ATP-dependent regulation of inwardly rectifying K+ current in bovine retinal pigment epithelial cells

Inwardlyrectifying K⁺ current(I_Kir) infreshly isolated bovine retinal pigment epithelial (RPE) cells wasstudied in the whole cell recording configuration of the patch-clamptechnique. When cells were dialyzed with pipette solution containing noATP, I_Kir randown completely in <10 min [half time(t_1/2) = 1.9 min]. In contrast, dialysis with 2 mM ATP sustainedI_Kir for 10 min or more. Rundown was also prevented with 4 mM GTP or ADP. When 0.5 mMATP was used,I_Kir ran down by~71%. Mg²⁺ was a criticalcofactor because rundown occurred when the pipette solution contained 4 mM ATP but no Mg²⁺(t_1/2 = 1.8 min).I_Kir also randown when the pipette solution contained 4 mMMg²⁺ + 4 mM5'-adenylylimidodiphosphate(t_1/2 = 2.7 min)or 4 mM adenosine 5'-O-(3-thiotriphosphate)(t_1/2 = 1.9 min),nonhydrolyzable and poorly hydrolyzable ATP analogs, respectively. Weconclude that the sustained activity ofI_Kirin bovine RPE requires intracellular MgATP and that the underlyingmechanism may involve ATP hydrolysis.

     

Apoptosis repressor with caspase domain inhibits cardiomyocyte apoptosis by reducing K+ currents

Cell shrinkageis an early prerequisite in programmed cell death, and cytoplasmicK⁺ is a dominant cation that controls intracellular ionhomeostasis and cell volume. Blockade of K⁺ channelsinhibits apoptotic cell shrinkage and attenuates apoptosis. We examined whether apoptotic repressor with caspase recruitment domain (ARC), an antiapoptotic protein, inhibits cardiomyocyte apoptosis by reducing K⁺ efflux throughvoltage-gated K⁺ (Kv) channels. In heart-derived H9c2cells, whole cell Kv currents (I_K(V)) wereisolated by using Ca²⁺-free extracellular (bath) solutionand including 5 mM ATP and 10 mM EGTA in the intracellular (pipette)solution. Extracellular application of 5 mM 4-aminopyridine (4-AP), ablocker of Kv channels, reversibly reduced I_K(V)by 50-60% in H9c2 cells. The remaining currents during 4-APtreatment may be generated by K⁺ efflux through4-AP-insensitive K⁺ channels. Overexpression of ARC inheart-derived H9c2 cells significantly decreasedI_K(V), whereas treatment with staurosporine, apotent apoptosis inducer, enhanced I_K(V)in wild-type cells. The staurosporine-induced increase inI_K(V) was significantly suppressed and thestaurosporine-mediated apoptosis was markedly inhibited incells overexpressing ARC compared with cells transfected with thecontrol neomycin vector. These results suggest that theantiapoptotic effect of ARC is, in part, due to inhibition of Kvchannels in cardiomyocytes.

     

Inducible expression of erythrocyte band 3protein

A permanent cell line with inducible expression of the humananion exchanger protein 1 (hAE1) was constructed in a derivative ofhuman embryonic kidney cells (HEK-293). In the absence of the inducer,muristerone A, the new cell line had no detectable hAE1 protein byWestern analysis or additional³⁶Cl flux. Increasing dose andincubation time with muristerone A increased the amount of protein(both unglycosylated and glycosylated). The4,4'-dinitrostilbene-2,2'-disulfonate(DNDS)-inhibitable rapid Cl exchange flux was increased up to40-fold in induced cells compared with noninduced cells. There was noDNDS-inhibitable rapid flux component in noninduced cells. This resultdemonstrates inducible expression of a new rapid Cl transport pathwaythat is DNDS sensitive. The additional transport of³⁶Cl and³⁵SO₄had the characteristics of hAE1-mediated transport in erythrocytes: 1) inhibition by 250 µM DNDS,2) activation of³⁶Cl efflux by external Cl with aconcentration producing half-maximal effect of 4.8 mM,3) activation of³⁶Cl efflux by external anionsthat was selective in the orderNO₃ = Cl > Br > I, and4) activation of³⁵SO₄influx by external protons. Under the assumption that the turnovernumbers of hAE1 were the same as in erythrocytes, there was good agreement (±3-fold) between the number of copies ofglycosylated hAE1 and the induced tracer fluxes. This is the firstexpression of hAE1 in a mammalian system to track the kineticcharacteristics of the native protein.

