Two-way arginine transport in human endothelial cells: TNF-alpha stimulation is restricted to system y+

Humanumbilical vein endothelial cells transport arginine through twoNa⁺-independent systems. System y⁺L isinsensitive to N-ethylmaleimide (NEM), inhibited byL-leucine in the presence of Na⁺, and referableto the expression of SLC7A6/y⁺LAT2,SLC7A7/y⁺LAT1, and SLC3A2/4F2hc. System y⁺ isreferable to the expression of SLC7A1/CAT1 and SLC7A2/CAT2B. Tumornecrosis factor- (TNF-) and bacterial lipopolysaccharide induce atransient stimulation of arginine influx and efflux through systemy⁺. Increased expression of SLC7A2/CAT2B is detectable from3 h of treatment, while SLC7A1 expression is inhibited at latertimes of incubation. System y⁺L activity and expressionremain unaltered. Nitric oxide synthase type 2 mRNA is not detected inthe absence or presence of TNF-, while the latter condition lowersnitric oxide synthase type 3 expression at the mRNA and the proteinlevel. Nitrite accumulation is comparable in cytokine-treated andcontrol cells up to 48 h of treatment. It is concluded thatmodulation of endothelial arginine transport by TNF- orlipopolysaccharide occurs exclusively through changes in CAT2B and CAT1expression and is dissociated from stimulation of nitric oxide production.

     

Allele-specific differences in ryanodine receptor 1 mRNA expression levels may contribute to phenotypic variability in malignant hyperthermia

Background

In the recessive aminoaciduria Lysinuric Protein Intolerance (LPI), mutations of SLC7A7/y+LAT1 impair system y⁺L transport activity for cationic amino acids. A severe complication of LPI is a form of Pulmonary Alveolar Proteinosis (PAP), in which alveolar spaces are filled with lipoproteinaceous material because of the impaired surfactant clearance by resident macrophages. The pathogenesis of LPI-associated PAP remains still obscure. The present study investigates for the first time the expression and function of y+LAT1 in monocytes and macrophages isolated from a patient affected by LPI-associated PAP. A comparison with mesenchymal cells from the same subject has been also performed.

Methods

Monocytes from peripheral blood were isolated from a 21-year-old patient with LPI. Alveolar macrophages and fibroblastic-like mesenchymal cells were obtained from a whole lung lavage (WLL) performed on the same patient. System y⁺L activity was determined measuring the 1-min uptake of [³H]-arginine under discriminating conditions. Gene expression was evaluated through qRT-PCR.

Results

We have found that: 1) system y⁺L activity is markedly lowered in monocytes and alveolar macrophages from the LPI patient, because of the prevailing expression of SLC7A7/y+LAT1 in these cells; 2) on the contrary, fibroblasts isolated from the same patient do not display the transport defect due to compensation by the SLC7A6/y+LAT2 isoform; 3) in both normal and LPI monocytes, GM-CSF induces the expression of SLC7A7, suggesting that the gene is a target of the cytokine; 4) GM-CSF-induced differentiation of LPI monocytes is comparable to that of normal cells, demonstrating that GM-CSF signalling is unaltered; 5) general and respiratory conditions of the patient, along with PAP-associated parameters, markedly improved after GM-CSF therapy through aerosolization.

Conclusions

Monocytes and macrophages, but not fibroblasts, derived from a LPI patient clearly display the defect in system y⁺L-mediated arginine transport. The different transport phenotypes are referable to the relative levels of expression of SLC7A7 and SLC7A6. Moreover, the expression of SLC7A7 is regulated by GM-CSF in monocytes, pointing to a role of y+LAT1 in the pathogenesis of LPI associated PAP.     

