Characterization of inorganic phosphate transport in osteoclast-like cells

Osteoclasts possess inorganic phosphate (P_i) transport systems to take up external P_i during bone resorption. In the present study, we characterized P_i transport in mouse osteoclast-like cells that were obtained by differentiation of macrophage RAW264.7 cells with receptor activator of NF-B ligand (RANKL). In undifferentiated RAW264.7 cells, P_i transport into the cells was Na⁺ dependent, but after treatment with RANKL, Na⁺-independent P_i transport was significantly increased. In addition, compared with neutral pH, the activity of the Na⁺-independent P_i transport system in the osteoclast-like cells was markedly enhanced at pH 5.5. The Na⁺-independent system consisted of two components with K_m of 0.35 mM and 7.5 mM. The inhibitors of P_i transport, phosphonoformic acid, and arsenate substantially decreased P_i transport. The proton ionophores nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone as well as a K⁺ ionophore, valinomycin, significantly suppressed P_i transport activity. Analysis of BCECF fluorescence indicated that P_i transport in osteoclast-like cells is coupled to a proton transport system. In addition, elevation of extracellular K⁺ ion stimulated P_i transport, suggesting that membrane voltage is involved in the regulation of P_i transport activity. Finally, bone particles significantly increased Na⁺-independent P_i transport activity in osteoclast-like cells. Thus, osteoclast-like cells have a P_i transport system with characteristics that are different from those of other Na⁺-dependent P_i transporters. We conclude that stimulation of P_i transport at acidic pH is necessary for bone resorption or for production of the large amounts of energy necessary for acidification of the extracellular environment. Na⁺-dependent phosphate cotransporter; RAW264.7; phosphate uptake     

Deciphering PiT transport kinetics and substrate specificity using electrophysiology and flux measurements

Members of the SLC20 family or type III Na⁺-coupled P_i cotransporters (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform a housekeeping function for intracellular P_i homeostasis. Previous studies have shown that PiT-1 and PiT-2 mediate electrogenic P_i cotransport when expressed in Xenopus oocytes, but only limited kinetic characterizations were made. To address this shortcoming, we performed a detailed analysis of SLC20 transport function. Three SLC20 clones (Xenopus PiT-1, human PiT-1, and human PiT-2) were expressed in Xenopus oocytes. Each clone gave robust Na⁺-dependent ³²P_i uptake, but only Xenopus PiT-1 showed sufficient activity for complete kinetic characterization by using two-electrode voltage clamp and radionuclide uptake. Transport activity was also documented with Li⁺ substituted for Na⁺. The dependence of the P_i-induced current on P_i concentration was Michaelian, and the dependence on Na⁺ concentration indicated weak cooperativity. The dependence on external pH was unique: the apparent P_i affinity constant showed a minimum in the pH range 6.2–6.8 of 0.05 mM and increased to 0.2 mM at pH 5.0 and pH 8.0. Xenopus PiT-1 stoichiometry was determined by dual ²²Na-³²P_i uptake and suggested a 2:1 Na⁺:P_i stoichiometry. A correlation of ³²P_i uptake and net charge movement indicated one charge translocation per P_i. Changes in oocyte surface pH were consistent with transport of monovalent P_i. On the basis of the kinetics of substrate interdependence, we propose an ordered binding scheme of Na⁺:H₂PO₄^–:Na⁺. Significantly, in contrast to type II Na⁺-P_i cotransporters, the transport inhibitor phosphonoformic acid did not inhibit PiT-1 or PiT-2 activity. Na⁺-P_i cotransport; two-electrode voltage clamp; surface pH electrode; SLC20; retroviral receptor     

Participation of Inorganic Orthophosphate in Regulation of the Ribulose-1,5-bisphosphate Carboxylase Activity in Response to Changes in the Photosynthetic Source-Sink Balance