     

Interactions of NH4+ and L-glutamate with NO3- transport processes of non-mycorrhizal Fagus sylvatica roots

The processes of NO₃^– uptake and transport and the effectsof NH₄⁺ or L-glutamate on these processes were investigatedwith excised non-mycorrhizal beech (Fagus sylvatica L.) roots.NO₃^– net uptake followed uniphasic Michaelis-Menten kineticsin a concentration range of 10µM to 1 mM with an apparentK_m of 9.2 µM and a V_max of 366 nmol g^–1 FW h^–1.NH₄⁺, when present in excess to NO₃^–, or 10 mM L-glutamateinhibited the net uptake of NO₃^– Apparently, part of NO₃^–taken up was loaded into the xylem. Relative xylem loading ofNO₃^– ranged from 3.21.6 to 6.45.1% of NO₃^– netuptake. It was not affected by treatment with NH₄⁺ or L-glutamate.¹⁶N/¹³N double labelling experiments showed that NO₃^–efflux from roots increased with increasing influx of NO₃^–and, therefore, declined if influx was reduced by NH₄⁺ or L-glutamateexposure. From these results it is concluded that NO₃^–net uptake by non-mycorrhizal beech roots is reduced by NH₄⁺or L-glutamate at the level of influx and not at the level ofefflux. Key words: Nitrate transport, net uptake, influx, efflux, ammonium, Fagus, Fagaceae     

Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]o regulation

The neuronal K-Cl cotransporter isoform (KCC2) was functionallyexpressed in human embryonic kidney (HEK-293) cell lines. Two stablytransfected HEK-293 cell lines were prepared: one expressing anepitope-tagged KCC2 (KCC2-22T) and another expressing theunaltered KCC2 (KCC2-9). The KCC2-22T cells produced aglycoprotein of ~150 kDa that was absent from HEK-293 control cells.The ⁸⁶Rb influx in both cell lineswas significantly greater than untransfected control HEK-293 cells. TheKCC2-9 cells displayed a constitutively active⁸⁶Rb influx that could beincreased further by 1 mMN-ethylmaleimide (NEM) but not by cellswelling. Both furosemide [inhibition constant (K_i) ~25µM] and bumetanide (K_i~55 µM) inhibited the NEM-stimulated ⁸⁶Rb influx in the KCC2-9cells. This diuretic-sensitive⁸⁶Rb influx in theKCC2-9 cells, operationally defined as KCC2 mediated, required external Clbut not external Na⁺ and exhibiteda high apparent affinity for externalRb⁺(K⁺)[Michaelis constant(K_m) = 5.2 ± 0.9 (SE) mM; n = 5] but alow apparent affinity for externalCl(K_m >50 mM). Onthe basis of thermodynamic considerations as well as the unique kineticproperties of the KCC2 isoform, it is hypothesized that KCC2 may servea dual function in neurons: 1) themaintenance of low intracellularCl concentration so as toallow Cl influx vialigand-gated Cl channelsand 2) the buffering of externalK⁺ concentration([K⁺]_o) in the brain.

