Characteristics of L-lactic acid transport in basal membrane vesicles of human placental syncytiotrophoblast

Thecharacteristics of L-lactic acid transport across thetrophoblast basal membrane were investigated and compared with those across the brush-border membrane by using membrane vesicles isolated from human placenta. The uptake ofL-[¹⁴C]lactic acid into basal membranevesicles was Na⁺ independent, and an uphill transport wasobserved in the presence of a pH gradient([H⁺]_out > [H⁺]_in).L-[¹⁴C]lactic acid uptake exhibitedsaturation kinetics with a K_m value of 5.89 ± 0.68 mM in the presence of a pH gradient.p-Chloromercuribenzenesulfonate and-cyano-4-hydroxycinnamate inhibited the initial uptake, whereas phloretin or 4,4'-diisothiocyanostilbene-2,2'-disulfonate did not.Mono- and dicarboxylic acids suppressed the initial uptake. Inconclusion, L-lactic acid transport in the basal membraneis H⁺ dependent and Na⁺ independent, as is alsothe case for the brush-border membrane transport, and itscharacteristics resemble those of monocarboxylic acid transporters.However, there were several differences in the effects of inhibitorsbetween basal and brush-border membrane vesicles, suggesting that thetransporter(s) involved in L-lactic acid transport in thebasal membrane of placental trophoblast may differ from those in thebrush-border membrane.

     

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.

     

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     

L-Carnitine transport in mouse renal and intestinal brush-border and basolateral membrane vesicles

We characterized the uptake of carnitine in brush-border membrane (BBM) and basolateral membrane (BLM) vesicles, isolated from mouse kidney and intestine. In kidney, carnitine uptake was Na⁺-dependent, showed a definite overshoot and was saturable for both membranes, but for intestine, it was Na⁺-dependent only in BLM. The uptake was temperature-dependent in BLM of both kidney and intestine. The BBM transporter in kidney had a high affinity for carnitine: apparent K_m=18.7 μM; V_max=7.85 pmol/mg protein/s. In kidney BLM, similar characteristics were obtained: apparent K_m=11.5 μM and V_max=3.76 pmol/mg protein/s. The carnitine uptake by both membranes was not affected within the physiological pH 6.5-8.5. Tetraethylammonium, verapamil, valproate and pyrilamine significantly inhibited the carnitine uptake by BBM but not by BLM. By Western blot analysis, the OCTN2 (a Na⁺-dependent high-affinity carnitine transporter) was localized in the kidney BBM, and not in BLM. Strong OCTN2 expression was observed in kidney and skeletal muscle, with no expression in intestine in accordance with our functional study. We conclude that different polarized carnitine transporters exist in kidney BBM and BLM. L-Carnitine uptake by mouse renal BBM vesicles involves a carrier-mediated system that is Na⁺-dependent and is inhibited significantly by specific drugs. The BBM transporter is likely to be OCTN2 as indicated by a strong reactivity with the anti-OCTN2 polyclonal antibody.     

Plant Growth in Relation to the Supply and Uptake of NO3-: A Comparison Between Relative Addition Rate and External Concentration as Driving Variables

Two approaches to quantifying relationships between nutrientsupply and plant growth were compared with respect to growth,partitioning, uptake and assimilation of NO₃^– by non-nodulatedpea (Pisum sativum L. cv. Marma). Plants grown in flowing solutionculture were supplied with NO₃^– at relative addition rates(RAR) of 0·03, 0·06, 0·12, and 0·18d^–1, or constant external concentrations ([NO₃^–)of 3, 10, 20, and 100 mmol m^–3 over 19 d. Following acclimation,relative growth rates (RGR)approached the corresponding RARbetween 0·03–0.12 d^-1, although growth was notlimited by N supply at RAR =0.18 d^-1. Growth rates showed littlechange with [NO₃–] between 10–100 mmol m^–3(RGR=0·15 –0·16 d^-1). The absence of growthlimitation over this range was suggested by high unit absorptionrates of NO₃^–, accumulation of NO₃^– in tissues andprogressive increases in shoot: root ratio. Rates of net uptakeof NO₃^– from 1 mol m^–3 solutions were assessed relativeto the growth-related requirement for NO₃^–, showing thatthe relative uptake capacity increased with RGR between 0·03–0·06d^–1 , but decreased thereafter to a theoretical minimumvalue at RGR     

