L-Carnitine transport in human placental brush-border membranes is mediated by the sodium-dependent organic cation transporter OCTN2

Maternofetal transport of L-carnitine, a molecule that shuttles long-chain fatty acids to the mitochondria for oxidation, is thought to be important in preparing the fetus for its lipid-rich postnatal milk diet. Using brush-border membrane (BBM) vesicles from human term placentas, we showed that L-carnitine uptake was sodium and temperature dependent, showed high affinity for carnitine (apparent K_m = 11.09 ± 1.32 µM; V_max = 41.75 ± 0.94 pmol·mg protein^–1·min^–1), and was unchanged over the pH range from 5.5 to 8.5. L-Carnitine uptake was inhibited in BBM vesicles by valproate, verapamil, tetraethylammonium, and pyrilamine and by structural analogs of L-carnitine, including D-carnitine, acetyl-D,L-carnitine, and propionyl-, butyryl-, octanoyl-, isovaleryl-, and palmitoyl-L-carnitine. Western blot analysis revealed that OCTN2, a high-affinity, Na⁺-dependent carnitine transporter, was present in placental BBM but not in isolated basal plasma membrane vesicles. The reported properties of OCTN2 resemble those observed for L-carnitine uptake in placental BBM vesicles, suggesting that OCTN2 may mediate most maternofetal carnitine transport in humans. membrane transport; valproate; maternofetal; xenobiotics; acylcarnitine     

Na+-dependent HCO3- uptake into the rat choroid plexus epithelium is partially DIDS sensitive

Several studies suggest the involvement of Na⁺ and HCO₃^– transport in the formation of cerebrospinal fluid. Two Na⁺-dependent HCO₃^– transporters were recently localized to the epithelial cells of the rat choroid plexus (NBCn1 and NCBE), and the mRNA for a third protein was also detected (NBCe2) (Praetorius J, Nejsum LN, and Nielsen S. Am J Physiol Cell Physiol 286: C601–C610, 2004). Our goal was to immunolocalize the NBCe2 to the choroid plexus by immunohistochemistry and immunogold electronmicroscopy and to functionally characterize the bicarbonate transport in the isolated rat choroid plexus by measurements of intracellular pH (pH_i) using a dual-excitation wavelength pH-sensitive dye (BCECF). Both antisera derived from COOH-terminal and NH₂-terminal NBCe2 peptides localized NBCe2 to the brush-border membrane domain of choroid plexus epithelial cells. Steady-state pH_i in choroidal cells increased from 7.03 ± 0.02 to 7.38 ± 0.02 (n = 41) after addition of CO₂/HCO₃^– into the bath solution. This increase was Na⁺ dependent and inhibited by the Cl^– and HCO₃^– transport inhibitor DIDS (200 µM). This suggests the presence of Na⁺-dependent, partially DIDS-sensitive HCO₃^– uptake. The pH_i recovery after acid loading revealed an initial Na⁺ and HCO₃^–-dependent net base flux of 0.828 ± 0.116 mM/s (n = 8). The initial flux in the presence of CO₂/HCO₃^– was unaffected by DIDS. Our data support the existence of both DIDS-sensitive and -insensitive Na⁺- and HCO₃^–-dependent base loader uptake into the rat choroid plexus epithelial cells. This is consistent with the localization of the three base transporters NBCn1, Na⁺-driven Cl^– bicarbonate exchanger, and NBCe2 in this tissue. bicarbonate metabolism; BCECF; cerebrospinal fluid; acid/base transport; ammonium prepulse     

Mechanism of thiamine uptake by human jejunal brush-border membrane vesicles

Thiamine, a water-soluble vitamin, is essential fornormal cellular functions, growth and development. Thiamine deficiency leads to significant clinical problems and occurs under a variety ofconditions. To date, however, little is known about the mechanism ofthiamine absorption in the native human small intestine. The objectiveof this study was, therefore, to characterize the mechanism of thiaminetransport across the brush-border membrane (BBM) of human smallintestine. With the use of purified BBM vesicles (BBMV) isolated fromthe jejunum of organ donors, thiamine uptake was found to be1) independent of Na⁺ but markedly stimulated byan outwardly directed H⁺ gradient (pH 5.5_in/pH7.5_out); 2) competitively inhibited by thecation transport inhibitor amiloride (inhibitor constant of 0.12 mM);3) sensitive to temperature and osmolarity of the incubation medium; 4) significantly inhibited by thiamine structuralanalogs (amprolium, oxythiamine, and pyrithiamine), but not byunrelated organic cations (tetraethylammonium,N-methylnicotinamide, or choline); 5) notaffected by the addition of ATP to the inside and outside of the BBMV;6) potential insensitive; and 7) saturable as afunction of thiamine concentration with an apparent Michaelis-Menten constant of 0.61 ± 0.08 µM and a maximal velocity of 1.00 ± 0.47 pmol · mg protein¹ · 10 s¹. Carrier-mediated thiamine uptake was also found inBBMV of human ileum. These data demonstrate the existence of aNa⁺-independent, pH-dependent, amiloride-sensitive,electroneutral carrier-mediated mechanism for thiamine absorption innative human small intestinal BBMV.