     

The effects of valinomycin on respiration and volume changes in plant mitochondria

The effects of valinomycin on the respiration and volume changeshave been studied with isolated mitochondria from bean hypocotyl(Phaseolus vulgaris L.) and cauliflower bud (Brassica oleraceaL.). In the presence of 10 mM K salts of chloride, acetate,phosphate, and sulfate respiration is stimulated by valinomycinconcomitant with osmotic swelling. When swelling declines respirationwith organic acid substrates also declines. In the presenceof the K salts of acetate and PO₄ but not Cl^– the terminationof respiration leads to contraction. The contraction in K-PO₄is inhibited by addition to the external medium of between 65to 100 mM K-PO₄. The results are interpreted to suggest thatvalinomycin in the presence of KCl facilitated the movementof K down an electrical gradient, with the Cl anion followingand osmotic swelling resulting. However, in a medium containingacetate or PO₄ the anions are actively transported against anelectrical gradient at the expense of metabolic energy. Valinomycinfacilitates the influx of K⁺ with the actively transported anionand swelling follows. When respiration terminates the activelytransported anions move passively back down their electrochemicalgradient and osmotic contraction follows. ¹ Present address: Department of Biology, Fort Lewis College,Durango, Colorado 81301, U.S.A. (Received July 21, 1972; )     

Effect of changes in respiratory blood parameters on equine red blood cell K-Cl cotransporter

K influx intoequine red blood cells (RBCs) was measured using⁸⁶Rb as a tracer for K underconditions designed to mimic the changes in respiratory bloodparameters that occur in vivo during strenuous exercise. The effects onK influx of physiological changes in pH, cell volume,O₂ tension(PO₂),CO₂ tension(PCO₂), and bicarbonate and lactateconcentrations were defined. Physiological PO₂ exerted a dominant controllinginfluence on the H⁺-stimulatedCl-dependent K influx, consistent with effects on the K-Clcotransporter; PO₂ required forhalf-maximal activity was 37 ± 3 mmHg (4.9 kPa). AlthoughRBCs were swollen at low pH, results showed explicitly that the volumechange per se had little effect on K influx. Lactate had no effect onvolume- or H⁺-stimulated Kinfluxes, nor did bicarbonate or PCO₂affect the magnitude of K influxes after these stimuli or aftertreatment with protein kinase/phosphatase inhibitors. These resultsrepresent the first detailed report ofO₂ dependence ofH⁺-stimulated K-Cl cotransport inRBCs from any mammalian species. They emphasize the importance ofPO₂ in control of RBC K-Clcotransport.

     

Riboflavin uptake by human-derived colonic epithelial NCM460 cells

Normal microflora ofthe large intestine synthesize a number of water-soluble vitaminsincluding riboflavin (RF). Recent studies have shown that colonicepithelial cells posses an efficient carrier-mediated mechanism forabsorbing some of these micronutrients. The aim of the present studywas to determine whether colonic cells also posses a carrier-mediatedmechanism for RF uptake and, if so, to characterize this mechanism andstudy its cellular regulation. Confluent monolayers of thehuman-derived nontransformed colonic epithelial cells NCM460 and[³H]RF were used in the study. Uptake of RF wasfound to be 1) appreciable and temperature and energydependent; 2) Na⁺ independent; 3) saturableas a function of concentration with an apparent K_mof 0.14 µM and V_max of 3.29 pmol · mgprotein¹ · 3 min¹; 4) inhibited by the structural analogslumiflavin and lumichrome (K_i of 1.8 and 14.1 µM,respectively) but not by the unrelated biotin; 5) inhibited ina competitive manner by the membrane transport inhibitor amiloride(K_i = 0.86 mM) but not by furosemide, DIDS, orprobenecid; 6) adaptively regulated by extracellular RF levels with a significant and specific upregulation and downregulation in RFuptake in RF-deficient and oversupplemented conditions, respectively;and 7) modulated by an intracellularCa²⁺/calmodulin-mediated pathway. These studies demonstratefor the first time the existence of a specialized carrier-mediatedmechanism for RF uptake in an in vitro cellular model system of humancolonocytes. This mechanism appears to be regulated by extracellularsubstrate level and by an intracellularCa²⁺/calmodulin-mediated pathway. It is suggested that theidentified transport system may be involved in the absorption ofbacterially synthesized RF in the large intestine and that this sourceof RF may contribute toward RF homeostasis, especially that of colonocytes.

