Effects of in Vivo Treatment with Abscisic Acid and/or Cytokinin on Activities of Vacuolar H+ Pumps of Tonoplast-Enriched Membrane Vesicles Prepared from Barley Roots

We investigated the effects of in vivo treatment (1 day) ofbarley roots with abscisic acid (ABA) and/or a cytokinin (6-benzyladenine;BA) on the ATP- and PP_i-dependent H⁺ transport activities oftonoplast-enriched membrane vesicles prepared from the roots.Treatment with ABA significantly increased the two H⁺ transportactivities. By contrast, treatment with BA significantly decreasedPP_i-dependent H⁺ transport activity, while the change in ATP-dependentH⁺ transport activity was small. Increases in the two H⁺ transportactivities caused by treatment with ABA were suppressed duringtreatment with ABA and BA. Changes in the NO^–-inhibitableATPase activity and the Na⁺-inhibitable PP_iase activity of membranevesicles after treatment of roots with phytohormone(s) (ABA,BA, ABA + BA) were similar to changes in the ATP- and PP_i dependentH⁺ transport activities of the membrane vesicles, respectively.Immunoblot analysis with antibodies raised against the functionalcatalytic subunits of the vacuolar H⁺ pumps (H⁺- ATPase andH⁺-PP_iase) of mung bean revealed that only the level of thefunctional catalytic subunit of the H⁺-PP_iase of the membranevesicles was significantly increased by treatment with ABA aloneand in combination with BA. These results suggest that treatmentwith ABA has a stimulatory effect on the activities of the twoH⁺ pumps of the vacuolar membrane of barley roots, with increasein the level of the catalytic subunit of the H⁺-PP_iase, andthat treatment with BA has an inhibitory effect on the two H⁺pump activities of the vacuolar membrane without changes inthe levels of the catalytic subunits of either H⁺ pump, withthe limitation that treatment with BA has an inhibitory effectonly when the activity of the H⁺-ATPase has been increased bytreatment with ABA. ³Present address: Department of Biology, Faculty of Science,Hirosaki University, Hirosaki, 036 Japan     

In vivo Treatments That Modulate PPi-Dependent H+ Transport Activity of Tonoplast-Enriched Membrane Vesicles from Barley Roots

The PP_i-dependent H⁺ transport activity of tonoplast-enrichedmembrane vesicles prepared from barley roots was greatly reducedwhen the plants were grown for 4 or 5 days with an additional3 raM KC1 in growth medium that contained only 0.1 mM CaCl₂in water. To characterize the mechanism of this reduction inactivity, we attempted to treat barley roots with K⁺ ions, Cl^-ions(or acetate), and A23187 [GenBank] (with or without Ca²⁺ ions), whichmight be expected to cause alkalization, acidification and mobilizationof Ca²⁺ ions in the cytoplasm, respectively. One-day treatmentof barley roots with K⁺ ions significantly decreased PP_i--dependentH⁺ transport activity of prepared tonoplast-enriched membranevesicles, while treatment with Cl^- ions or acetate significantlyincreased the activity. A similar increase in the activity alsooccurred by treatment with Ca²⁺ ions alone or in combinationwith A23187 [GenBank] . Determination of the PP_i-hydrolyzing activity ofmembrane vesicles showed that changes in this activity by thevarious treatments were similar to those in the PP_i-dependentH⁺ transport activity. The changes in ATP-dependent H⁺ transportactivity of membrane vesicles caused by these treatments weresmall. These results indicate that the in vivo treatments hadsignificant effects on the H⁺ transport activity of H⁺-PP_i-ase,one of the two active vacuolar H⁺-pumps (H⁺-PP_iase and H⁺-ATPase).In addition, these results suggest the possibility that changesin levels of cytoplasmic H⁺ or Ca²⁺ ions may be involved inmodulation of the H⁺ transport activity of the vacuolar H⁺-PP_iaseduring plant growth. (Received September 14, 1992; Accepted March 1, 1993)     

Rapid Reconstitution of Tonoplast H+-Translocating Pyrophosphatase from Cultured Rice Cells into Liposomes