Sink-limited conditions, defined as treatment with continuousillumination, cause a reduction in the rate of photosyntheticfixation of CO₂ in single-rooted leaves of soybean (Glycinemax. Merr. cv. Turunoko). We suggested previously that thisreduction is due to a deactivation of ribulose-1,5-bisphosphatecarboxylase (RuBPcase, E.C. 4.1.1.39 [EC] ) that is caused by a decreasein the level of P_i in the leaves [Sawada et al. (1989) PlantCell Physiol. 30: 691, Sawada et al. (1990) Plant Cell Physiol.31: 697]. In the present study, the mechanism of regulationof RuBPcase activity by P_i was examined. The activity of RuBPcasein the sink-limited leaves, exposed for 6 or 7 d to continuousillumination to alter the source/sink balance, was enhancedwith increasing concentrations of P_i, in a CO₂-free preincubationmedium in the presence of 5 mM MgCl₂ The maximum value [6.3µmole CO₂ (mg Chl)^–1 min^–1] was obtained atapproximately 5 mM P_i after a 5 min incubation, being 3 timesof the activity without the preincubation. The activity of acrude preparation of RuBPcase that had been deactivated by removalof CO₂ and Mg²⁺ ions by the gel filtration was 5.2–9.3nmole CO₂ (mg protein)^–1 min^–1 and was also enhancedby P_i plus Mg²⁺ ions. The maximum value [147–151 nmoleCO₂ (mg protein)^–1 min^–1] was attained at 5 mM P_iafter a 5 min incubation. The cycle of activation and inactivationof deactivated crude RuBPcase was perfectly reversible by additionof P_i to the enzyme and removal of P_i from the enzyme. Levelsof free P_i and of esterified phosphate in the sink-limited leaveswere 69% and 31% of the total phosphate, respectively. By contrast,in the control leaves, these values were 87% and 13%, respectively.These results support our previously stated hypothesis and indicatean important role for free P_i in the regulation of RuBPcaseactivity, in particular in sink-limited plants. (Received February 21, 1992; Accepted July 23, 1992)     

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     

Characterization of transport mechanisms and determinants critical for Na+-dependent Pi symport of the PiT family paralogs human PiT1 and PiT2

The general phosphate need in mammalian cells is accommodated by members of the P_i transport (PiT) family (SLC20), which use either Na⁺ or H⁺ to mediate inorganic phosphate (P_i) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na⁺-dependent P_i (NaP_i) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with ³²P_i as a traceable P_i source. For PiT1, the Michaelis-Menten constant for P_i was determined as 322.5 ± 124.5 µM. PiT2 was analyzed for the first time and showed positive cooperativity in P_i uptake with a half-maximal activity constant for P_i of 163.5 ± 39.8 µM. PiT1- and PiT2-mediated Na⁺-dependent P_i uptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na⁺ dependency patterns. However, only PiT2 was capable of Na⁺-independent P_i transport at acidic pH. Study of the impact of divalent cations Ca²⁺ and Mg²⁺ revealed that Ca²⁺ was important, but not critical, for NaP_i transport function of PiT proteins. To gain insight into the NaP_i cotransport function, we analyzed PiT2 and a PiT2 P_i transport knockout mutant using ²²Na⁺ as a traceable Na⁺ source. Na⁺ was transported by PiT2 even without P_i in the uptake medium and also when P_i transport function was knocked out. This is the first time decoupling of P_i from Na⁺ transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E⁵⁵ and E⁵⁷⁵ are responsible for linking P_i import to Na⁺ transport in PiT2. inorganic phosphate transport; retroviral receptor; SLC20     

Regulation of Ribulose-l,5-bisphosphate Carboxylase Activity in Response to Changes in the Source/Sink Balance in Single-Rooted Soybean Leaves: The Role of Inorganic Orthophosphate in Activation of the Enzyme

The results of our previous study [Sawada et al. (1989) PlantCell Physiol. 30: 691] implied that, under sink-limited conditions,a decrease in the activity of ribulose-l,5-bisphosphate carboxylase(EC 4.1.1.39 [EC] ) caused a reduction in the rate of photosyntheticfixation of CO₂ in single-rooted leaves of soybean (Glycinemax L. Merr. cv. Tsurunoko). This reduction in the rate of photosynthesisin source leaves seemed to correspond to a decrease in the demandby sink tissues for photoassimilates. In the present study,the activity of RuBPcase in vivo was estimated by measuringthe "initial" activity immediately after extraction from standardleaves (grown under a regime of 10 h of light and 14 h of darkness)and from sink-limited leaves (exposed for 6 or 7 d to continuouslight to alter the source/sink balance). The rate of photosynthesisin the sink-limited leaves decreased to 47% of that in the standardleaves. The "initial" activity of RuBPcase was 4.3 in the standardleaves but only 1.6 µmole CO₂.(mg Chl)^–1.min^–1in the sink-limited leaves. These results appear to indicatethat the reduction in photosynthetic activity under sink-limitedconditions was mostly due to a deactivation of RuBPcase. Theactivity of deactivated RuBPcase in the sink-limited leaveswas restored to 4.1 µmole CO₂.(mg Chl)^–1.min^–1by incubation of the enzyme in a medium that contained onlyNa₂HPO₄. This result suggests that free P_i in chloro-plastsplays an important role in the activation of the enzyme. Thelevel of P_i in the sink-limited leaves was 62% of that in thestandard leaves. On the basis of these various results, it appearsthat the deactivation of RuBPcase in the sink-limited leavesis the result of a decrease in the level of P_i. The role offree P_i in the activation of RuBPcase, in particular at atmosphericconcentrations of CO₂, was also investigated. (Received November 30, 1989; Accepted May 11, 1990)     