     

Orthophosphate Relations of Root: NO-3 Effects on Orthophosphate Influx, Accumulation and Secretion into the Xylem

Lamaze, T., Sentenac, H. and Grignon, C. 1987. Orthophosphaterelations of root: NO^–₃effects on orthophosphate influx,accumulation and secretion into the xylem.—J. exp. Bot.38: 923–934. Orthophosphate (Pi) accumulation by barley (Hordeum vulgareL.) roots was specifically inhibited by NO^–₃ as comparedto Cl^– and SO₄^{2 –}, and Pi secretion into the xylemwas stimulated. The inhibition of Pi accumulation by NO^–₃was also observed in roots of intact photosynthesizing horsebean(Vicia faba L.), rice (Oryza sativa L.) and soybean (Glycinemax L.) plants. NO^–₃ effects on Pi transport by rootswere more thoroughly investigated with corn (Zea mays L.). Theywere due to intracellular NO^–₃. Pi secretion was stillstimulated by NO^–₃ after Pi withdrawal from the absorptionsolution. ³²Pi influx decreased during Pi accumulation, supportingthe hypothesis that this ion allosterically regulated its owntransport system by feedback control. This control was modulatedby other anions: the decrease was more pronounced in the presenceof nitrate. Chronologically, the depressive effect of NO^–₃on ³²Pi influx appeared after the inhibition of Pi accumulation.Furthermore, under conditions where Pi accumulation was notaffected by NO^–₃, ³²Pi influx and Pi secretion into thexylem became insensitive to the presence of nitrate. Our hypothesisis that the stimulative effect of NO^–₃ on Pi secretionand the depressive one on ³²Pi influx are the repercussionsof an increase in the Pi cytosolic concentration due to an NO^–₃-induced decrease in Pi uptake by the vacuoles. Key words: Root, orthophosphate fluxes, orthophosphate accumulation, nitrate, ionic interaction     

Kinetic evidence that the Na-PO4 cotransporter is the molecular mechanism for Na/Li exchange in human red blood cells

The molecular basis for Na/Li exchange is unknown. Li can be transported by the Na pump, anion exchanger (AE1), a background leak, and the Na/Li exchanger. In vivo the intraerythrocyte concentration of Li results from the balance of passive entry, mostly on AE1, and the active extrusion on the Na/Li exchanger. Here we show that erythrocytes have Li-activated PO₄ transport that behaves as if it is mediated by the Na-PO₄ cotransporter (hBNP1) and provide evidence that this Na/Li-PO₄ cotransporter is also the mechanism for Na/Li exchange. First, external Li (>20 mM) activated PO₄ influx severalfold. Li activation of PO₄ influx was potentiated by the presence of external Na. Second, the ouabain-insensitive ²²Na efflux was stimulated by external Li and then inhibited by external PO₄. Third, phloretin inhibited Na- and Li-activated PO₄ flux with the same K_i, 0.25 mM. Fourth, external PO₄ (0.1–1.0 mM) inhibited ouabain-insensitive Li efflux only if external Na was present. Fifth, arsenate, a phosphate congener, inhibited both Na-PO₄ cotransport and Li-activated PO₄ flux with similar kinetics when Na or Li concentration was high but did not inhibit Li_out/Na_in exchange when Li_out concentration was low. The collective results suggest that both Na and Li are substrates for at least two sites on the same PO₄ cotransporter and that Na/Li exchange behaves as if it is mediated by this Na/Li-PO₄ cotransporter when only one cation is bound. Plasma and intracellular PO₄ concentrations may be important regulators of Li transport and its therapeutic effects. sodium/lithium exchange; sodium,lithium-phosphate cotransport; human erythrocytes; kinetic model     