We report the rapid and functional reconstitution of H⁺-pyrophosphatase(H⁺-PPase) from the tonoplast of cultured rice (Oryza sativaL.) cells to proteoliposomes. The CHAPS-solubilized H⁺-PPasewas incorporated into liposomes by gel-filtration. Both theactivities of PP_i-hy-drolysis and H⁺-pumping were influencedby the lipid-pro-tein ratio and cholesterol. (Received June 10, 1996; Accepted January 9, 1997)     

Characterization of Two Proton Transport Systems in the Tonoplast of Plasmalemma-Permeabilized Nitella Cells

ATP-dependent and PP_i-dependent H⁺-transport systems of thetonoplast were characterized in plasmalemma-permeabilized Nitellacells, where direct access to the protoplasmic surface of thetonoplast was possible. Since H⁺ transport across the tonoplastcan be measured in situ, the identity of the membrane responsiblefor H⁺ pumping is unequivocal. H⁺ transport was evaluated bythe accumulation of neutral red. While both transport systemswere obligately dependent on Mg²⁺, the two transport systemsshowed completely different sensitivity to NO₃^– and K⁺,suggesting the presence of two types of H⁺-pumps in Nitellatonoplast. NO₃^– applied to the protoplasmic surface, completelyand reversibly inhibited ATP-dependent transport but had noeffect on PP_i-dependent transport. By contrast, NO₃^– appliedinto the vacuole by the vacuolar perfusion technique did notinhibit ATP-dependent or PP_i-dependent H⁺ transport. Replacementof K⁺ with the organic cation, BTP, inhibited PP_i-dependenttransport but not the ATP-dependent one, indicating that PP_i-dependenttransport is K⁺ dependent. The sensitivities of the H⁺ transportsystems found in the tonoplast of Nitella are quite similarto those of higher plant tonoplasts. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received February 21, 1987; Accepted May 27, 1987)     

A vacuolar H-pyrophosphatase differential activation and energy coupling integrate the responses of weeds and crops to drought stress

Background

Cyperus rotundus L. is a C4 weed of large vegetative and reproductive vigor endowed with competitive advantages over most crop species mainly under adverse environmental conditions. Vacuole functions are critical for the mechanisms of drought resistance, and here the modulation of the primary system of vacuolar ion transport is investigated during a transient water stress imposed to this weed and to C4 crop species (Zea mays L.).

Methods

The vacuolar H⁺ pumps, the H⁺-ATPase and H⁺-PP_iase, expression, activities and the energy coupling were spectrophotometrically investigated as key elements in the differential drought-resistance mechanisms developed by weeds and crops.

Results

In C. rotundus tonoplasts, ATP hydrolysis was more sensitive to drought than its coupled H⁺ transport, which was in turn at least 3-folds faster than that mediated by the H⁺-PP_iase. Its PP_i hydrolysis was only slightly affected by severe water deficit, contrasting with the disruption induced in the PP_i-dependent H⁺-gradient. This effect was antagonized by plant rehydration as the H⁺-PP_iase activity was highly stimulated, reassuming a coupled PP_i-driven H⁺ pumping. Maize tonoplasts exhibited 2–4 times lower hydrolytic activities than that of C. rotundus, but were able to overactivate specifically PP_i-dependent H⁺ pumping in response to stress relief, resulting in an enhanced H⁺-pumps coupling efficiency.

Conclusion

These results together with immunoanalysis revealed profiles consistent with pre- and post-translational changes occurring on the tonoplast H⁺-pumps, which differ between weeds and crops upon water deficit.

General significance

The evidences highlight an unusual modulation of the H⁺-PP_iase energy coupling as a key biochemical change related to environmental stresses adaptive capacity of plants.     

A Ca2+-ATPase and a Mg2+/H+-antiporter are present on tonoplast membranes from roots of Zea mays L.