Phosphorus-31 NMR Studies of Wild-Type and NaCl-Tolerant Citrus Cultured Cells

Ben–Hayyim, G. and Navon, G. 1985. Phosphorus–31NMR studies of wild–type and NaCl–tolerant Citruscultured cells.—J. exp. Bot. 36: 1877–1888. Theinternal pH of the cytoplasm and vacuole and the relative distributionof internal P_i concentrations between those two cell compartmentshave been determined by ³¹P nuclear magnetic resonance spectroscopyin wild–type and NaCl–tolerant cell lines of Shamoutiorange (Citrus sinensis L. Osbeck). Wild–type cells accumulatehigher amounts of P_i than the NaCl–tolerant cells whenexposed to equal external P_i concentrations. This additionalP_i is located mainly in the vacuole. When both types of cellsare exposed to increasing external P_i concentrations, the internalP_i concentrations increase. The cytoplasmic P_i concentrationreaches saturation at a rather low external P_i concentrationwhile the vacuolar P_i can be increased by a large factor. Transferof cells from aerobic to anaerobic conditions causes an immediateincrease of P_i in the cytoplasm and a slow acidification. Exposureof cells to NaCl during the period of their growth results inan increase in total P_i concentration with a large increasein the ratio of vacuolar to cytoplasmic P_i levels. When thesecells are exposed to NaCl for a short time, total internal P_iconcentration docs not change significantly but its proportionschange in favour of the vacuole. pH values of the cytoplasmand the vacuole under all these conditions are rather constant,the value being 5.8–6.0 for the vacuole and 7.4–7.6for the cytoplasm. Moreover, subjecting these cells to a widerange of external pH values does not change their intracellularpH. These results indicate that a strong regulation of internalpH is operating in both types of cells. The presence of a phosphorylatedmetabolite with an unusual pH titration curve, located in thevacuole, is also reported. Key words: Citrus, callus, ³¹P-NMR, NaCI tolerance, intracellular pH     

Phosphate Transport and Apoplastic Phosphate Homeostasis in Barley Leaves

Levels of apoplastic inorganic phosphate (P_i) in leaves andP_i-transport activities of mesophyll cells were measured insitu in control and P_i-deficient plants. When detached leaveswere fed a solution that contained 10 mM P_i, the apoplasticP_i levels, as measured by an infiltration method, remained almostconstant. When the leaves were immersed in pure water, the apoplasticP_i level gradually decreased. With 50 mM P_i in the feeding solution,the level increased dramatically. The apoplastic P_i levels inP_i-deficient leaves were somewhat, but not very much lower thanthose in controls. When the immersion medium was changed topure water 60 mm after feeding with 10 mM P_i, the apoplasticP_i levels started to decrease and then returned to the initiallevel. It is suggested that intracellular P_i may be transportedback to the apoplast to maintain the apoplastic P_i levels ata constant value. Changes in cytoplasmic pH were measured during feeding of P_ito the leaves by use of the pH-sensitive fluorescent dye, pyranineafter Yin et al. (l990a, b). On feeding of P_i the cytoplasmicpH decreased in P_i-deficient plants as a result of co-transportof P_i and protons in situ. After removal of P_i from the immersionmedium, the cytoplasmic pH returned to the original value. ³ Present address: Institute für Biochemische Pflanzenpathologie,GSF-München, D-8042 Neuherberg, Germany.     