y+LAT1 and y+LAT2 contribution to arginine uptake in different human cell models: Implications in the pathophysiology of Lysinuric Protein Intolerance

y+LAT1 (encoded by SLC7A7), together with y+LAT2 (encoded by SLC7A6), is the alternative light subunits composing the heterodimeric transport system y+L for cationic and neutral amino acids. SLC7A7 mutations cause lysinuric protein intolerance (LPI), an inherited multisystem disease characterized by low plasma levels of arginine and lysine, protein‐rich food intolerance, failure to thrive, hepatosplenomegaly, osteoporosis, lung involvement, kidney failure, haematologic and immunological disorders. The reason for the heterogeneity of LPI symptoms is thus far only poorly understood. Here, we aimed to quantitatively compare the expression of SLC7A7 and SLC7A6 among different human cell types and evaluate y+LAT1 and y+LAT2 contribution to arginine transport. We demonstrate that system y+L‐mediated arginine transport is mainly accounted for by y+LAT1 in monocyte‐derived macrophages (MDM) and y+LAT2 in fibroblasts. The kinetic analysis of arginine transport indicates that y+LAT1 and y+LAT2 share a comparable affinity for the substrate. Differences have been highlighted in the expression of SLC7A6 and SLC7A7 mRNA among different cell models: while SLC7A6 is almost equally expressed, SLC7A7 is particularly abundant in MDM, intestinal Caco‐2 cells and human renal proximal tubular epithelial cells (HRPTEpC). The characterization of arginine uptake demonstrates that system y+L is operative in renal cells and in Caco‐2 where, at the basolateral side, it mediates arginine efflux in exchange with leucine plus sodium. These findings explain the defective absorption/reabsorption of arginine in LPI. Moreover, y+LAT1 is the prevailing transporter in MDM sustaining a pivotal role in the pathogenesis of immunological complications associated with the disease.     

Development and polarization of cationic amino acid transporters and regulators in the human placenta

We have investigatedL-arginine transport systems in the human placentalsyncytiotrophoblast across gestation using purified microvillous (MVM)and basal (BM) plasma membrane vesicles. In MVM from first-trimesterand term placentas, L-arginine transport was by systemsy⁺ and y⁺L. In BM (term placentas), however,there was evidence for system y⁺L only. The Michaelisconstant of system y⁺L was significantly lower (P < 0.05) in first-trimester compared with term MVM and lower in termMVM compared with BM (P < 0.05). There was no functionalevidence for system b⁰⁺ in term MVM or BM. Cationic aminoacid transporter (CAT) 1, CAT 4, and 4F2hc were detected using RT-PCRin placentas throughout gestation. rBAT was not detected in termplacentas. An ~85-kDa and an ~135-kDa protein was detected byWestern blotting in MVM under reducing and nonreducing conditions,respectively, consistent with the 4F2hc monomer and the 4F2hc-lightchain dimer, and their expression was significantly higher (P < 0.05) in term compared with first-trimester MVM. These proteinswere not detected in BM despite functional evidence for systemy⁺L. These data suggest different roles for 4F2hc in thedevelopment and polarization of cationic amino acid transporters in the syncytiotrophoblast.

     

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     

INFgamma stimulates arginine transport through system y+L in human monocytes

Freshly isolated human monocytes transport L-arginine mostly through a sodium independent, NEM insensitive pathway inhibited by L-leucine in the presence, but not in the absence of sodium. Interferon-gamma (IFNgamma) stimulates this pathway, identifiable with system y+L, and markedly enhances the expression of SLC7A7, the gene that encodes for system y+L subunit y+LAT1, but not of SLC7A6, that codes for the alternative subunit y+LAT2. System y+ plays a minor role in arginine uptake by monocytes and the expression of system y+-related genes, SLC7A1 and SLC7A2, is not changed by IFNgamma. These results demonstrate that system y+L is sensitive to IFNgamma.     

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     

Interaction of reactive oxygen species with ion transport mechanisms

The use ofelectrophysiological and molecular biology techniques has shed light onreactive oxygen species (ROS)-induced impairment of surface andinternal membranes that control cellular signaling. These deleteriouseffects of ROS are due to their interaction with various ion transportproteins underlying the transmembrane signal transduction, namely,1) ion channels, such asCa²⁺ channels (includingvoltage-sensitive L-type Ca²⁺currents, dihydropyridine receptor voltage sensors, ryanodine receptorCa²⁺-release channels, andD-myo-inositol1,4,5-trisphosphate receptor Ca²⁺-release channels),K⁺ channels (such asCa²⁺-activatedK⁺ channels, inward and outwardK⁺ currents, and ATP-sensitiveK⁺ channels),Na⁺ channels, andCl channels;2) ion pumps, such as sarcoplasmicreticulum and sarcolemmal Ca²⁺pumps,Na⁺-K⁺-ATPase(Na⁺ pump), andH⁺-ATPase(H⁺ pump);3) ion exchangers such as theNa⁺/Ca²⁺exchanger andNa⁺/H⁺exchanger; and 4) ion cotransporterssuch asK⁺-Cl,Na⁺-K⁺-Cl,andP_i-Na⁺cotransporters. The mechanism of ROS-induced modificationsin ion transport pathways involves1) oxidation of sulfhydryl groups located on the ion transport proteins,2) peroxidation of membrane phospholipids, and 3) inhibition ofmembrane-bound regulatory enzymes and modification of the oxidativephosphorylation and ATP levels. Alterations in the ion transportmechanisms lead to changes in a second messenger system, primarilyCa²⁺ homeostasis, which furtheraugment the abnormal electrical activity and distortion of signaltransduction, causing cell dysfunction, which underlies pathologicalconditions.