     

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

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

     

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.

     

Characterization of L-glutamine transport by a human neuroblastoma cell line

This study characterized theNa⁺-dependent transport of L-glutamine by ahuman neuroblastoma cell line, SK-N-SH. The Na⁺-dependentcomponent represented >95% of the total glutamine uptake. Kineticstudies showed a single saturable high-affinity carrier with aMichaelis constant (K_m) of 163 ± 23 µMand a maximum transport velocity (V_max) of13,713 ± 803 pmol · mgprotein¹ · min¹. Glutamine uptakewas markedly inhibited in the presence of L-alanine, L-asparagine, and L-serine. Li⁺ didnot substitute for Na⁺. These data show thatL-glutamine is predominantly taken up through systemASC. Glutamine deprivation resulted in the decrease of glutamine transport by a mechanism that decreasedV_max without affectingK_m. The expression of the system ASC subtypeASCT2 decreased in the glutamine-deprived group, whereas glutaminedeprivation did not induce changes in system ASC subtype ASCT1 mRNAexpression. Adaptive increases in Na⁺-dependent glutamate,Na⁺-dependent 2-(methylamino)isobutyric acid, andNa⁺-independent leucine transport were observed underglutamine-deprived conditions, which were completely blocked byactinomycin D and cycloheximide. These mechanisms may allow cells tosurvive and even grow under nutrient-deprived conditions.

     

Contribution of Na(+)-K(+)-Cl(-) cotransporter to high-[K(+)](o)- induced swelling and EAA release in astrocytes

We hypothesized that highextracellular K⁺ concentration([K⁺]_o)-mediated stimulation ofNa⁺-K⁺-Cl cotransporter isoform 1 (NKCC1) may result in a net gain of K⁺ and Cland thus lead to high-[K⁺]_o-induced swellingand glutamate release. In the current study, relative cell volumechanges were determined in astrocytes. Under 75 mM[K⁺]_o, astrocytes swelled by 20.2 ± 4.9%. This high-[K⁺]_o-mediated swelling wasabolished by the NKCC1 inhibitor bumetanide (10 µM, 1.0 ± 3.1%; P < 0.05). Intracellular³⁶Cl accumulation was increased from acontrol value of 0.39 ± 0.06 to 0.68 ± 0.05 µmol/mgprotein in response to 75 mM [K⁺]_o. Thisincrease was significantly reduced by bumetanide (P < 0.05). Basal intracellular Na⁺ concentration([Na⁺]_i) was reduced from 19.1 ± 0.8 to16.8 ± 1.9 mM by bumetanide (P < 0.05).[Na⁺]_i decreased to 8.4 ± 1.0 mM under75 mM [K⁺]_o and was further reduced to5.2 ± 1.7 mM by bumetanide. In addition, the recovery rate of[Na⁺]_i on return to 5.8 mM[K⁺]_o was decreased by 40% in the presenceof bumetanide (P < 0.05). Bumetanide inhibitedhigh-[K⁺]_o-induced ¹⁴C-labeledD-aspartate release by ~50% (P < 0.05).These results suggest that NKCC1 contributes tohigh-[K⁺]_o-induced astrocyte swelling andglutamate release.

     

Regulation of intracellular calcium in N1E-115 neuroblastoma cells: the role of Na(+)/Ca(2+) exchange