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)     

Effects of Some Metabolic Phosphorus Compounds on Rates of Photosynthesis of detached Phosphorus-deficient Subterranean Clover Leaves

Rates of photosynthesis (net CO₂ uptake in saturating light)of leaves sampled from phosphorusdeficient subterranean cloverplants (Trifolium subterraneum L. cv. Mt. Barker) were lowerthan those of non-deficient leaves. When comparable deficientleaves were placed in solutions containing 0.13 mM P_i¹, therewere no responses in photosynthesis, even though earlier resultshad established these solutions as optimal for responses forintact deficient plants. Deficient leaves, placed for the first12 h after detachment in solutions of increasing P_i¹ concentrations(0.15, 0.70, 2.0, and 6.0 mM) and then in distilled water, showedmarked increases in photosynthesis in the three higher phosphatetreatments on the first day after detachment. During the following6 d the decline in photosynthesis was less the higher the initialphosphate treatment. By contrast, non-deficient leaves in thesame treatments showed a decline in photosynthesis with increasingphosphate levels, due to leaf damage in the two highest treatments(phosphorus toxicity). Rates of photosynthesis of deficient leaves kept for 3 h in3 or 6 mM FDP¹ or G-6-P¹ increased within 24 h and remainedhigher than those for corresponding leaves in 0.13 mM P_i ordistilled water. There were no differences between the sametreatments for non-deficient leaves, thus enabling a clear distinctionbetween leaves that were deficient and those that were not.There was no leaf damage in these solutions, even after 48 h.AMP¹ or ADP¹ had no effect. ATP¹ and 3-PGA¹ caused toxicitysymptoms. Fructose itself (6 mM) had no effect on photosynthesis.     

Pyridoxal 5-phosphate, Phenyl Phosphate and Acetyl Phosphate, as Inhibitors of Photophosphorylation Competitive with Phosphate

Pyridoxal 5-phosphate, phenyl phosphate and acetyl phosphate,as well as rß-naphthyl monophosphate, inhibited photophosphorylationof spinach chloroplasts competitively with P_i and noncompetitivelywith ADP. The apparent dissociation constant of the inhibitor-enzymecomplex (K_i) values of pyridoxal 5-phosphate, phenyl phosphateand acetyl phosphate for the P_i site were 1.1, 3.8 and 2.4 _mM,respectively. These organic phosphates inhibited Ca²⁺-ATPaseof the isolated coupling factor 1 (CF₁) (EC 3.6.1.3 [EC] ) noncompetitivelywith ATP. AMP, creatine phosphate, fructose 1,6-bisphosphate,glucose 6-phosphate, 3-phosphoglyceric acid, ribose 5-phosphateand PP_i did not significantly inhibit photophosphorylation.Like rß-naphthyl monophosphate, pyridoxal 5-phosphateand phenyl phosphate inhibited photophosphorylation and thecoupled electron transport, but were almost without effect onthe basal electron transport. On the other hand, acetyl phosphateconsiderably inhibited photophosphorylation, but had almostno effect on the coupled electron transport rate and the basalrate. The results suggest that these organic phosphates inhibitphotophosphorylation by binding at the P_i site on the activecenter of CF₁ and that their binding inhibits the ATPase activityof isolated CF₁. These four organic phosphates which inhibited photophosphorylationcompetitively with Pi could not substitute for ADP or ATP ininhibiting ferricyanide photoreduction by decreasing H⁺-permeabilitythrough CF₁ and in protecting the ATPase of isolated CF₁ againstcold-anion inactivation. ¹ This work was supported in part by Grants-in-Aid for ScientificResearch from the Ministry of Education, Science and Culture,Japan to H.S. (Received May 25, 1981; Accepted September 28, 1981)     

In squid nerves intracellular Mg(2+) promotes deactivation of the ATP-upregulated Na(+)/Ca(2+) exchanger

We investigated the role of intracellular Mg²⁺(Mg_i²⁺) on the ATP regulation ofNa⁺/Ca²⁺ exchanger in squid axons and bovineheart. In squid axons and nerve vesicles, the ATP-upregulated exchangerremains activated after removal of cytoplasmic Mg²⁺, evenin the absence of ATP. Rapid and complete deactivation of theATP-stimulated exchange occurs upon readmission ofMg_i²⁺. At constant ATP concentration, the effectof intracellular Mg²⁺ concentration([Mg²⁺]_i) on the ATP regulation of exchangeris biphasic: activation at low [Mg²⁺]_i,followed by deactivation as [Mg²⁺]_i isincreased. No correlation was found between the above results and thelevels of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] measured innerve membrane vesicles. Incorporation ofPtdIns(4,5)P₂ into membrane vesicles activates Na⁺/Ca²⁺ exchange in mammalian heart but not insquid nerve. Moreover, an exogenous phosphatase prevents MgATPactivation in squid nerves but not in mammalian heart. It is concludedthat 1) Mg_i²⁺ is an essentialcofactor for the deactivation part of ATP regulation of the exchangerand 2) the metabolic pathway of ATP upregulation of theNa⁺/Ca²⁺ exchanger is different in mammalianheart and squid nerves.

