the alkalinization of a weakly buffered medium (pH 6.13) following the addition of ATP to a suspension of membrane vesicles of Beta vulgaris L. Initial rates of ATP hydrolysis were measured in an assay where ATP hydrolysis is coupled to NADH oxidation and monitored spectrophotometrically (A₃₄₀) or by monitoring the release of ³²P from [γ-³²P]ATP. Inasmuch as this anion-sensitive H⁺-ATPase is strongly inhibited by NO₃⁻, initial rates of H⁺ influx and ATP hydrolysis were measured in the absence and presence of NO₃⁻ to account for ATPase activity not involved in H⁺ transport. The NO₃⁻-sensitive activities were calculated and used to estimate the ratio of H⁺ transported to ATP hydrolyzed. These measurements resulted in an estimate of the H⁺/ATP stoichiometry of 1.96 ± 0.14 suggesting that the actual stoichiometry is 2 H⁺ transported per ATP hydrolyzed. When compared with the reported values of the electrochemical potential gradient for H⁺ across the tonoplast measured in vivo, our result suggests that the H⁺-ATPase does not operate near equilibrium but is regulated by cellular factors other than energy supply.     

Chloroplast-generated ROS dominate NaCl- induced K+ efflux in wheat leaf mesophyll

Mesophyll K⁺ retention ability has been recently reported as an important component of salinity stress tolerance in wheat. In order to investigate the role of ROS in regulating NaCl^-induced K⁺ efflux in wheat leaf mesophyll, a series of pharmacological experiments was conducted using MV (methyl viologen, superoxide radical inducer), DPI (an inhibitor of NADPH oxidase), H₂O₂ (to mimic apoplastic ROS), and EGCG ((−)-Epigallocatechin gallate, ROS scavenger). Mesophyll pre-treatment with 10 μM MV resulted in a significantly higher NaCl^-induced K⁺ efflux in leaf mesophyll, while 50 μM EGCG pre-treatment alleviated K⁺ leakage under salt stress. No significant change in NaCl^-induced K⁺ efflux in leaf mesophyll was found in specimens pre-treated by H₂O₂ and DPI, compared with the control. The highest NaCl^-induced H⁺ efflux in leaf mesophyll was also found in samples pre-treated with MV, suggesting a futile cycle between increased H⁺-ATPase activity and ROS-induced K⁺ leak. Overall, it is suggested that, under saline stress, K⁺ efflux from wheat mesophyll is mediated predominantly by non-selective cation channels (NSCC) regulated by ROS produced in chloroplasts, at least in bread wheat.     

Differential expression of P-type ATPases in intestinal epithelial cells: Identification of putative new atp1a1 splice-variant

P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the membrane. Ten different subtypes have been described. In mammalia, 15 genes of P-type ATPases from subtypes II-A, II-B and II-C, that transport low-atomic-weight cations (Ca²⁺, Na⁺, K⁺ and H⁺), have been reported. They include reticulum and plasma-membrane Ca²⁺-ATPases, Na⁺/K⁺-ATPase and H⁺/K⁺-ATPases. Enterocytes and colonocytes show functional differences, which seem to be partially due to the differential expression of P-type ATPases. These enzymes have 9 structural motifs, being the phosphorylation (E) and the Mg²⁺ATP-binding (H) motifs the most preserved. These structural characteristics permitted developing a Multiplex-Nested-PCR (MN-PCR) for the simultaneous identification of different P-type ATPases. Thus, using MN-PCR, seven different cDNAs were cloned from enterocytes and colonocytes, including SERCA3, SERCA2, Na⁺/K⁺-ATPase α1-isoform, H⁺/K⁺-ATPase α2-isoform, PMCA1, PMCA4 and a cDNA-fragment that seems to be a new cassette-type splice-variant of the atp1a1 gen. PMCA4 in enterocytes and H⁺/K⁺-ATPase α2-isoform in colonocytes were differentially expressed. This cell-specific expression pattern is related with the distinctive enterocyte and colonocyte functions.     