Ca2+ influx through alpha1S DHPR may play a role in regulating Ca2+ release from RyR1 in skeletal muscle

Skeletal muscle fiber types differ in their contents of total phosphate, which includes inorganic phosphate (P_i) and high-energy organic pools of ATP and phosphocreatine (PCr). At steady state, uptake of P_i into the cell must equal the rate of efflux, which is expected to be a function of intracellular P_i concentration. We measured ³²P-labeled P_i uptake rates in different muscle fiber types to determine whether they are proportional to cellular P_i content. P_i uptake rates in isolated, perfused rat hindlimb muscles were linear over time and highest in soleus (2.42 ± 0.17 µmol·g^–1·h^–1), lower in red gastrocnemius (1.31 ± 0.11 µmol·g^–1·h^–1), and lowest in white gastrocnemius (0.49 ± 0.06 µmol·g^–1·h^–1). Reasonably similar rates were obtained in vivo. P_i uptake rates at plasma P_i concentrations of 0.3–1.7 mM confirm that the P_i uptake process is nearly saturated at normal plasma P_i levels. P_i uptake rate correlated with cellular P_i content (r = 0.99) but varied inversely with total phosphate content. Sodium-phosphate cotransporter (PiT-1) protein expression in soleus and red gastrocnemius were similar to each other and seven- to eightfold greater than PiT-1 expression in white gastrocnemius. That the PiT-1 expression pattern did not match the pattern of P_i uptake across fiber types implies that other factors are involved in regulating P_i uptake in skeletal muscle. Furthermore, fractional turnover of the cellular P_i pool (0.67, 0.57, and 0.33 h^–1 in soleus, red gastrocnemius, and white gastrocnemius, respectively) varies among fiber types, indicating differential management of intracellular P_i, likely due to differences in resistance to P_i efflux from the fiber. inorganic phosphate; sodium-inorganic phosphate transporters; PiT-2; inorganic phosphate efflux     

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     

Impaired trafficking of choline transporter-like protein-1 at plasma membrane and inhibition of choline transport in THP-1 monocyte-derived macrophages

The present study investigates choline transport processes and regulation of choline transporter-like protein-1 (CTL1) in human THP-1 monocytic cells and phorbol myristate 13-acetate (PMA)-differentiated macrophages. Choline uptake is saturable and therefore protein-mediated in both cell types, but its transport characteristics change soon after treatments with PMA. The maximal rate of choline uptake intrinsic to monocytic cells is greatly diminished in differentiated macrophages as demonstrated by alterations in V_max values from 1,973 ± 118 to 380 ± 18 nmol·mg^–1·min^–1, when the binding affinity did not change significantly (K_m values 56 ± 8 and 53 ± 6 µM, respectively). Treatments with hemicholinim-3 effectively inhibit most of the choline uptake, establishing that a choline-specific transport protein rather than a general transporter is responsible for the observed kinetic parameters. mRNA screening for the expression of various transporters reveals that CTL1 is the most plausible candidate that possesses the described kinetic and inhibitory properties. Fluorescence-activated cell sorting analyses at various times after PMA treatments further demonstrate that the disappearance of CTL1 protein from the cell surface follows the same trend as the reduction in choline uptake. Importantly, the loss of functional CTL1 from the cell surface occurs without significant changes in total CTL1 protein or its mRNA level indicating that an impaired CTL1 trafficking is the key contributing factor to the reduced choline uptake, subsequent to the PMA-induced THP-1 differentiation to macrophages. protein trafficking     

Nitrogen-13 Studies of Nitrate Fluxes in Barley Roots: II. EFFECT OF PLANT N-STATUS ON THE KINETIC PARAMETERS OF NITRATE INFLUX