     

Characterization of two Mg2+ transporters in sealed plasma membrane vesicles from rat liver

The plasmamembrane of mammalian cells possesses rapidMg²⁺ transport mechanisms. Theidentity of Mg²⁺ transporters isunknown, and so are their properties. In this study,Mg²⁺ transporters werecharacterized using a biochemically and morphologically standardizedpreparation of sealed rat liver plasma membranes (LPM) whoseintravesicular content could be set and controlled. The system has theadvantages that it is not regulated by intracellular signalingmachinery and that the intravesicular ion milieu can be designed. Theresults indicate that 1) LPM retaintrapped intravesicular total Mg²⁺with negligible leak; 2) theaddition of Na⁺ orCa²⁺ induces a concentration- andtemperature-dependent efflux corresponding to 30-50% of theintravesicular Mg²⁺;3) the rate of flux is very rapid(137.6 and 86.8 nmol total Mg²⁺ · µm² · min¹after Na⁺ andCa²⁺ addition, respectively);4) coaddition of maximalconcentrations of Na⁺ andCa²⁺ induces an additiveMg²⁺ efflux;5) bothNa⁺- andCa²⁺-stimulatedMg²⁺ effluxes are inhibited byamiloride, imipramine, or quinidine but not by vanadate orCa²⁺ channel blockers;6) extracellularNa⁺ orCa²⁺ can stimulateMg²⁺ efflux in the absence ofMg²⁺ gradients; and7)Mg²⁺ uptake occurs in LPM loadedwith Na⁺ but not withCa²⁺, thus indicating thatNa⁺/Mg²⁺but notCa²⁺/Mg²⁺exchange is reversible. These data are consistent with the operation oftwo distinct Mg²⁺ transportmechanisms and provide new information on rates of Mg²⁺ transport, specificity of thecotransported ions, and reversibility of the transport.

     

Volume-dependent taurine release from cultured astrocytes requires permissive [Ca2+]i and calmodulin

Cell swelling results in regulatory activation of multipleconductive anion pathways permeable toward a broad spectrum of intracellular organic osmolytes. Here, we explore the involvement ofextracellular and intracellularCa²⁺ in volume-dependent[³H]taurine effluxfrom primary cultured astrocytes and compare theCa²⁺ sensitivity of this efflux inslow (high K⁺ medium induced) andfast (hyposmotic medium induced) cell swelling. NeitherCa²⁺-free medium norCa²⁺-channel blockers prevented thevolume-dependent[³H]taurine release.In contrast, loading cells with the membrane-permeable Ca²⁺ chelator1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM suppressed[³H]taurine efflux by65-70% and 25-30% underhigh-K⁺ and hyposmotic conditions,respectively. Fura 2 measurements confirmed that BAPTA-AM, but notCa²⁺-free media, significantlyreduced resting intracellular Ca²⁺concentration([Ca²⁺]_i).The calmodulin antagonists trifluoperazine and fluphenazine reversiblyand irreversibly, respectively, inhibited thehigh-K⁺-induced[³H]taurine release,consistent with their known actions on calmodulin. In hyposmoticconditions, the effects were less pronounced. These data suggest thatvolume-dependent taurine release requires minimal basal[Ca²⁺]_iand involves calmodulin-dependent step(s). Quantitative differences inCa²⁺/calmodulin sensitivity ofhigh-K⁺-induced and hyposmoticmedium-induced taurine efflux are due to both the effects of theinhibitors on high-K⁺-induced cellswelling and their effects on transport systems and/or signalingmechanisms determining taurine efflux.