Specific modification of gastric K+-stimulated ATPase activity by thimerosal

Treatment of hog gastric microsomes with the sulfhydryl reagent, thimerosal (ethylmercurithiosalicylate), produced differential effects on the K⁺-ATPase and the K⁺-stimulated p-nitrophenylphosphatase activities. For example, exposure to 2 mM thimerosal for 3 min severely reduced the activity of K⁺-stimulated ATPase, while K⁺-p-nitrophenylphosphatase activity was enhanced 2- to 3-fold. Higher concentration of thimerosal, or longer incubation times, also led to inhibition of K⁺-p-nitrophenylphosphatase. The activated state of p-nitrophenylphosphatase could be sustained by a 20-fold, or greater, dilution of treated membranes, and could be reversed by reduction of membrane SH groups by exogenous thiols. Significant activation of K⁺-p-nitrophenylphosphatase was not produced by p-chloromercuribenzene sulfonate, p-chloromercuribenzoate or mersalyl; however, ethyl mercuric chloride had qualitatively similar activity effects as thimerosal. Kinetics of K⁺-p-nitrophenylphosphatase for thimerosal-treated membranes were altered as follows: V increased; K_m for p-nitrophenylphosphate unchanged for K_a for K⁺ increased. ATP, which is a potent inhibitor of K⁺-p-nitrophenylphosphatase activity in native membranes (K_I ≈ 200 μM). These data suggest that there are multiple SH groups which differentially influence the gastric K⁺-stimulated ATPase activity. Defined treatments with thimerosal are interpreted as an uncoupling of the K⁺-stimulated phosphatase component of the enzyme (for which p-nitrophenylphosphatase is a presumed model reaction). Such differential modifications can be usefully applied to the study of partial reactions of the enzyme and their specific role in the related H⁺-transport reaction.     

UV-Stimulated K Efflux from Rose Cells: Counterion and Inhibitor Studies

Irradiation of a washed suspension of cultured rose (Rosa damascena var. Gloire de Guilan) cells with about 1,680 joules per square meter of short wave ultraviolet (UV) light (254 nanometers) caused K⁺ to appear in the external medium. Short-term tracer (⁸⁶Rb⁺) experiments confirmed the earlier suggestion (Wright, Murphy 1978 Plant Physiol 61: 434-436) that UV increases the efflux of K⁺; there was also a small decrease in influx of K⁺. There was a partial recovery of fluxes from the effects of UV radiation, but no net accumulation of K⁺ within 16 to 18 hours after the irradiation. The K⁺ appearing in the medium was matched by an equivalent amount of HCO₃⁻; it was suggested that HCO₃⁻ was the principal counterion for the K⁺ flux induced by UV. Inhibitors of ATP synthesis (10⁻⁵ molar carbonyl cyanide m-chlorophenyl hydrazone; 0.05 millimolar KCN plus 0.75 millimolar salicylhydroxamic acid) strongly reduced the UV-stimulated K⁺ leakage, suggesting that the leakage was dependent in some way on ATP concentration inside the cells. The UV-induced K⁺ leakage was also dependent on temperature and the presence of Ca²⁺ in the external medium.     

ATP synthesis catalyzed by a V-ATPase: an alternative pathway for energy conservation operating in plant vacuoles?

The electrochemical H⁺ gradient generated in tonoplast vesicles isolated from maize seeds was found to be able to drive the reversal of the catalytic cycle of both vacuolar H⁺-pumps (Façanha and de Meis, 1998). Here we describe the reversibility of the vacuolar V-type H⁺-ATPase (V-ATPase) even in the absence of the H⁺ gradient in a water-Me₂SO co-solvent mixture, resulting in net synthesis of [γ-³²P]ATP from [³²P]P_i and ADP. The water-Me₂SO (5 to 20 %) media promoted inhibition of both PP_i hydrolysis and synthesis reactions whereas it slightly affected the ATP hydrolysis and clearly stimulated the ATP synthesis, which was unaffected by uncoupling agents (FCCP, Triton X-100 or NH₄⁺). This effect of Me₂SO on the ATP⇔³²P exchange reaction seems to be related to a decrease of the apparent K_m of the V-ATPase for P_i. The results are in accordance to the concept that the energetics of ATP synthesis catalysis depends on the solvation energies interacting in the enzyme microenvironment. A possible physiological significance of this phenomenon for the metabolism of desiccation-tolerant plant cells is discussed.Key words: bind energy, proton pumps, proton gradient, DMSO, corn seeds, V₁V₀-ATPase, membrane bound H⁺-pyrophosphatase     