Influx of nitrate into the roots of intact barley plants wasfollowed over periods of 1–15 min using nitrogen-13 asa tracer. Based on measurements taken over 15 min from a rangeof external nitrate concentrations (0·2–250 mmolm^–3), the kinetic parameters of influx, I_max and K_m, werecalculated. Compared with plants grown in the presence of nitrate throughout,plants that had been starved of N for 3 d showed a significantlygreater value ofI_max for ¹³N-nitrate influx (by a factor of1·4–1·8), but a similar value of K_m (12–14mmol m^–3). Pre-treating N-starved plants with nitratefor about 5 h further increased the subsequent rate of ¹³N-nitrateinflux, but had little effect in the unstarved controls. Allowingfor this induction of additional nitrate transport, the differencein rates of nitrate influx in control and N-starved plants wassufficient to account for the previously-observed differencein net uptake by the two groups of plants. In barley plants grown without any exposure to nitrate, butwith ammonium as N-source, both I_max and K_m for subsequent ¹³N-nitrateinflux were significantly decreased (by about one-half) comparedwith the corresponding nitrate-grown controls. The importance of changes in the rate of influx in the regulationof net uptake of nitrate is discussed. Key words: Ion transport, nitrate, influx, kinetic parameters, N-deficiency     

Chloride Transport in Chara corallina and the Electrochemical Potential Difference for Hydrogen Ions

The pH of the cytoplasm of Chara corallina cells has been measuredwith the weak acid 5,5-dimethyloxazolidine-2,4-dione (DM0).Over an external pH range 4·5–9·5 the resultsfit the regression equation pH_cytoplasm=6·28+0·22pH_out. Using measured values of the electric potential difference acrossthe plasmalemma we have calculated the electrochemical potentialdifference across this membrane for H⁺ and Cl^–. Thesedata are used to test the hypothesis that the inward transportof Cl^– is coupled to the inthix of H⁺ or, which comesto the same thing, efflux of OH^–. One-for-one couplingwill not give net Cl^– uptake from solutions with pH greaterthan about 7·2, unless the cytoplasmic Cl^– concentrationis lower than 10 mM, or the pH just outside the membrane islower than that in the bulk solution. It is shown that net Cl^–uptake proceeds from solutions with pH up to 9. The alternative possibility is that Cl^– transport is broughtabout by co-transport of two H⁺ for each Cl^–; this isnot ruled out by the results reported. Such a mechanism mightbe detectable by its electrogenic effect: although such effectshave not been detected, it is shown that they would be smallunder most conditions. Other possible mechanisms are discussed.     

Dexamethasone treatment causes resistance to insulin-stimulated cellular potassium uptake in the rat

Patients treated with glucocorticoids have elevated skeletal muscle ouabain binding sites. The major Na⁺-K⁺-ATPase (NKA) isoform proteins found in muscle, ₂ and ₁, are increased by 50% in rats treated for 14 days with the synthetic glucocorticoid dexamethasone (DEX). This study addressed whether the DEX-induced increase in the muscle NKA pool leads to increased insulin-stimulated cellular K⁺ uptake that could precipitate hypokalemia. Rats were treated with DEX or vehicle via osmotic minipumps at one of two doses: 0.02 mg·kg^–1·day^–1 for 14 days (low DEX; n = 5 pairs) or 0.1 mg·kg^–1·day^–1 for 7 days (high DEX; n = 6 pairs). Insulin was infused at a rate of 5 mU·kg^–1·min^–1 over 2.5 h in conscious rats. Insulin-stimulated cellular K⁺ and glucose uptake rates were assessed in vivo by measuring the exogenous K⁺ infusion () and glucose infusion (G_inf) rates needed to maintain constant plasma K⁺ and glucose concentrations during insulin infusion. DEX at both doses decreased insulin-stimulated glucose uptake as previously reported. G_inf (in mmol·kg^–1·h^–1) was 10.2 ± 0.6 in vehicle-treated rats, 5.8 ± 0.8 in low-DEX-treated rats, and 5.2 ± 0.6 in high-DEX-treated rats. High DEX treatment also reduced insulin-stimulated K⁺ uptake. (in mmol·kg^–1·h^–1) was 0.53 ± 0.08 in vehicle-treated rats, 0.49 ± 0.14 in low-DEX-treated rats, and 0.27 ± 0.08 in high-DEX-treated rats. DEX treatment did not alter urinary K⁺ excretion. NKA ₂-isoform levels in the low-DEX-treated group, measured by immunoblotting, were unchanged, but they increased by 38 ± 15% (soleus) and by 67 ± 3% (gastrocnemius) in the high-DEX treatment group. The NKA ₁-isoform level was unchanged. These results provide novel evidence for the insulin resistance of K⁺ clearance during chronic DEX treatment. Insulin-stimulated cellular K⁺ uptake was significantly depressed despite increased muscle sodium pump pool size. skeletal muscle; sodium pump; Na⁺-K⁺-ATPase     