The primary or secondary energized transport of Ca²⁺, Mg²⁺ and H⁺ into tonoplast membrane vesicles from roots of Zea mays L. seedlings was studied photometrically by using the fluorescent Ca²⁺ indicator Indo 1 and the pH indicator neutral red. The localization of an ATP-dependent, vanadate-sensitive Ca²⁺ pump on tonoplast-type vesicles was demonstrated by the co-migration of the Ca²⁺-pumping and tonoplast H⁺-pyrophosphatase (PP_iase) activity on continuous sucrose density gradients. In ER-membrane fractions, only a low Ca²⁺-pumping activity could be detected. The ATP-dependent Ca²⁺ uptake into tonoplast vesicles (using Ca²⁺ concentrations from 0.8–1 μM) was completely inhibited by the Ca²⁺ ionophore ionomycin (1 μM) whereas the protonophore nigericin (1 μM) which eliminates ATP-dependent intravesicular H⁺ accumulation had no effect. Vanadate (IC₅₀ = 43 μM) and diethylstilbesterol (IC₅₀ = 5.2 μM) were potent inhibitors of this type of Ca²⁺ transport. The nucleotides GTP, UTP, ITP, and ADP gave 27%–50% of the ATP-dependent activity (K _m = 0.41 mM). From these results, it was suggested that this ATP-dependent high-affinity Ca²⁺ transport mechanism is the only functioning Ca²⁺ transporter of the tonoplast under in-vivo conditions i.e. under the low cytosolic Ca²⁺ concentration. In contrast, the secondary energized Ca²⁺-transport mechanism of the tonoplast, the low-affinity Ca²⁺/H⁺-antiporter, which was reported to allow the uptake of Ca²⁺ in exchange for H⁺, functions chiefly as an Mg²⁺ transporter under physiological conditions because cytosolic Mg²⁺ is several orders of magnitude higher than the Ca²⁺ concentration. This conclusion was deduced from experiments showing that Mg²⁺ ions in a concentration range of 0.01 to 1 mM triggered a fast efflux of H⁺ from acid-loaded vesicles. Furthermore, the proton-pumping activity of the tonoplast H⁺-ATPase and H⁺-PP_iase was found to be influenced by Ca²⁺ differently from and independently of the Mg²⁺ concentration. Calcium was a strong inhibitor for the H⁺-PP_iase (IC₅₀ = 18 μM, Hill coefficient nH = 1.7) but a weak one for the H⁺-ATPase (IC₅₀ = 330 μM, nH = 1). From these results it is suggested that at the tonoplast membrane a functional interaction exists between (i) the Ca²⁺-and Mg²⁺-regulated H⁺-PP_iase, (ii) the newly described high-affinity Ca²⁺-AT-Pase, (iii) the low-affinity Mg²⁺(Ca²⁺)/H⁺-antiporter and (iv) the H²⁺-ATPase.     

SEA-0400, a potent inhibitor of the Na+/Ca2+ exchanger, as a tool to study exchanger ionic and metabolic regulation

The effects of a new, potent, and selective inhibitor of the Na⁺/Ca²⁺ exchange, SEA-0400 (SEA), on steady-state outward (forward exchange), inward (reverse exchange), and Ca²⁺/Ca²⁺ transport exchange modes were studied in internally dialyzed squid giant axons from both the extra- and intracellular sides. Inhibition by SEA takes place preferentially from the intracellular side of the membrane. Its inhibition has the following characteristics: it increases synergic intracellular Na⁺ (Na_i⁺) + intracellular H⁺ (H_i⁺) inactivation, is antagonized by ATP and intracellular alkalinization, and is enhanced by intracellular acidification even in the absence of Na⁺. Inhibition by SEA is still present even after 1 h of its removal from the experimental solutions, whereas removal of the cointeracting agents of inhibition, Na_i⁺ and H_i⁺, even in the continuous presence of SEA, releases inhibition, indicating that SEA facilitates the reversible attachment of the natural H_i⁺ and Na_i⁺ synergic inhibitors. On the basis of a recent model of squid Na⁺/Ca²⁺ exchange regulation (DiPolo R and Beaugé L. J Physiol 539: 791–803, 2002), we suggest that SEA acts on the H_i⁺ + Na_i⁺ inactivation process and can interact with the Na⁺-free and Na⁺-bound protonized carrier. Protection by ATP concurs with the antagonism of the nucleotide by H_i⁺ + Na_i⁺ synergic inhibition. ionic-metabolic interactions     