     

Inhibition of Ion Transport in Excised Barley Roots by Abscisic Acid; Relation to Water Permeability of the Roots

Previous papers have shown that abscisic acid can inhibit transportof ions across the root to the xylem vessels, resulting in reducedexudation from excised roots or inhibiting guttation from intactplants. However, it has not been established whether the inhibitionwas due to a reduction in salt transport (J_s) or in permeabilityof the roots to water (L_p). This paper investigates the effectof ABA on L_p and J_s separately. It is shown that L_p increasedin ABA and then fell, but was about the same as in control rootswhen transport was inhibited. The effect of ABA on exudationtherefore appeared to be mainly due to reduction in J_s. Inhibitionof J_s was also present in intact, transpiring plants and sowas not due to reduced water flow. The inhibition of ion releaseto the xylem affected Na⁺, Mg²⁺, Ca²⁺, and phosphate as wellas the major ion in the exudate, K⁺. It is concluded that ABAinhibits salt transport to the shoot by acting on ion transportinto the xylem, and not by reducing water flow coupled withsalt transport.     

A Sodium Pump in the Plasma Membrane of the Marine Alga Heterosigma akashiwo

ATP-dependent transport of ²²Na⁺ into liposomes reconstitutedfrom plasma membrane proteins of Heterosigma akashiwo was examined.The apparent K^m values for transport of Na⁺ were 400 µMfor ATP and 7 mM for Na⁺. ATP-dependent transport of ²²Na⁺ wasnot inhibited by a protonophore or a membrane-permeable cationbut was inhibited by an inhibitor of P-type ATPases. (Received October 2, 1995; Accepted February 1, 1996)     

Effects of Some Amino Acid Analogues on the Uptake and Transport of K+ and Ca2+ in Wheat and Mung Bean Seedlings: I. CORTICAL CELL FLUXES AND TRANSPORT TO THE STELE IN EXCISED ROOT SEGMENTS, AS AFFECTED BY p-FLUOROPHENYLALANINE

Effluxes of K⁺ and Ca²⁺ from root segments of both wheat, Triticunaestivum L. cv. Capelle and mung bean, Vigna radiata (L.) Wilczek,were measured in the presence or absence of 20 mol m^–3para-fluorophenylalanine (p-FPA). The results were used to estimatethe compartment contents and transmembrane K⁺ and Ca²⁺ fluxesin root cortex cells. Using the Ussing-Teorell flux equationas the criterion, it was concluded that entry of K⁺ from theoutside solution to the cytoplasm, and from the cytoplasm tothe vacuole were active in both wheat and mung bean. Also, inboth species, Ca²⁺ entered the cytoplasm passively across theplasmalemma and was actively pumped back to the external solution.However, interpretation of the direction of active transportacross the tonoplast depends upon an assumption about Ca²⁺ activityin the cytoplasm. The only qualitative effect of p-FPA was to alter the drivingforce for K⁺ influx, across the plasmalemma in wheat, from anactive to a passive one. Quantitative effects of the analoguewere seen for K⁺ fluxes in both wheat and mung bean and forCa²⁺ fluxes in wheat. The p-FPA reduced transport of K⁺ in bothspecies, while transport of Ca²⁺ was unaffected. The implicationsof these results for the ‘two pump hypothesis’ arediscussed. Key words: Triticum aestivum, Vigna radiata, Two pump hypothesis     

Functional significance of channels and transporters expressed in the inner ear and kidney