Role of extracellular [Ca2+] in fatigue of isolated mammalian skeletal muscle

The possiblerole of altered extracellular Ca²⁺concentration([Ca²⁺]_o)in skeletal muscle fatigue was tested on isolated slow-twitch soleusand fast-twitch extensor digitorum longus muscles of the mouse. Thefollowing findings were made. 1) Achange from the control solution (1.3 mM[Ca²⁺]_o)to 10 mM[Ca²⁺]_o,or to nominally Ca²⁺-freesolutions, had little effect on tetanic force in nonfatigued muscle.2) Almost complete restoration oftetanic force was induced by 10 mM[Ca²⁺]_oin severely K⁺-depressed muscle(extracellular K⁺ concentration of10-12 mM). This effect was attributed to a 5-mV reversal of theK⁺-induced depolarization andsubsequent restoration of ability to generate action potentials(inferred by using the twitch force-stimulation strength relationship).3) Tetanic force depressed bylowered extracellular Na⁺concentration (40 mM) was further reduced with 10 mM[Ca²⁺]_o.4) Tetanic force loss at elevatedextracellular K⁺ concentration (8 mM) and lowered extracellular Na⁺concentration (100 mM) was partially reversed with 10 mM[Ca²⁺]_oor markedly exacerbated with low[Ca²⁺]_o.5) Fatigue induced by using repeatedtetani in soleus was attenuated at 10 mM[Ca²⁺]_o(due to increased resting and evoked forces) and exacerbated at low[Ca²⁺]_o.These combined results suggest, first, that raised[Ca²⁺]_oprotects against fatigue rather than inducing it and, second, that aconsiderable depletion of[Ca²⁺]_oin the transverse tubules may contribute to fatigue.

     

Ovine male genital duct epithelial cells differentiate in vitro and express functional CFTR and ENaC

To investigate the biology of the malegenital duct epithelium, we have established cell cultures from theovine vas deferens and epididymis epithelium. These cells develop tightjunctions, high transepithelial electrical resistance, and alumen-negative transepithelial potential difference as a sign of activetransepithelial ion transport. In epididymis cultures the equivalentshort-circuit current (I_sc) averaged 20.8 ± 0.7 µA/cm² (n = 150) and was partially inhibited byapical application of amiloride with an inhibitor concentration of 0.64 µM. In vas deferens cultures, I_sc averaged 14.4 ± 1.1 µA/cm² (n = 18) and was also inhibited byapical application of amiloride with a half-maximal inhibitorconcentration (K_i) of 0.68 µM. The remainingamiloride-insensitive I_sc component in epididymisand vas deferens cells was partially inhibited by apical application ofthe Cl channel blocker diphenylamine-2-carboxylicacid (1 mM). It was largely dependent on extracellularCl and, to a lesser extent, on extracellularHCO₃. It was further stimulated bybasolateral application of forskolin (10⁵ M), which increasedI_sc by 3.1 ± 0.3 µA/cm² (n=65) in epididymis and 0.9 ± 0.1 µA/cm² (n =11) in vas deferens. These findings suggest that cultured ovine vasdeferens and epididymis cells absorb Na⁺ viaamiloride-sensitive epithelial Na⁺ channels (ENaC) andsecrete Cl and HCO₃via apical cystic fibrosis transmembrane conductance regulator (CFTR)Cl channels. This interpretation is supported byRT-PCR data showing that vas deferens and epididymis cells express CFTRand ENaC mRNA.

     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.