In fura 2-loaded N1E-115 cells, regulationof intracellular Ca²⁺ concentration([Ca²⁺]_i) following a Ca²⁺ loadinduced by 1 µM thapsigargin and 10 µM carbonylcyanidep-trifluoromethyoxyphenylhydrazone (FCCP) wasNa⁺ dependent and inhibited by 5 mM Ni²⁺. Incells with normal intracellular Na⁺ concentration([Na⁺]_i), removal of bath Na⁺,which should result in reversal of Na⁺/Ca²⁺exchange, did not increase [Ca²⁺]_i unlesscell Ca²⁺ buffer capacity was reduced. When N1E-115 cellswere Na⁺ loaded using 100 µM veratridine and 4 µg/mlscorpion venom, the rate of the reverse mode of theNa⁺/Ca²⁺ exchanger was apparently enhanced,since an ~4- to 6-fold increase in [Ca²⁺]_ioccurred despite normal cell Ca²⁺ buffering. In SBFI-loadedcells, we were able to demonstrate forward operation of theNa⁺/Ca²⁺ exchanger (net efflux ofCa²⁺) by observing increases (~ 6 mM) in[Na⁺]_i. These Ni²⁺ (5 mM)-inhibited increases in [Na⁺]_i could onlybe observed when a continuous ionomycin-induced influx ofCa²⁺ occurred. The voltage-sensitive dyebis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used tomeasure changes in membrane potential. Ionomycin (1 µM) depolarizedN1E-115 cells (~25 mV). This depolarization was Na⁺dependent and blocked by 5 mM Ni²⁺ and 250-500 µMbenzamil. These data provide evidence for the presence of anelectrogenic Na⁺/Ca²⁺ exchanger that is capableof regulating [Ca²⁺]_i after release ofCa²⁺ from cell stores.

     

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     

Regulation of the epithelial Na+ channel by intracellular Na+

The hypothesis that the intracellularNa⁺ concentration([Na⁺]_i)is a regulator of the epithelialNa⁺ channel (ENaC) was tested withthe Xenopus oocyte expression systemby utilizing a dual-electrode voltage clamp.[Na⁺]_iaveraged 48.1 ± 2.2 meq (n = 27)and was estimated from the amiloride-sensitive reversal potential.[Na⁺]_iwas increased by direct injection of 27.6 nl of 0.25 or 0.5 MNa₂SO₄.Within minutes of injection,[Na⁺]_istabilized and remained elevated at 97.8 ± 6.5 meq(n = 9) and 64.9 ± 4.4 (n = 5) meq 30 min after theinitial injection of 0.5 and 0.25 MNa₂SO₄,respectively. This increase of[Na⁺]_icaused a biphasic inhibition of ENaC currents. In oocytes injected with0.5 MNa₂SO₄(n = 9), a rapid decrease of inwardamiloride-sensitive slope conductance(g_Na) to 0.681 ± 0.030 of control within the first 3 min and a secondary, slowerdecrease to 0.304 ± 0.043 of control at 30 min were observed.Similar but smaller inhibitions were also observed with the injectionof 0.25 MNa₂SO₄.Injection of isotonicK₂SO₄(70 mM) or isotonicK₂SO₄made hypertonic with sucrose (70 mMK₂SO₄-1.2M sucrose) was without effect. Injection of a 0.5 M concentration ofeitherK₂SO₄,N-methyl-D-glucamine (NMDG) sulfate, or 0.75 M NMDG gluconate resulted in a much smaller initial inhibition (<14%) and little or no secondary decrease. Thusincreases of[Na⁺]_ihave multiple specific inhibitory effects on ENaC that can betemporally separated into a rapid phase that was complete within 2-3 min and a delayed slow phase that was observed between 5 and 30 min.

     

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.

     

Endogenous expression of the renal high-affinity H+-peptide cotransporter in LLC-PK1 cells

The reabsorption of filtered di- andtripeptides as well as certain peptide mimetics from the tubular lumeninto renal epithelial cells is mediated by anH⁺-coupledhigh-affinity transport process. Here we demonstrate for the first timeH⁺-coupled uptake of dipeptidesinto the renal proximal tubule cell lineLLC-PK₁. Transport was assessed1) by uptake studies using theradiolabeled dipeptideD-[³H]Phe-L-Ala,2) by cellular accumulation of the fluorescent dipeptide D-Ala-Lys-AMCA, and3) by measurement of intracellularpH (pH_i) changes as aconsequence of H⁺-coupleddipeptide transport. Uptake ofD-Phe-L-Alaincreased linearly over 11 days postconfluency and showed all thecharacteristics of the kidney cortex high-affinity peptide transporter,e.g., a pH optimum for transport ofD-Phe-L-Alaof 6.0, an apparent K_m value forinflux of 25.8 ± 3.6 µM, and affinities of differently chargeddipeptides or the -lactam antibiotic cefadroxil to the binding sitein the range of 20-80 µM.pH_i measurements established thepeptide transporter to induce pronounced intracellular acidification inLLC-PK₁ cells and confirm itspostulated role as a cellular acid loader.

     

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     

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.

     

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.