Stimulation of the Extrusion of Protons and H+-ATPase Activities with the Decline in Pyrophosphatase Activity of the Tonoplast in Intact Mung Bean Roots under High-NaCl Stress and Its Relation to External Levels of Ca2+ Ions

Extrusion of protons as a response to high-NaCl stress in intactmung bean roots was investigated at different external concentrationsof Ca²⁺ ions ([Ca²⁺]_ex). The extrusion of protons was graduallyenhanced in the roots exposed to 100 mM NaCl, and high [Ca²⁺]_exdiminished this enhancement of the extrusion. Vesicles of plasmalemmaand tonoplast were prepared from the roots and the H⁺-translocatingATPase (H⁺-ATPase) activities associated with the two typesof membrane and the H⁺-pyrophosphatase (H⁺-PPase) activity ofthe tonoplast were assayed. The plasmalemma ATPase was stimulatedin parallel with dramatic increases in the intracellular concentrationof Na⁺([Na⁺]_in). High [Ca²⁺]ex prevented the increase in [Na⁺]inand diminished the stimulation of ATPase activity. The tonoplastATPase showed a rapid response to salt stress and was similarlystimulated even at high [Ca²⁺]M. The activities of both ATPaseswere, however, insensitive to concentrations of Na⁺ ions upto 100 HIM. By contrast, H⁺-PPase activity of the tonoplastwas severely inhibited with increasing [Na⁺]in under salt stressand recovered with high [Ca²⁺]ex. These findings suggest thathigh-NaCl stress increases the intracellular concentration ofNa⁺ ions in mung bean roots, which inhibits the tonoplast H⁺-PPase,and the activity of the plasmalemma H⁺-ATPase is thereby stimulatedand regulates the cytoplasmic pH. (Received March 26, 1991; Accepted December 13, 1991)     

Ion Effects on the H+-Translocating Adenosine Triphosphatase and Pyrophosphatase Associated with the Tonoplast of Chora corallina

H⁺-translocating ATPase and pyrophosphatase (PPase) associatedwith the tonoplast of Chara corallina were isolated with theaid of a perfusion technique, and the effects of ions on theiractivities were studied. All the alkali metal cations testedstimulated the ATPase and ATPdependent H⁺ pumping activitiesonly by 10 to 40%. Anions, on the other hand, strongly affectedthe activities. Potassium salts of Cl^- and Br^- stimulated them,while F^- and NO₃^- inhibited them. By contrast, the H⁺-translocatingPPase was insensitive to anions but sensitive to cations. Theorder of cation stimulation was Rb⁺=K⁺>Cs⁺>Na⁺=Li⁺>choline⁺.NO₃^- (50 mil), thought to be a specific inhibitor of the tonoplast-typeH⁺-ATPase, inhibited the ATPdependent H⁺ pumping almost completelybut the ATPase activity by only about 50%. Na⁺ inhibited thePP₁-dependent H⁺ pumping (I_5O=5OmM) in the presence of 50 mMKCl but not the ATP-dependent one. The PPase was more sensitiveto F^- (I₅₀=400µM) than the ATPase. Both the H⁺-ATPaseand the H⁺-PPase required Mg²⁺ for their activities, althoughan excess was inhibitory to both. The different sensitivitiesof the PP₁-dependent and the ATP-dependent H⁺- pumping enzymesto ions correspond to the tonoplast enzymes of higher plantsand may be used as "markers" to distinguish between these enzymesin characean cells (Received October 2, 1987; Accepted May 18, 1988)     

Intracellular pH homeostasis in cultured human placental syncytiotrophoblast cells: recovery from acidification