     

Kinetic Characterization of Two Phosphate Uptake Systems with Different Affinities in Suspension-Cultured Catharanthus roseus Protoplasts

We studied the kinetics of inorganic phosphate (P₁) uptake from0.1–1,000 µM P₁ by protoplasts from suspension-culturedcells of Catharanthus roseus (L.) G. Don. Concentration dependenceof [³²P]P₁ uptake revealed two kinetically different uptakesystems, a high-affinity system and a low-affinity system, withK_m values of 3.0 and 47 µM, respectively. Protoplastsfrom cells grown in P_i-rich media had a medium level of thelow-affinity activity and a very low level of the high-affinityactivity. It appeared low-affinity system is expressed constitutively,while the high-affinity system is regulated by the availabilityof P_i. When cells grown in a P_i-rich media were transferredto P_i-depleted media, the high-affinity activity increased significantlyafter 2 d, but the low-affinity activity was barely changed.Upon addition of 10 mM P_i, the high level of the high-affinityactivity fell to almost undetectable level in 1d. Both uptakesystems exhibited maximum activity between pH 5 and 6. ¹ Present address: Tokyo Research Laboratories, Kyowa HakkoKogyo Co., Ltd., 3-6-6 Asahi-cho, Machida, Tokyo, 194 Japan.     

Hormonal regulation of ENaCs: insulin and aldosterone

Although a variety of hormones and other agents modulate renalNa⁺ transport acting by way of theepithelial Na⁺ channel (ENaC), themode(s), pathways, and their interrelationships in regulation of thechannel remain largely unknown. It is likely that several hormones maybe present concurrently in vivo, and it is, therefore, important tounderstand potential interactions among the various regulatory factorsas they interact with the Na⁺transport pathway to effect modulation ofNa⁺ reabsorption in distal tubulesand other native tissues. This study represents specifically adetermination of the interaction between two hormones, namely,aldosterone and insulin, which stimulate Na⁺ transport by entirelydifferent mechanisms. We have used a noninvasive pulse protocol ofblocker-induced noise analysis to determine changes in single-channelcurrent (i_Na),channel open probability (P_o), andfunctional channel density(N_T) ofamiloride-sensitive ENaCs at various time points following treatmentwith insulin for 3 h of unstimulated control and aldosterone-pretreatedA6 epithelia. Independent of threefold differences of baseline values of transport caused by aldosterone, 20 nM insulin increased by threefold and within 10-30 min the density of the pool of apical membrane ENaCs(N_T) involvedin transport. The very early (10 min) increases of channel density wereaccompanied by relatively small decreases ofi_Na(10-20%) and decreases ofP_o (28%) in the aldosterone-pretreated tissues but not the control unstimulated tissues. The early changes ofi_Na,P_o, andN_T weretransient, returning very slowly over 3 h toward their respectivecontrol values at the time of addition of insulin. We conclude thataldosterone and insulin act independently to stimulate apicalNa⁺ entry into the cells of A6epithelia by increase of channel density.

     

Increase in cytosolic Ca2+ induced by elevation of extracellular Ca2+ in skeletal myogenic cells

Cytoplasmic Ca²⁺concentration ([Ca²⁺]_i) variation is akey event in myoblast differentiation, but the mechanism by which itoccurs is still debated. Here we show that increases of extracellular Ca²⁺ concentration ([Ca²⁺]_o)produced membrane hyperpolarization and a concentration-dependent increase of [Ca²⁺]_i due to Ca²⁺influx across the plasma membrane. Responses were not related toinositol phosphate turnover and Ca²⁺-sensing receptor.[Ca²⁺]_o-induced[Ca²⁺]_i increase was inhibited byCa²⁺ channel inhibitors and appeared to be modulated byseveral kinase activities. [Ca²⁺]_i increasewas potentiated by depletion of intracellular Ca²⁺ storesand depressed by inactivation of the Na⁺/Ca²⁺exchanger. The response to arginine vasopressin (AVP), which inducesinositol 1,4,5-trisphosphate-dependent[Ca²⁺]_i increase in L6-C5 cells, was notmodified by high [Ca²⁺]_o. On the contrary,AVP potentiated the [Ca²⁺]_i increase in thepresence of elevated [Ca²⁺]_o. Other clones ofthe L6 line as well as the rhabdomyosarcoma RD cell line and thesatellite cell-derived C2-C12 line expressed similar responses to high[Ca²⁺]_o, and the amplitude of the responseswas correlated with the myogenic potential of the cells.