Rubidium Transport in Membrane Vesicles from the Halophyte Suaeda maritima

The uptake and efflux of Rb⁺ by membrane vesicles isolated fromshoots of the halophyte Suaeda maritima have been investigated.Uptake came to an apparent equilibrium after 1 h and the initialrate of uptake was considerably slower than that reported forbacterial membrane vesicles Additions of ATP reduced both Rb⁺uptake and the half-time for loss in efflux experiments, althoughthis effect was not specific for ATP and probably was not associatedwith energy transfer The permeability coefficient for Rb⁺ wascalculated to be between 0 1 and 0 3 x 10^–2 cm s^–1.The value of membrane vesicles in ion transport studies in plantsis discussed. Suaeda maritima, seablite, halophyte, membrane vesicles, ion transport, rubidium     

Na(+)-coupled transport of L-carnitine via high-affinity carnitine transporter OCTN2 and its subcellular localization in kidney

The mechanism of Na(+)-dependent transport of L-carnitine via the carnitine/organic cation transporter OCTN2 and the subcellular localization of OCTN2 in kidney were studied. Using plasma membrane vesicles prepared from HEK293 cells that were stably transfected with human OCTN2, transport of L-carnitine via human OCTN2 was characterized. Uptake of L-[(3)H]carnitine by the OCTN2-expressing membrane vesicles was significantly increased in the presence of an inwardly directed Na(+) gradient, with an overshoot, while such transient uphill transport was not observed in membrane vesicles from cells that were mock transfected with expression vector pcDNA3 alone. The uptake of L-[(3)H]carnitine was specifically dependent on Na(+) and the osmolarity effect showed that Na(+) significantly influenced the transport rather than the binding. Changes of inorganic anions in the extravesicular medium and of membrane potential by valinomycin altered the initial uptake activity of L-carnitine by OCTN2. In addition, the fluxes of L-carnitine and Na(+) were coupled with 1:1 stoichiometry. Accordingly, it was clarified that Na(+) is coupled with flux of L-carnitine and the flux is an electrogenic process. Furthermore, OCTN2 was localized on the apical membrane of renal tubular epithelial cells. These results clarified that OCTN2 is important for the concentrative reabsorption of L-carnitine after glomerular filtration in the kidney.     

Characterization of regulatory mechanisms and states of human organic cation transporter 2

Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37°C. After subtraction of unspecific uptake determined in WT at 37°C or in hOCT2 at 8°C saturable specific uptake of both substrates was measured. K_m values of hOCT2-mediated uptake of 95 µM amiloride and 24 µM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC₅₀ (in µM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca²⁺/CaM complex (–55 ± 5%, n = 10 and –63 ± 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (–54 ± 5%, n = 14, and –31 ± 6%, n = 26) and PKA (–16 ± 5%, n = 16, and –18 ± 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 ± 6%, n = 8, and +55 ± 17%, n = 16). Inhibition of Ca²⁺/CaM complex resulted in a significant decrease of V_max (160–99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (–22 ± 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca²⁺/CaM complex causes changes in transport capacity via hOCT2 trafficking. organic cation transport; fluorescence measurement; 4-[4-(dimethylamino)-styryl]-n-methylpyridinium; amiloride     

L-Carnitine transport in mouse renal and intestinal brush-border and basolateral membrane vesicles.