Resting or basal intracellular pH (pH_i) measured in cultured human syncytiotrophoblast cells was 7.26 ± 0.04 (without HCO₃^–) or 7.24 ± 0.03 (with HCO₃^–). Ion substitution and inhibitor experiments were performed to determine whether common H⁺-transporting species were operating to maintain basal pH_i. Removal of extracellular Na⁺ or Cl^– or addition of amiloride or dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H₂DIDS) had no effect. Acidification with the K⁺/H⁺ exchanger nigericin reduced pH_i to 6.25 ± 0.15 (without HCO₃^–) or 6.53 ± 0.10 (with HCO₃^–). In the presence of extracellular Na⁺, recovery to basal pH_i was prompt and occurred at similar rates in the absence and presence of HCO₃^–. Ion substitution and inhibition experiments were also used to identify the species mediating the return to basal pH_i after acidification. Recovery was inhibited by removal of Na⁺ or addition of amiloride, whereas removal of Cl^– and addition of H₂DIDS were ineffective. Addition of the Na⁺/H⁺ exchanger monensin to cells that had returned to basal pH_i elicited a further increase in pH_i to 7.48 ± 0.07. Analysis of recovery data showed that there was a progressive decrease in pH per minute as pH_i approached the basal level, despite the continued presence of a driving force for H⁺ extrusion. These data show that in cultured syncytial cells, in the absence of perturbation, basal pH_i is preserved despite the absence of active, mediated pH maintenance. They also demonstrate that an Na⁺/H⁺ antiporter acts to defend the cells against acidification and that it is the sole transporter necessary for recovery from an intracellular acid load. sodium/hydrogen antiporter; pH regulation; fluorescence; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein     

Shrinkage-induced activation of Na+/H+ exchange in rat renal mesangial cells

Using thepH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF),we examined the effect of hyperosmolar solutions, which presumablycaused cell shrinkage, on intracellular pH(pH_i) regulation in mesangialcells (single cells or populations) cultured from the rat kidney. Thecalibration of BCECF is identical in shrunken and unshrunken mesangialcells if the extracellular K⁺concentration ([K⁺])is adjusted to match the predicted intracellular[K⁺]. ForpH_i values between ~6.7 and~7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH₂O) is threefold larger than in unshrunken cells (~300mosmol/kgH₂O). In the nominalabsence ofCO₂/HCO₃,exposing cell populations to a HEPES-buffered solution supplementedwith ~300 mM mannitol (600 mosmol/kgH₂O) causes steady-statepH_i to increase by ~0.4. The pH_i increase is due to activationofNa⁺/H⁺exchange because, in single cells, it is blocked in the absence ofexternal Na⁺ or in the presence of50 µM ethylisopropylamiloride (EIPA). Preincubating cells in aCl-free solution for atleast 14 min inhibits the shrinkage-induced pH_i increase by 80%. Wecalculated the pH_i dependence oftheNa⁺/H⁺exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) thepH_i dependence of the totalacid-extrusion rate and 2) thepH_i dependence of theEIPA-insensitive acid-loading rate. Shrinkage alkali shifts thepH_i dependence ofNa⁺/H⁺exchange by ~0.7 pH units.     

Glucose activates H(+)-ATPase in kidney epithelial cells

The vacuolar H⁺-ATPase (V-ATPase) acidifies compartments of the vacuolar system of eukaryotic cells. In renal epithelial cells, it resides on the plasma membrane and is essential for bicarbonate transport and acid-base homeostasis. The factors that regulate the H⁺-ATPase remain largely unknown. The present study examines the effect of glucose on H⁺-ATPase activity in the pig kidney epithelial cell line LLC-PK₁. Cellular pH was measured by performing ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. Intracellular acidification was induced with NH₃/NH₄⁺ prepulse, and rates of intracellular pH (pH_i) recovery (after in situ calibration) were determined by the slopes of linear regression lines during the first 3 min of recovery. The solutions contained 1 µM ethylisopropylamiloride and were K⁺ free to eliminate Na⁺/H⁺ exchange and H⁺-K⁺-ATPase activity. After NH₃/NH₄⁺-induced acidification, LLC-PK₁ cells had a significant pH_i recovery rate that was inhibited entirely by 100 nM of the V-ATPase inhibitor concanamycin A. Acute removal of glucose from medium markedly reduced V-ATPase-dependent pH_i recovery activity. Readdition of glucose induced concentration-dependent reactivation of V-ATPase pH_i recovery activity within 2 min. Glucose replacement produced no significant change in cell ATP or ADP content. H⁺-ATPase activity was completely inhibited by the glycolytic inhibitor 2-deoxy-D-glucose (20 mM) but only partially inhibited by the mitochondrial electron transport inhibitor antimycin A (20 µM). The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (500 nM) abolished glucose activation of V-ATPase, and activity was restored after wortmannin removal. Glucose activates V-ATPase activity in kidney epithelial cells through the glycolytic pathway by a signaling pathway that requires PI3K activity. These findings represent an entirely new physiological effect of glucose, linking it to cellular proton secretion and vacuolar acidification. proton secretion; glycolysis; intracellular pH; concanamycin A     