     

Furosemide: a Specific Inhibitor of Pi Transport across the Plasma Membrane of Plant Cells

A search was made for inhibitors of P_i uptake that act directlyon the P_i transporter in the plasma membranes of Catharanthusroseus cells to inhibit P_i uptake without inhibition of protonpumping. Using standard electrodes, we monitored changes inpH and in the concentration of K⁺ ions, as well as the rateof P_i uptake, when an inhibitor to be tested was applied tothe cells in unbuffered medium. A9C (28 µM), a blockerof anion channels, inhibited P_i uptake but it also inhibitedthe proton pump. However, a structurally similar inhibitor,furosemide, inhibited P_i uptake without inhibiting proton pumping. It is suggested that the carboxylic group of these inhibitorsinteracts with the P_i-binding site (probably an amino group)of the P_i transporter in the plasma membrane and that the hydrophobicstructure of these inhibitors facilitates their accumulationin the plasma membrane. ³Present address: Department of Biology, Hitotsubashi University,2-1 Naka, Kunitachi, Tokyo, 186 Japan     

Long-Term Phosphate Starvation and Respiratory Metabolism in Suspension-Cultured Catharanthus roseus Cells

In order to clarify the metabolic adaptation of respiratorypathways in plants to limited levels of P_i, the effects of long-termstarvation of P_i on the activities of various enzymes relatedto respiratory metabolism were examined in suspension-culturedCatharanthus roseus cells. When the activities were expressedas units per g fresh weight, only those of phosphoenolpyruvate-hydrolyzing(PEP-hydrolyzing) enzyme (which may possibly be equivalent tothe acid phosphatase activity derived from vacuoles) and PEPcarboxylase were higher in the Pⁱ-starved cells than in controlcells. Activities of other enzymes in the Pⁱ-starved cells werelower than or similar to those of the control cells. Time-coursestudies indicated that PEP-hydrolyzing activity was inducibleby starvation of Pⁱ. However, in contrast to the results reportedby Duff et al. [(1989a) Plant Physiol. 90: 1275.], fluctuationsin the activity of PP¹:fructose-6-phosphate 1-phosphotransferaseduring starvation of Pⁱ were similar to those in levels of phosphofructokinaseand 6-phosphogluconate dehydrogenase. These data suggest thatthe concept of the phosphate starvation-inducible ‘bypasses’,which are engineered via the coarse control (i.e., induction)of specified enzymes and were proposed initially by Duff etal. in Brassica nigra cells, is not directly applicable to Catharanthusroseus cells in suspension. Tracer experiments using [U-¹⁴C]glutamineindicated that a significant proportion of respiratory substratescould be supplied from the enlarged pool of amino acids duringstarvation of Pⁱ. These assumptions are supported by the observedfluctuations in levels of free amino acids and of protein inP¹-fed and P¹-deficient Catharanthus roseus cells. ¹Part 41 in the series ‘Metabolic Regulation in PlantCell Cultrue’ ²Present Address: Morinaga Mild Industry, 5-1-83, Higashihara,Zamma-shi, Kanagawa, 228 Japan     