We characterized the uptake of carnitine in brush-border membrane (BBM) and basolateral membrane (BLM) vesicles, isolated from mouse kidney and intestine. In kidney, carnitine uptake was Na(+)-dependent, showed a definite overshoot and was saturable for both membranes, but for intestine, it was Na(+)-dependent only in BLM. The uptake was temperature-dependent in BLM of both kidney and intestine. The BBM transporter in kidney had a high affinity for carnitine: apparent K(m)=18.7 microM; V(max)=7.85 pmol/mg protein/s. In kidney BLM, similar characteristics were obtained: apparent K(m)=11.5 microM and V(max)=3.76 pmol/mg protein/s. The carnitine uptake by both membranes was not affected within the physiological pH 6.5-8.5. Tetraethylammonium, verapamil, valproate and pyrilamine significantly inhibited the carnitine uptake by BBM but not by BLM. By Western blot analysis, the OCTN2 (a Na(+)-dependent high-affinity carnitine transporter) was localized in the kidney BBM, and not in BLM. Strong OCTN2 expression was observed in kidney and skeletal muscle, with no expression in intestine in accordance with our functional study. We conclude that different polarized carnitine transporters exist in kidney BBM and BLM. L-Carnitine uptake by mouse renal BBM vesicles involves a carrier-mediated system that is Na(+)-dependent and is inhibited significantly by specific drugs. The BBM transporter is likely to be OCTN2 as indicated by a strong reactivity with the anti-OCTN2 polyclonal antibody.     

Guanine nucleotide transport by atractyloside-sensitive and -insensitive carriers in isolated heart mitochondria

Inprevious work (McKee EE, Bentley AT, Smith RM Jr, and Ciaccio CE,Biochem Biophys Res Commun 257: 466-472, 1999), the transport of guanine nucleotides into the matrix of intact isolated heart mitochondria was demonstrated. In this study, the time course andmechanisms of guanine nucleotide transport are characterized. Twodistinct mechanisms of transport were found to be capable of movingguanine nucleotides across the inner membrane. The first carrier wassaturable, displayed temperature dependence, preferred GDP to GTP, anddid not transport GMP or IMP. When incubated in the absence ofexogenous ATP, this carrier had a V_max of946 ± 53 pmol · mg¹ · min¹ with aK_m of 2.9 ± 0.3 mM for GDP. However,transport of GTP and GDP on this carrier was completely inhibited byphysiological concentrations of ATP, suggesting that this carrier wasnot involved with guanine nucleotide transport in vivo. Becausetransport on this carrier was also inhibited by atractyloside, thiscarrier was consistent with the well-characterized ATP/ADP translocase. The second mechanism of guanine nucleotide uptake was insensitive toatractyloside, displayed temperature dependence, and was capable oftransporting GMP, GDP, and GTP at approximately equal rates but did nottransport IMP, guanine, or guanosine. GTP transport via this mechanismwas slow, with a V_max of 48.7 ± 1.4 pmol · mg¹ · min¹ and aK_m = 4.4 ± 0.4 mM. However, becausethe requirement for guanine nucleotide transport is low in nondividingtissues such as the heart, this transport process is neverthelesssufficient to account for the matrix uptake of guanine nucleotides andmay represent the physiological mechanism of transport.