     

Apoplastic Solute Concentrations of Organic Acids and Mineral Nutrients in the Leaves of Several Fagaceae

Ion chromatographic methods determined organic acids and mainnutrient minerals in the apoplastic solution from leaves ofseveral Fagaceae (Quercus ilex L., Quercus cerris L., Quercusvirgiliana (Ten.) Ten, and Fagus sylvatica L.). The anions oforganic acids found in high amounts (250 to 650 µM) werequinate, malate, and oxalate. Lactate, pyruvate, formate andacetate were detected in relatively low amounts with concentrationsbetween 20 and 200 µM. The total concentration of organicacids in the apoplastic sap ranged between 1.5 and 2 mM. Thetotal concentration of inorganic cations (K⁺, Mg²⁺, NH₄⁺, Ca²⁺,Na⁺) and anions (C1^–, NO₃^–, SO^2–₄ and PO^3–₄)in the apoplastic sap varied between 5 and 10 mM, and 0.35 and1.8 mM, respectively. We conclude that the concentration oforganic acid ions in the leaf apoplast depends mainly on theexchange with the leaf cells and is influenced by the electrochemicalgradient between the symplast and the apoplast in relation tothe water potential of the leaf. The determination of formateand acetate in the apoplastic compartment of leaves lend weightto the argument that the production of these acids by treesis a important emission source to the atmosphere. (Received June 9, 1998; Accepted April 8, 1999)     

Protein kinase D inhibits plasma membrane Na+/H+ exchanger activity

The regulation of plasma membraneNa⁺/H⁺exchanger (NHE) activity by protein kinase D (PKD), a novel proteinkinase C- and phorbol ester-regulated kinase, was investigated. Todetermine the effect of PKD on NHE activity in vivo, intracellular pH(pH_i) measurements were made inCOS-7 cells by microepifluorescence using the pH indicator cSNARF-1.Cells were transfected with empty vector (control), wild-type PKD, orits kinase-deficient mutant PKD-K618M, together with green fluorescentprotein (GFP). NHE activity, as reflected by the rate of acid efflux(J_H), wasdetermined in single GFP-positive cells following intracellularacidification. Overexpression of wild-type PKD had no significanteffect on J_H(3.48 ± 0.25 vs. 3.78 ± 0.24 mM/min in control atpH_i 7.0). In contrast,overexpression of PKD-K618M increasedJ_H (5.31 ± 0.57 mM/min at pH_i 7.0;P < 0.05 vs. control). Transfectionwith these constructs produced similar effects also in A-10 cells,indicating that native PKD may have an inhibitory effect on NHE in bothcell types, which is relieved by a dominant-negative action ofPKD-K618M. Exposure of COS-7 cells to phorbol ester significantlyincreased J_H in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M(because basal J_Hwas already near maximal). A fusion protein containing the cytosolicregulatory domain (amino acids 637-815) of NHE1 (the ubiquitousNHE isoform) was phosphorylated in vitro by wild-type PKD, but with lowstoichiometry. These data suggest that PKD inhibits NHE activity,probably through an indirect mechanism, and represents a novel pathwayin the regulation of the exchanger.

     

Flexible Coupling of Phosphate Uptake in Dunaliella acidophila at Extremely Low pH Values