Effects of Extracellular pH on UV-Induced K Efflux from Cultured Rose Cells

Ultraviolet (UV) light causes a specific leakage of K⁺ from cultured rose cells (Rosa damascena). During K⁺ efflux, there is also an increase in extracellular HCO₃⁻ and acidification of the cell interior. We hypothesized that the HCO₃⁻ originated from intracellular hydration of respiratory CO₂ and served as a charge balancing mechanism during K⁺ efflux, the K⁺ and HCO₃⁻ being cotransported out of the cell through specific channels. An alternative hypothesis which would yield similar results would be the countertransport of K⁺ and H⁺. To test these hypotheses, we studied the effect of a range of external pH values (pH 5-9), regulated by various methods (pH-stat, 100 millimolar Tris-Mes buffer, or CO₂ partial pressure), on the UV-induced K⁺ efflux. Both UV-C (<290 nanometers) and UV-B (290-310 nanometers) induced K⁺ efflux with a minimum at about pH 6 to 7, and greater efflux at pH values of 5, 8, and 9. Since pH values of 8 and 9 increased instead of reduced the efflux of K⁺, these data are not consistent with the notion that the efflux of K⁺ is dependent on an influx of H⁺, a process that would be sensitive to external H⁺ concentration. We suggest that the effect of pH on K⁺ efflux may be mediated through the titration of specific K⁺-transporting proteins or channels in the plasma membrane. Since we could not detect the presence of carbonic anhydrase activity in cell extracts, we could not use the location of this enzyme to aid in our interpretation regarding the site of hydration of CO₂.     

Splice Cassette II of Na+,HCO3? Cotransporter NBCn1 (slc4a7) Interacts with Calcineurin A: IMPLICATIONS FOR TRANSPORTER ACTIVITY AND INTRACELLULAR pH CONTROL DURING RAT ARTERY CONTRACTIONS*

Activation of Na⁺,HCO₃⁻ cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH_i) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na⁺,HCO₃⁻ cotransporter NBCn1 (slc4a7) and the Ca²⁺/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pH_i control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineurin Aβ. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aβ co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na⁺,HCO₃⁻ cotransport activity was investigated in VSMCs of mesenteric arteries after an NH₄⁺ prepulse. During depolarization with 50 mm extracellular K⁺ to raise intracellular [Ca²⁺], Na⁺,HCO₃⁻ cotransport activity was inhibited 20–30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na⁺,HCO₃⁻ cotransport activity in VSMCs when cytosolic [Ca²⁺] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aβ. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aβ and the Na⁺/H⁺ exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aβ and cassette II of NBCn1. Intracellular Ca²⁺ activates Na⁺,HCO₃⁻ cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.     

NH(4)(+)-stimulated low-K(+) uptake is associated with the induction of H(+) extrusion by the plasma membrane H(+)-ATPase in sorghum roots under K(+) deficiency

The effect of external inorganic nitrogen and K⁺ content on K⁺ uptake from low-K⁺ solutions and plasma membrane (PM) H⁺-ATPase activity of sorghum roots was studied. Plants were grown for 15 days in full-nutrient solutions containing 0.2 or 1.4 mM K⁺ and inorganic nitrogen as NO₃^-, NO₃^-/NH₄⁺ or NH₄⁺ and then starved of K⁺ for 24, 48 and 72 h. NH₄⁺ in full nutrient solution significantly affected the uptake efficiency and accumulation of K⁺, and this effect was less pronounced at the high K⁺ concentration. In contrast, the translocation rate of K⁺ to the shoot was not altered. Depletion assays showed that plants grown with NH₄⁺ more efficiently depleted the external K⁺ and reached higher initial rates of low-K⁺ uptake than plants grown with NO₃^-. One possible influence of K⁺ content of shoot, but not of roots, on K⁺ uptake was evidenced. Enhanced K⁺-uptake capacity was correlated with the induction of H⁺ extrusion by PM H⁺-ATPase. In plants grown in high K⁺ solutions, the increase in the active H⁺ gradient was associated with an increase of the PM H⁺-ATPase protein concentration. In contrast, in plants grown in solutions containing 0.2 mM K⁺, only the initial rate of H⁺-pumping and ATP hydrolysis were affected. Under these conditions, two specific isoforms of PM H⁺-ATPase were detected, independent of the nitrogen source and deficiency period. No change in enzyme activity was observed in NO₃^--grown plants. The results suggest that K⁺ homeostasis in NH₄⁺-grown sorghum plants may be regulated by a high capacity for K⁺ uptake, which is dependent upon the H⁺-pumping activity of PM H⁺-ATPase.