A number of ion channels and transporters are expressed in both the inner ear and kidney. In the inner ear, K⁺ cycling and endolymphatic K⁺, Na⁺, Ca²⁺, and pH homeostasis are critical for normal organ function. Ion channels and transporters involved in K⁺ cycling include K⁺ channels, Na⁺-2Cl^–-K⁺ cotransporter, Na⁺/K⁺-ATPase, Cl^– channels, connexins, and K⁺/Cl^– cotransporters. Furthermore, endolymphatic Na⁺ and Ca²⁺ homeostasis depends on Ca²⁺-ATPase, Ca²⁺ channels, Na⁺ channels, and a purinergic receptor channel. Endolymphatic pH homeostasis involves H⁺-ATPase and Cl^–/HCO₃^– exchangers including pendrin. Defective connexins (GJB2 and GJB6), pendrin (SLC26A4), K⁺ channels (KCNJ10, KCNQ1, KCNE1, and KCNMA1), Na⁺-2Cl^–-K⁺ cotransporter (SLC12A2), K⁺/Cl^– cotransporters (KCC3 and KCC4), Cl^– channels (BSND and CLCNKA + CLCNKB), and H⁺-ATPase (ATP6V1B1 and ATPV0A4) cause hearing loss. All these channels and transporters are also expressed in the kidney and support renal tubular transport or signaling. The hearing loss may thus be paralleled by various renal phenotypes including a subtle decrease of proximal Na⁺-coupled transport (KCNE1/KCNQ1), impaired K⁺ secretion (KCNMA1), limited HCO₃^– elimination (SLC26A4), NaCl wasting (BSND and CLCNKB), renal tubular acidosis (ATP6V1B1, ATPV0A4, and KCC4), or impaired urinary concentration (CLCNKA). Thus, defects of channels and transporters expressed in the kidney and inner ear result in simultaneous dysfunctions of these seemingly unrelated organs. cochlea; vestibular labyrinth; stria vascularis; deafness; renal tubule     

Na(+) entry via store-operated channels modulates Ca(2+) signaling in arterial myocytes

In manynonexcitable cells, hormones and neurotransmitters activateNa⁺ influx and mobilizeCa²⁺ from intracellular stores.The stores are replenished by Ca²⁺influx via "store-operated"Ca²⁺ channels (SOC). The mainroutes of Na⁺ entry in these cellsare unresolved, and no role forNa⁺ in signaling has beenrecognized. We demonstrate that the SOC are a majorNa⁺ entry route in arterialmyocytes. Unloading of the Ca²⁺stores with cyclopiazonic acid (a sarcoplasmic reticulumCa²⁺ pump inhibitor) and caffeineinduces a large externalNa⁺-dependent rise in thecytosolic Na⁺ concentration. Onecomponent of this rise in cytosolicNa⁺ concentration is likely due toNa⁺/Ca²⁺exchange; it depends on elevation of cytosolicCa²⁺ and is insensitive to 10 mMMg²⁺ and 10 µMLa³⁺. Another component isinhibited by Mg²⁺ andLa³⁺, blockers of SOC; thiscomponent persists in cells preloaded with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid to buffer Ca²⁺ transients andpreventNa⁺/Ca²⁺exchange-mediated Na⁺ entry. ThisNa⁺ entry apparently is mediatedby SOC. The Na⁺ entry influencesNa⁺ pump activity andNa⁺/Ca²⁺exchange and has unexpectedly large effects on cell-wideCa²⁺ signaling. The SOC pathwaymay be a general mechanism by which Na⁺ participates in signaling inmany types of cells.

     

Characterization of ATPase Activity Associated with Plasma Membrane from Vigna Hypocotyls

A plasma membrane fraction was isolated from the hypocotylsof cowpea {Vigna unguiculata) by a combination of differentialcentrifugation and sucrose density gradient centrifugation.The ATPase activity of this fraction was dependent on divalentcations (Mn²⁺>Mg²⁺>Co²⁺>Ca²⁺>Fe²⁺>Zn²⁺>Ni²⁺)but was not further stimulated by monovalent cations (K⁺ and/orNa⁺). The pH optimum for the activation of ATPase by Mg²⁺ was7.0. This fraction hydrolyzed ATP or UTP as a substrate andthe ATPase activity obeyed a Michaelis-Menten type of kinetics.The K_m for MgATP ranged from 0.65 to 1.1 mM. The ATPase activitywas inhibited by inhibitors such as N, N'- dicyclohexylcarbodiimide,diethylstilbestrol and triphenyltin chloride, all of which arereported to block proton (H⁺) transport in plant cells, butwas insensitive to those of mitochondrial ATPase such as oligomycinand sodium azide. The ATPase activity was not stimulated bytreatment with ionophores (e.g., carbonyl cyanide p-trifluoromethoxyphenylhydrazone,3,5-di-ter-butyl-4-hydroxybenzilidenemalononitrile and valinomycin⁺KCl)which would be expected to dissipate the electrochemical potentialdifference of H⁺ or the membrane potential difference. The characteristics of the ATPase are compared with those ofplasma membrane ATPases of other plants and its possible rolein H⁺-transport is discussed. ¹ Present address: Institute of Applied Biochemistry, Yagi MemorialPark, Mitake, Gifu 505-01, Japan or Laboratory for Plant EcologicalStudies, Faculty of Science, Kyoto University, Kyoto 606, Japan. (Received April 20, 1984; Accepted August 14, 1984)     

sgk: an essential convergence point for peptide and steroid hormone regulation of ENaC-mediated Na+ transport