     

Three distinct mechanisms of HCO3- secretion in rat distal colon

HCO₃^– secretion has long been recognized in the mammalian colon, but it has not been well characterized. Although most studies of colonic HCO₃^– secretion have revealed evidence of lumen Cl^– dependence, suggesting a role for apical membrane Cl^–/HCO₃^– exchange, direct examination of HCO₃^– secretion in isolated crypt from rat distal colon did not identify Cl^–-dependent HCO₃^– secretion but did reveal cAMP-induced, Cl^–-independent HCO₃^– secretion. Studies were therefore initiated to determine the characteristics of HCO₃^– secretion in isolated colonic mucosa to identify HCO₃^– secretion in both surface and crypt cells. HCO₃^– secretion was measured in rat distal colonic mucosa stripped of muscular and serosal layers by using a pH stat technique. Basal HCO₃^– secretion (5.6 ± 0.03 µeq·h^–1·cm^–2) was abolished by removal of either lumen Cl^– or bath HCO₃^–; this Cl^–-dependent HCO₃^– secretion was also inhibited by 100 µM DIDS (0.5 ± 0.03 µeq·h^–1·cm^–2) but not by 5-nitro-3-(3-phenylpropyl-amino)benzoic acid (NPPB), a Cl^– channel blocker. 8-Bromo-cAMP induced Cl^–-independent HCO₃^– secretion (and also inhibited Cl^–-dependent HCO₃^– secretion), which was inhibited by NPPB and by glibenclamide, a CFTR blocker, but not by DIDS. Isobutyrate, a poorly metabolized short-chain fatty acid (SCFA), also induced a Cl^–-independent, DIDS-insensitive, saturable HCO₃^– secretion that was not inhibited by NPPB. Three distinct HCO₃^– secretory mechanisms were identified: 1) Cl^–-dependent secretion associated with apical membrane Cl^–/HCO₃^– exchange, 2) cAMP-induced secretion that was a result of an apical membrane anion channel, and 3) SCFA-dependent secretion associated with an apical membrane SCFA/HCO₃^– exchange. chloride/bicarbonate exchange; short-chain fatty acid/bicarbonate exchange; anion channel; pH stat     

Excitable Active Electrogenic Ion-Transport Units in the Plasmalemma of Nitellopsis obtusa

With the use of voltage clamp and current clamp techniques thesupposition was proved that during the hyperpolarizing response(HR) N. obtusa cells generate active electromotive force (emf)at the expense of metabolic energy. Threshold inward currentsent through the plasmalemma of the cell which was depolarizedwith 100 mol m^–3 KG resulted in the HR with the transferof the membrane's excitable units from the high-conductive stateto the low-conductive state. During the HR the membrane potentialV_m increased from –135±10 mV to –290±15mV, the membrane resistance increased from 3.3±1.5 kOhmcm² to 5.8±1.2 kOhm cm² and the membrane emf E_m increasedfrom –20±4 mV to –93± 15 mV. Changesin the external concentration of K^–, Na⁺, Cl^– andH^– did not affect the patterns of HR. Cells which weredepolarized by light also generated HR (in normal medium) whichwas accompanied with the increase of V_m, R_m and E_m. The highvalue of Em generated during the HR can be explained only withthe involvement of active electrogenic charge transfer acrossthe membrane. 0.05 mol m^–3 DCCD added to the externalmedium inhibited the HR in both cases. Key words: Active ion transport, Hyperpolarizing response, Nitellopsis obtusa     

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     

The Carbon Economy of Rubus chamaemorus L. II. Respiration

Respiratory activity and seasonal changes in carbohydrate contentof the storage organs of Rubus chamaemorus L. have been investigated.Leaf dark respiration rate increases in a non-linear mannerfrom 0·7 mg CO₂ evolved dm^–2 h^–1 at 0 °Cto 4·6 rng CO₂ evolved dm^–2 hh^–1 at 30 °C.Root and rhizome respiration rates increase from 1 µ1O₂ uptake g^–1 fresh weight h^–1 at 0.7 ° C to10 µ10, uptake g^–1 f. wt h^–1 at 20 °C.Rhizome carbohydrate reserves decline from a September peakof 33 per cent alcohol insoluble d. wt to 16 per cent in May. The circumpolar distribution of R. chamaemorus is discussedin relation to the evidence presented here and in the precedingpaper of the series.