Unique uptake and efflux systems of inorganic phosphate in osteoclast-like cells

During bone resorption, a large amount of inorganic phosphate (P_i) is generated within the osteoclast hemivacuole. The mechanisms involved in the disposal of this P_i are not clear. In the present study, we investigated the efflux of P_i from osteoclast-like cells. P_i efflux was activated by acidic conditions in osteoclast-like cells derived by the treatment of RAW264.7 cells with receptor activator of nuclear factor-B ligand. Acid-induced P_i influx was not observed in renal proximal tubule-like opossum kidney cells, osteoblast-like MC3T3-E1 cells, or untreated RAW264.7 cells. Furthermore, P_i efflux was stimulated by extracellular P_i and several P_i analogs [phosphonoformic acid (PFA), phosphonoacetic acid, arsenate, and pyrophosphate]. P_i efflux was time dependent, with 50% released into the medium after 10 min. The efflux of P_i was increased by various inhibitors that block P_i uptake, and extracellular P_i did not affect the transport of [¹⁴C]PFA into the osteoclast-like cells. Preloading of cells with P_i did not stimulate P_i efflux by PFA, indicating that the effect of P_i was not due to transstimulation of P_i transport. P_i uptake was also enhanced under acidic conditions. Agents that prevent increases in cytosolic free Ca²⁺ concentration, including acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 2-aminoethoxydiphenyl borate, and bongkrekic acid, significantly inhibited P_i uptake in the osteoclast-like cells, suggesting that P_i uptake is regulated by Ca²⁺ signaling in the endoplasmic reticulum and mitochondria of osteoclast-like cells. These results suggest that osteoclast-like cells have a unique P_i uptake/efflux system and can prevent P_i accumulation within osteoclast hemivacuoles. phosphate transporter; RAW264.7; proton dependent; acidification     

Sodium gradient-dependent transport of magnesium in rat ventricular myocytes

Cytoplasmic concentration of Mg²⁺([Mg²⁺]_i) was measured with a fluorescentindicator furaptra in ventricular myocytes enzymatically dissociatedfrom rat hearts (25°C). To study Mg²⁺ transport acrossthe cell membrane, cells were treated with ionomycin inCa²⁺-free (0.1 mM EGTA) and high-Mg²⁺ (10 mM)conditions to facilitate passive Mg²⁺ influx. Rate of riseof [Mg²⁺]_i due to the net Mg²⁺influx was significantly smaller in the presence of 130 mMextracellular Na⁺ than in its absence. We also tested theextracellular Na⁺ dependence of the net Mg²⁺efflux from cells loaded with Mg²⁺. After[Mg²⁺]_i was raised by ionomycin and highMg²⁺ to the level 0.5-0.6 mM above the basal value(~0.7 mM), washout of ionomycin and lowering extracellular[Mg²⁺] to 1.2 mM caused rapid decline of[Mg²⁺]_i in the presence of 140 mMNa⁺. This net efflux of Mg²⁺ was completelyinhibited by withdrawal of extracellular Na⁺ and waslargely attenuated by imipramine, a known inhibitor of Na⁺/Mg²⁺ exchange, with 50% inhibition at 79 µM. The relation between the rate of net Mg²⁺ efflux andextracellular Na⁺ concentration([Na⁺]_o) had a Hill coefficient of 2 and[Na⁺]_o at half-maximal rate of 82 mM. Theseresults demonstrate the presence of Na⁺ gradient-dependentMg²⁺ transport, which is consistent withNa⁺/Mg²⁺ exchange, in cardiac myocytes.

     

Estimation of Cytoplasmic Free Mg2+ Levels and Phosphorylation Potentials in Mung Bean Root Tips by In Vivo 31P NMR Spectroscopy

The cytoplasmic [MgATP]/[ATP]_free ratios, free Mg²⁺ concentrations,and phosphorylation potentials in mung bean [Vigna mungo (L.)Hepper] root tip cells were investigated by ³¹P nuclear magneticresonance spectroscopy. ³¹P NMR spectra show well defined peaksdue to G6P, cytoplasmic P_i, vacuolar P_i, ATP, UDP-glucose andnicotinamide adenine nucleotides. The concentrations of phosphorusmetabolites were determined from quantitative ³¹P NMR spectra.The [MgATP]/[ATP]_free ratio was 9.45. Accordingly, about 90%of the cytoplasmic ATP was complexed to Mg²⁺. Utilizing thedissociation constant (K_d) determined for MgATP, the cytoplasmicfree Mg²⁺ concentration was estimated to be 0.4mM. The NMR-derivedphosphorylation potential, [ATP]/([ADP][P_i]), was 960 M^-1. Thesodium azide treatment decreased the [ATP]/[ADP] ratio and thephosphorylation potential, and increased the [Mg²⁺]^free. Metabolicinhibition may have been enhanced by an increase in [Mg^2+freeand a decrease in the free energy change for ATP hydrolysis,which resulted due to a decrease in the ATP level. ¹Present address: National Food Research Institute, TsukubaCity, Ibaraki 305, Japan. (Received February 8, 1988; Accepted June 1, 1988)