     

Palytoxin-induced cell death cascade in bovine aortic endothelial cells

The plasmalemmal Na⁺-K⁺-ATPase (NKA) pump is the receptor for the potent marine toxin palytoxin (PTX). PTX binds to the NKA and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca²⁺. However, the ability of PTX to directly increase cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) via Na⁺ pump channels and to initiate Ca²⁺ overload-induced oncotic cell death has not been examined. Thus the purpose of this study was to determine the effect of PTX on [Ca²⁺]_i and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3–100 nM) produced a graded increase in [Ca²⁺]_i that was dependent on extracellular Ca²⁺. The increase in [Ca²⁺]_i initiated by 100 nM PTX was blocked by pretreatment with ouabain with an IC₅₀ < 1 µM. The elevation in [Ca²⁺]_i could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). These results suggest that the PTX-induced rise in [Ca²⁺]_i occurs via the Na⁺ pump. Subsequent to the rise in [Ca²⁺]_i, PTX also caused a concentration-dependent increase in uptake of the vital dye ethidium bromide (EB) but not YO-PRO-1. EB uptake was also blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently expressed green fluorescent protein (molecular mass 27 kDa) and rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca²⁺ or by adding ouabain after PTX, 2) blocked by the cytoprotective amino acid glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca²⁺ overload. necrosis; vital dyes; membrane blebs; time-lapse video microscopy; fura-2     

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

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

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.

     

cAMP-sensitive endocytic trafficking in A6 epithelia

Blocker-induced noise analysis and laser scanning confocalmicroscopy were used to test the idea that cAMP-mediated vesicle exocytosis/endocytosis may be a mechanism for regulation of functional epithelial Na⁺ channels (ENaCs) at apical membranes of A6epithelia. After forskolin stimulation of Na⁺ transport andlabeling apical membranes with the fluorescent dyeN-(3-triethylammoniumpropyl)4-(6-4 diethylaminophenyl)hexatrienyl pyridinium dibromide (FM 4-64), ENaC densities(N_T) decreased exponentially (time constant~20 min) from mean values of 320 to 98 channels/cell within 55 minduring washout of forskolin. Two populations of apical membrane-labeledvesicles appeared in the cytosol within 55 min, reaching mean valuesnear 18 vesicles/cell, compared with five vesicles per cell in control,unstimulated tissues. The majority of cAMP-dependent endocytosedvesicles remained within a few micrometers of the apical membranes forthe duration of the experiments. A minority of vesicles migrated to >5µm below the apical membrane. Because steady states require identicalrates of endocytosis and exocytosis, and because forskolin increased endocytic rates by fivefold or more, cAMP/protein kinase A acts kinetically not only to increase rates of cycling of vesicles at theapical membranes, but also principally to increase exocytic rates.These observations are consistent with and support, but do not prove,that vesicle trafficking is a mechanism for cAMP-mediated regulation ofapical membrane channel densities in A6 epithelia.

     

Functional characterization of human NBC4 as an electrogenic Na+-HCO3- cotransporter (NBCe2)

We havefunctionally characterized Na⁺-driven bicarbonatetransporter (NBC)4, originally cloned from human heart by Pushkin etal. (Pushkin A, Abuladze N, Newman D, Lee I, Xu G, and Kurtz I. Biochem Biophys Acta 1493: 215-218, 2000). Of the fourNBC4 variants currently present in GenBank, our own cloning efforts yielded only variant c. We expressed NBC4c (GenBank accession no.AF293337) in Xenopus laevis oocytes and assayed membrane potential (V_m) and pH regulatory function withmicroelectrodes. Exposing an NBC4c-expressing oocyte to a solutioncontaining 5% CO₂ and 33 mM HCOelicited a large hyperpolarization, indicating that the transporter iselectrogenic. The initial CO₂-induced decrease inintracellular pH (pH_i) was followed by a slow recovery thatwas reversed by removing external Na⁺. Two-electrodevoltage clamp of NBC4c-expressing oocytes revealed largeHCO- and Na⁺-dependent currents. When wevoltage clamped V_m far from NBC4c's estimatedreversal potential (E_rev), the pH_irecovery rate increased substantially. Both the currents andpH_i recovery were blocked by 200 µM4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). We estimatedthe transporter's HCO:Na⁺ stoichiometryby measuring E_rev at different extracellularNa⁺ concentration ([Na⁺]_o)values. A plot of E_rev againstlog[Na⁺]_o was linear, with a slope of 54.8 mV/log[Na⁺]_o. This observation, as well asthe absolute E_rev values, are consistent with a2:1 stoichiometry. In conclusion, the behavior of NBC4c, which wepropose to call NBCe2-c, is similar to that of NBCe1, the firstelectrogenic NBC.