To studythe role of sgk (serum, glucocorticoid-induced kinase) inhormonal regulation of Na⁺ transport mediated by theepithelial Na⁺ channel (ENaC), clonal cell lines stablyexpressing human sgk, an S422A sgk mutant, or aD222A sgk mutant were created in the background of the A6model renal epithelial cell line. Expression of normal sgkresults in a 3.5-fold enhancement of basal transport and potentiationof the natriferic response to antidiuretic hormone (ADH). Transfectionof a S422A mutant form of sgk, which cannot bephosphorylated by phosphatidylinositol-dependent kinase (PDK)-2, results in a cell line that is indistinguishable from the parent linein basal and hormone-stimulated Na⁺ transport. The D222Asgk mutant, which lacks kinase activity, functions as adominant-negative mutant inhibiting basal as well as peptide- andsteroid hormone-stimulated Na⁺ transport. Thussgk activity is necessary for ENaC-mediated Na⁺transport. Phosphorylation and activation by PDK-2 are necessary forsgk stimulation of ENaC. Expression of normal sgkover endogenous levels results in a potentiated natriferic response toADH, suggesting that the enzyme is a rate-limiting step for the hormoneresponse. In contrast, sgk does not appear to be therate-limiting step for the cellular response to aldosterone or insulin.

     

Time-dependent stimulation by aldosterone of blocker-sensitive ENaCs in A6 epithelia

To study and define the early time-dependent response (6 h) ofblocker-sensitive epithelial Na⁺channels (ENaCs) to stimulation ofNa⁺ transport by aldosterone, weused a new modified method of blocker-induced noise analysis todetermine the changes of single-channel current (i_Na) channel open probability(P_o), andchannel density(N_T) undertransient conditions of transport as measured by macroscopic short-circuit currents(I_sc). In threegroups of experiments in which spontaneous baseline rates of transportaveraged 1.06, 5.40, and 15.14 µA/cm², stimulation of transportoccurred due to increase of blocker-sensitive channels.N_T variedlinearly over a 70-fold range of transport (0.5-35µA/cm²). Relatively small andslow time-dependent but aldosterone-independent decreases ofP_o occurredduring control (10-20% over 2 h) and aldosterone experimentalperiods (10-30% over 6 h). When theP_o of control andaldosterone-treated tissues was examined over the 70-fold extendedrange of Na⁺ transport,P_o was observedto vary inversely withI_sc, falling from~0.5 to ~0.15 at the highest rates ofNa⁺ transport or ~25% per3-fold increase of transport. Because decreases ofP_o from anysource cannot explain stimulation of transport by aldosterone, it isconcluded that the early time-dependent stimulation ofNa⁺ transport in A6 epithelia isdue exclusively to increase of apical membraneN_T.

     

Effects of Some Amino Acid Analogues on the Uptake and Transport of K+ and Ca2+ in Wheat and Mung Bean Seedlings: II. UPTAKE AND TRANSLOCATION IN WHOLE SEEDLING PLANTS

The effects of some amino acid analogues (o-, m- and p-fluorophenylalanineand azetidine-2-carboxylic acid) on uptake of⁴²K and ⁴⁵Ca intothe roots and transport to the shoots of whole wheat and mungbean seedlings were measured. The effect of each analogue oneither K⁺ or Ca²⁺ movement could be placed into one of fourcategories: (1) No effect on either ion uptake or transport;(2) No effect on ion uptake, but a reduction in transport; (3)Similar reductions in ion uptake and transport; (4) A relativelygreater reduction in ion transport than in uptake. At leasttwo independent sites of protein involvement in ion movementwere required to account for all four types of analogue effectobserved; one site of protein involvement was probably at theplasmalemma of root cortex cells and the second site, involvinga protein that turned over more quickly, was within the stele.Some evidence was found that Ca²⁺ transport is a passive process.Light did not stimulate uptake. Key words: Triticum aestivum, Vigna radiata, Two pump hypothesis