The acidophilic alga Dunaliella acidophila exhibits optimalgrowth at pH 1. We have investigated the regulation of phosphateuptake by this alga using tracer techniques and by performingintracellular phosphate measurements under different growthconditions including phosphate limitation. In batch culturewith 2·2 mol m^–3 phosphate in the medium the uptakeof phosphate at micromolar phosphate concentrations followeda linear time dependence in the range of minutes and rates werein the range of 1 µmol phosphate mg^–1 chl h^–1,only. However, under discontinuous phosphate-limited growthconditions, tracer influx revealed a biphasic pattern at micromolarphosphate concentrations: An initial burst phase resulted ina 10⁴-fold internal phosphate accumulation and levelled offafter about 10 s. A double reciprocal plot of the initial influxrates obtained for phosphate-limited and unlimited algae exhibitedMichaelis-Menten kinetics. Phosphate limitation caused a significantactivation of the maximum velocity of uptake, yielding V_maxup to 1 mmol mg^–1 chl h^–1 as compared to valuesin the order of 50 µmol phosphate mg^–1 chl h^–1for the second phase (this magnitude is also representativefor non-limited batch cultures). Concomitantly the Michaelisconstant was altered from 4 mmol m^–3 to 0·7 mmolm^–3. The rapid uptake of phosphate was inhibited by arsenateand FCCP and was not stimulated by Na⁺. The pH dependence oftracer accumulation and measurements of the intracellular phosphatepool under different growth conditions indicate that at lowpH and low external phosphate concentrations the high protongradient present under these conditions is utilized for a H₃PO₄uptake or a H⁺/H₂PO^–4 cotransport. However, when the externalphosphate concentration was increased to levels sufficientlyhigh for transport to be driven by the positive membrane potential(10 mol m^–3 phosphate), the pH dependence of phosphateuptake was more complex, but could be explained by the uptakeof H₃PO₄ or a H⁺/H₂PO^–₄-cotransport at low pH and a differenttype H₂PO^–4-transport (with unknown type of ion coupling)at high pH-values. It is suggested that this flexible couplingof phosphate transport is of essential importance for the acidresistance of Dunaliella acidophila. Key words: Acid resistance, Dunaliella acidophila, phosphate cotransport, phosphate limitation, plasma membrane, sodium     

Separate entry pathways for phosphate and oxalate in rat brain microsomes

ATP-dependent ⁴⁵Ca uptake in rat brainmicrosomes was measured in intracellular-like media containingdifferent concentrations of PO₄ and oxalate. In the absenceof divalent anions, there was a transient ⁴⁵Caaccumulation, lasting only a few minutes. Addition of PO₄did not change the initial accumulation but added a second stage that increased with PO₄ concentration. Accumulation during thesecond stage was inhibited by the following anion transport inhibitors: niflumic acid (50 µM),4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS; 250 µM),and DIDS (3-5 µM); accumulation during the initial stage wasunaffected. Higher concentrations of DIDS (100 µM), however,inhibited the initial stage as well. Uptake was unaffected by 20 mM Na,an activator, or 1 mM arsenate, an inhibitor of Na-PO₄ cotransport. An oxalate-supported ⁴⁵Ca uptake was larger,less sensitive to DIDS, and enhanced by the catalytic subunit ofprotein kinase A (40 U/ml). Combinations of PO₄ and oxalatehad activating and inhibitory effects that could be explained byPO₄ inhibition of an oxalate-dependent pathway, but notvice versa. These results support the existence of separate transportpathways for oxalate and PO₄ in brain endoplasmic reticulum.

     

Regulation of KCa current by store-operated Ca2+ influx depends on internal Ca2+ release in HSG cells

This study examines theCa²⁺ influx-dependent regulationof the Ca²⁺-activatedK⁺ channel(K_Ca) in human submandibulargland (HSG) cells. Carbachol (CCh) induced sustained increases in theK_Ca current and cytosolic Ca²⁺ concentration([Ca²⁺]_i),which were prevented by loading cells with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Removal of extracellularCa²⁺ and addition ofLa³⁺ orGd³⁺, but notZn²⁺, inhibited the increases inK_Ca current and[Ca²⁺]_i.Ca²⁺ influx during refill (i.e.,addition of Ca²⁺ to cells treatedwith CCh and then atropine inCa²⁺-free medium) failed to evokeincreases in the K_Ca current but achieved internal Ca²⁺ storerefill. When refill was prevented by thapsigargin,Ca²⁺ readdition induced rapidactivation of K_Ca. These dataprovide further evidence that intracellularCa²⁺ accumulation provides tightbuffering of[Ca²⁺]_iat the site of Ca²⁺ influx (H. Mogami, K. Nakano, A. V. Tepikin, and O. H. Petersen. Cell 88: 49-55, 1997). We suggestthat the Ca²⁺ influx-dependentregulation of the sustained K_Cacurrent in CCh-stimulated HSG cells is mediated by the uptake ofCa²⁺ into the internalCa²⁺ store and release via theinositol 1,4,5-trisphosphate-sensitive channel.