Characterization of the tonoplast proton pumps in Cucumis sativus L. root cells

Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was obtained after centrifugation of microsome pellet (10,000 – 80,000 g) on discontinuous sucrose density gradient (20, 28, 32 and 42 %). Lack of PEP carboxylase (cytosol marker) and cytochrome c oxidase (mitochondrial marker) together with a slight activity of VO₄-ATPase (plasma membrane marker) and NADH-cytochrome c reductase (ER marker) in tonoplast preparation confirmed its high purity. Using latency of nitrate-inhibited ATPase and H⁺ pumping as criteria it was established that the majority of tonoplast vesicles were sealed and oriented right(cytoplasmic)-side-out. Strong acidification of the interior of vesicles observed at the presence of both, ATP and PP_i, confirmed that obtained tonoplast contains two classes of proton pumps: V-ATPase and H⁺PP_iase. To examine and characterise of proton-transport systems in tonoplast, the effect of various inhibitors on H⁺ pumping and hydrolytic activities of ATPase and PP_iase were measured. ATP-dependent activities (H⁺ flux and ATP hydrolysis) were specifically decreased by nitrate and bafilomycin A₁, whereas the PP_iase activities were reduced in the presence of fluoride and Na⁺ ions. Both enzymes showed a similar sensitivity to DCCD and DES. The results of experiments with KCl and NaCl suggested that the vacuolar ATPase was stimulated by Cl⁻, whereas the vacuolar Pp_iase requires K⁺ ions for its activity.     

Aluminum Stress Increases K+ Efflux and Activities of ATP- and PPi- Dependent H+ Pumps of Tonoplast-Enriched Membrane Vesicles from Barley Roots

Aluminum stress significantly stimulated K⁺ effux from barleyroots that had been preloaded with K⁺ The stress also increasedPP₁- and ATP-dependent H⁺ pump activities of tonoplast enrichedmembrane vesicles from the roots. Ca²⁺ ions reduced increasesin K⁺ effux and both H⁺ pump activities under these conditions. (Received April 25, 1992; Accepted August 4, 1992)     

Differential stimulation by Cl- of H+ transport and ATPase activity in purified plasma membrane vesicles from maize (Zea mays L.) roots

Maize (Zea mays L.) root plasma membranes purified by the aqueouspolymer two-phase technique have previously been shown to bevery low in tonoplast H⁺ -ATPase and H⁺ -PPase activities. Westernblots of a similar preparation showed that, compared to a microsomalfraction, there was practically no reaction with antibodiesto the tonoplast enzymes, but a strong reaction with an antibodyto the plasma membrane H⁺ -ATPase. Freeze/thaw treatment ofthe plasma membrane vesicles increased the proportion with aninsideout orientation to about 40%. This preparation was usedto demonstrate that substitution of KCl for K₂S0₄ resulted ina 14-fold stimulation of H⁺ transport, but an increase in ATPaseactivity of less than 10%. In contrast to its effect on tonoplastvesicles, Cl^– had only a small effect on the membranepotential of plasma membrane vesicles, assayed by oxonol V fluorescencequench recovery. To account for the apparent variability inthe H⁺/ATP coupling ratio, it may be necessary to devise a modelthat takes into consideration the possibility of non-linearbehaviour with respect to the membrane potential of the protonleak and/or of slip in the ATPase. Key words: ATPase, plasma membrane, anion stimulation, proton transport     