     

Structure, function, and genomic organization of human Na(+)-dependent high-affinity dicarboxylate transporter

We have clonedand functionally characterized the human Na⁺-dependenthigh-affinity dicarboxylate transporter (hNaDC3) from placenta. ThehNaDC3 cDNA codes for a protein of 602 amino acids with 12 transmembrane domains. When expressed in mammalian cells, the clonedtransporter mediates the transport of succinate in the presence ofNa⁺ [concentration of substrate necessary for half-maximaltransport (K_t) for succinate = 20 ± 1 µM]. Dimethylsuccinate also interacts with hNaDC3. TheNa⁺-to-succinate stoichiometry is 3:1 and concentration ofNa⁺ necessary for half-maximal transport(K^Na+_0.5) is 49 ± 1 mM as determined by uptake studies withradiolabeled succinate. When expressed in Xenopuslaevis oocytes, hNaDC3 induces Na⁺-dependent inwardcurrents in the presence of succinate and dimethylsuccinate. At amembrane potential of 50 mV,K^Suc_0.5 is 102 ± 20 µM andK^Na+_0.5 is 22 ± 4 mM as determined by the electrophysiological approach. Simultaneous measurements of succinate-evoked charge transfer andradiolabeled succinate uptake in hNaDC3-expressing oocytes indicate acharge-to-succinate ratio of 1:1 for the transport process, suggestinga Na⁺-to-succinate stoichiometry of 3:1. pH titration ofcitrate-induced currents shows that hNaDC3 accepts preferentially thedivalent anionic form of citrate as a substrate. Li⁺inhibits succinate-induced currents in the presence of Na⁺.Functional analysis of rat-human and human-rat NaDC3 chimeric transporters indicates that the catalytic domain of the transporter lies in the carboxy-terminal half of the protein. The humanNaDC3 gene is located on chromosome20q12-13.1, as evidenced by fluorescent in situ hybridization. Thegene is >80 kbp long and consists of 13 exons and 12 introns.

     

Ca2+ depolarizes adrenal cortical cells through selective inhibition of an ATP-activated K+ current

Bovine adrenalzona fasciculata cells (AZF) express a noninactivatingK⁺ current(I_AC) whoseinhibition by adrenocorticotropic hormone and ANG II may be coupled tomembrane depolarization andCa²⁺-dependentcortisol secretion. We studiedI_ACinhibition byCa²⁺ and theCa²⁺ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette)Ca²⁺concentration([Ca²⁺]_i)buffered to 0.02 µM,I_AC reachedmaximum current density of 25.0 ± 5.1 pA/pF(n = 16); raising[Ca²⁺]_ito 2.0 µM reduced it 76%. In inside-out patches, elevated[Ca²⁺]_idramatically reducedI_AC channelactivity. Ionomycin inhibited I_AC by 88 ± 4% (n = 14) without altering rapidlyinactivating A-type K⁺ current.Inhibition of I_ACby ionomycin was unaltered by adding calmodulin inhibitory peptide tothe pipette or replacing ATP with its nonhydrolyzable analog5'-adenylylimidodiphosphate.I_AC inhibition byionomycin was associated with membrane depolarization. When[Ca²⁺]_iwas buffered to 0.02 µM with 2 and 11 mM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ionomycin inhibitedI_AC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively (n = 4). ANG II inhibitedI_AC significantlymore effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising[Ca²⁺]_iinhibits I_ACthrough a mechanism not requiring calmodulin or protein kinases,suggesting direct interaction withI_AC channels. ANGII may inhibitI_AC anddepolarize AZF by activating parallel signaling pathways, one of whichuses Ca²⁺ asa mediator.