Age-related differences in Na+-dependent Ca2+ accumulation in rabbit hearts exposed to hypoxia and acidification

In this study, we test the hypothesisthat in newborn hearts (as in adults) hypoxia and acidificationstimulate increased Na⁺ uptake, in part via pH-regulatoryNa⁺/H⁺ exchange. Resulting increases inintracellular Na⁺ (Na_i) alter the force drivingthe Na⁺/Ca²⁺ exchanger and lead to increasedintracellular Ca²⁺. NMR spectroscopy measuredNa_i and cytosolic Ca²⁺ concentration([Ca²⁺]_i) and pH (pH_i) inisolated, Langendorff-perfused 4- to 7-day-old rabbit hearts. AfterNa⁺/K⁺ ATPase inhibition, hypoxic hearts gainedNa⁺, whereas normoxic controls did not [19 ± 3.4 to139 ± 14.6 vs. 22 ± 1.9 to 22 ± 2.5 (SE) meq/kg drywt, respectively]. In normoxic hearts acidified using theNH₄Cl prepulse, pH_i fell rapidly and recovered,whereas Na_i rose from 31 ± 18.2 to 117.7 ± 20.5 meq/kg dry wt. Both protocols caused increases in [Ca]_i;however, [Ca]_i increased less in newborn hearts than inadults (P < 0.05). Increases in Na_i and[Ca]_i were inhibited by theNa⁺/H⁺ exchange inhibitormethylisobutylamiloride (MIA, 40 µM; P < 0.05), aswell as by increasing perfusate osmolarity (+30 mosM) immediately before and during hypoxia (P < 0.05). The data supportthe hypothesis that in newborn hearts, like adults, increases inNa_i and [Ca]_i during hypoxia and afternormoxic acidification are in large part the result of increased uptakevia Na⁺/H⁺ and Na⁺/Ca²⁺exchange, respectively. However, for similar hypoxia and acidification protocols, this increase in [Ca]_i is less in newborn thanadult hearts.

     

Functional upregulation of H+-ATPase by lethal acid stress in cultured inner medullary collecting duct cells

The response ofH⁺-ATPase to lethal acid stress isunknown. A mutant strain (called NHE2d) was derived from cultured inner medullary collecting duct cells (mIMCD-3 cells) following three cyclesof lethal acid stress. Cells were grown to confluence on coverslips,loaded with2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, andmonitored for intracellular pH(pH_i) recovery from an acid load. The rate of Na⁺-independentpH_i recovery from an acid load inmutant cells was approximately fourfold higher than in parent cells(P < 0.001). TheNa⁺-independentH⁺ extrusion was ATP dependent and K⁺ independent and wascompletely inhibited in the presence of diethylstilbestrol, N, N'-dicyclohexylcarbodiimide,or N-ethylmaleimide. Theseresults indicate that theNa⁺-independentH⁺ extrusion in cultured medullarycells is mediated via H⁺-ATPaseand is upregulated in lethal acidosis. Northern hybridization experiments demonstrated that mRNA levels for the 16- and 31-kDa subunits of H⁺-ATPase remainedunchanged in mutant cells compared with parent cells. We propose thatlethal acid stress results in increased H⁺-ATPase activity in innermedullary collecting duct cells. Upregulation ofH⁺-ATPase could play a protectiverole against cell death in severe intracellular acidosis.

     

Light-Induced H+ Accumulation in the Vacuole of Nitellopsis obtusa

The effects of light on the pH in the vacuole and the electricpotential difference across the plasmalemma and the tonoplastof Nitellopsis obtusa were investigated by means of conventionaland H⁺-specific glass or antimony microelectrodes. Illuminationis found to bring about a decrease in the pH of the vacuolarsap by 0.1–0.5 units concomitant with a depolarizationof the cell. The light-induced changes of the potential differenceand the vacuolar pH depend in different ways on the pH of theexternal medium (pH_o). At pH_o 9.0 cells exhibit great light-inducedpotential changes (up to 100 mV), but only small pH changesof the vacuolar sap. At neutral or slightly acidic pH_o valuesthe amplitude of the light-induced pH changes in the vacuoleincreases up to 0.3–0.5 pH units, but the amplitudes ofthe potential changes at the plasmalemma are relatively small.At pH_o 9.0 a transient acidification of the medium is observedupon illumination whereas at lower pH values light-induced alkalinizationwas only seen. Transfer of the cells from pH_o 9.0 to pH_o 7.5results in a cell hyperpolarization by 60–80 mV and adecrease of the vacuolar pH by 0.4–0.5 units under lightconditions but has no significant effect on the potential andthe vacuolar pH in the darkness. It is proposed that mechanismsof active H⁺ extrusion from the cytoplasm are located both inthe plasmalemma and the tonoplast. The observed acidificationin the vacuole appears to be determined by a light-induced increaseof the concentration of H⁺ in the cytoplasm. The H⁺ conductionof the plasmalemma seems to increase on illumination. The patternof the light-induced H⁺ fluxes across the tonoplast and theplasmalemma depends crucially on the extent of the light-inducedchanges in the H⁺ conductance and on the electrochemical gradientfor H⁺ at the plasmalemma.     

Homeostasis and Transport of Inorganic Phosphate in Plants

In this review, homeostasis of inorganic phosphate (P_i) in plantsis discussed in terms of membrane transport of P_i. Phosphatehomeostasis is observed in plant systems at various levels.The cytoplasmic level of P_i is kept almost constant by exploitationof the vacuole as a reservoir of P_i. The vacuole also seemsto maintain the apoplastic level of P_i at a quasi-constant level.During P_i deficiency, P_i is re-translocated from the older tothe younger leaves. The concentration of P_i in young leaves,thus, is kept at a higher level without a supply of P_i fromthe root. The phenomenon can be referred to as leaf-level P_ihomeostasis. All these phenomena are related to membrane P_itransport activities. P_i uptake activities of both the plasmamembrane and the tonoplast change in response to the supplyof P_i. P_i transport across the plasma membrane is controlledby the activities of both the P_i transporter and the H⁺ pump,the activity of which is modulated by P_i itself. ¹Recipient of the JSPP Young Investigator Award, 1994.     

Differences in regulation of pH(i) in large (>/=10 nuclei) and small (</=5 nuclei) osteoclasts

Osteoclasts aremultinucleated cells that resorb bone by extrusion of protons andproteolytic enzymes. They display marked heterogeneity in cell size,shape, and resorptive activity. Because high resorptive activity invivo is associated with an increase in the average size of osteoclastsin areas of greater resorption and because of the importance of protonextrusion in resorption, we investigated whether the activity of thebafilomycin A₁-sensitive vacuolar-typeH⁺-ATPase (V-ATPase) and amiloride-sensitiveNa⁺/H⁺ exchanger differed between large andsmall osteoclasts. Osteoclasts were obtained from newborn rabbit bones,cultured on glass coverslips, and loaded with the pH-sensitiveindicator2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF).Intracellular pH (pH_i) was recorded in single osteoclasts by monitoring fluorescence. Large (10 nuclei) and small (5 nuclei) osteoclasts differed in that large osteoclasts had a higher basal pH_i, their pH_i was decreased by bafilomycinA₁ addition or removal of extracellular Na⁺,and the realkalinization upon readdition of Na⁺ wasbafilomycin A₁ sensitive. After acid loading, asubpopulation of large osteoclasts (40%) recovered by V-ATPaseactivity alone, whereas all small osteoclasts recovered byNa⁺/H⁺ exchanger activity. Interestingly, in60% of the large osteoclasts, pH_i recovery was mediated byboth the Na⁺/H⁺ exchanger and V-ATPaseactivity. Our results show a striking difference betweenpH_i regulatory mechanisms of large and small osteoclaststhat we hypothesize may be associated with differences in the potentialresorptive activity of these cells.