Calcium transport in intact human erthrocytes

Intact human erythrocytes can be readily loaded with calcium by incubation in hypersomotic media at alkaline pH. Erythrocyte calcium content increases from 15-20 to 120-150 nmol/g hemoglobin after incubation for 2 h at 20 degree C in a 400 mosmol/kg, pH 7.8 solution containing 100 mM sodium chloride, 90 mM tetramethylammonium chloride, 1 mM potassium chloride, and 10 mM calcium chloride. Calcium uptake is a time-dependent process that is associated with an augmented efflux of potassium. The ATP content in these cells remains at more than 60% of normal and is not affected by calcium. Calcium uptake is influenced by the cationic composition of the external media. The response to potassium is diphasic. With increasing potassium concentrations, the net accumulation of calcium initially increases, becoming maximal at 1 mM potassium, then diminishes, falling below basal levels at concentrations above 3 mM potassium. Ouabain inhibits the stimulatory effect of low concentrations of potassium. The inhibitory effects of higher concentrations of potassium are ouabain insensitive and independent of the external calcium concentration. Sodium also inhibits calcium uptake but this inhibition can be modified by altering the external concentration of calcium. The effux of calcium from loaded erythrocytes is not significantly altered by changes in osmolality, medium ion composition, or ouabain. It is concluded that hypertonicity increases the net uptake of calcium by increasing the influx of calcium and that some part of the sodium potassium transport system is involved in this influx process.     

Crisis placement

The effect of the phosphatase inhibitor calyculin A (cal A) on the kinetic parameters of the Na+-coupled taurine uptake via the taurine transporter in the Ehrlich ascites tumour cells has been investigated. Preincubation with cal A (100 nM) reduces the initial taurine influx by about 20%, but has no effect on the diffusional component of the taurine influx or on the taurine release from cells suspended in isotonic or in hypotonic medium. Thus, cal A-sensitive phosphatases only affect taurine transport mediated by the Na+-dependent taurine transporter. Cal A increases the Michaelis-Menten constant for binding of taurine to the transporter from 31+/-6 to 45+/-4 microM and reduces the taurine transport capacity from 210+/-20 to 170+/-10 nmol x g dry wt(-1) x min(-1) [corrected]. The Michaelis-Menten constant for binding of Na+ to the taurine transporter is concomitantly increased from 96+/-11 to 129+/-8 mM and the Na+:taurine coupling ratio for activation of the transport cycle is reduced from 3.3+/-0.6 to 2.4+/-0.2. This suggests that cal A-sensitive phosphatases maintain a high affinity of the taurine transporter towards Na+ and taurine as well as a high taurine transport capacity in unpertubated Ehrlich cells.     

Opioid receptors on peripheral sensory axons

Enzyme-modified amperometric microsensors have been utilized in the investigation of acetylcholine and choline diffusion in solution and choline uptake and diffusion in rat brains. A small amount of the substance of interest was introduced by pressure injection and transport to the sensor was monitored. The apparent diffusion coefficients for acetylcholine and choline in agarose gel perfused with physiological solutions were determined to be 5.2 +/- 0.7 x 10(-6) cm2/s and 6.1 +/- 0.8 x 10(-6) cm2/s, respectively. Choline transport was monitored in two brain regions: the caudate and anterior hypothalamus. The transport time of choline in the caudate was concentration dependent, but was unaffected by the presence of a competitive, high-affinity uptake inhibitor, hemicholinium-3. The apparent diffusion coefficient (D) and uptake rate (k) for choline in the caudate and anterior hypothalamus were calculated using a model for point source diffusion coupled with first-order uptake kinetics. The effect of the sensors' response time on the measurements was removed by deconvolution. The D and k were 1.8 +/- 0.1 x 10(-6) cm2/s and 2.0 +/- 0.1 x 10(-2) s-1 in the caudate and 1.9 +/- 0.1 x 10(-6) cm2/s and 3.2 +/- 0.6 x 10(-2) s-1 in the anterior hypothalamus. The reduced diffusion coefficient determined in brain tissue compared to agar gel is consistent with the increased tortuosity of the brain microenvironment. A substance in brain tissue, presumably acetylcholinesterase, prevents the use of differential measurements of acetylcholine because choline sensors became sensitive to acetylcholine.     

Lactate uptake by skeletal muscle sarcolemmal vesicles decreases after 4 wk of hindlimb unweighting in rats

We investigated the effects of 4 wk of hypodynamia on the rate of lactate transport in skeletal muscle sarcolemmal vesicles from control and hindlimb-suspended rats. Characterization of the sarcolemmal preparations was achieved with a marker enzyme (K+-p-nitrophenylphosphatase) and measurement of 1 mM [U-14C]lactate transport activity under zero-trans conditions with or without a pH gradient or the transport inhibitor alpha-hydroxycinnamate. Preparations from the two groups were not significantly different concerning yield and purification. Based on these results, we used this model to analyze the lactate transport activity after hypodynamia by tail suspension. Hindlimb suspension caused a shift from slow to fast myosin heavy chain isoforms in soleus muscles with a 40% decrease in the citrate synthase activity (from 35.3 +/- 3.7 to 21.4 +/- 2.1 mu mol x g-1 x min-1; P < 0.05). Lactate (1 mM) uptake in vesicles from the two groups was a function of time, and the rate after hindlimb suspension was significantly decreased in the suspended compared with the control group (2.25 +/- 0.44 and 3.50 +/- 0.26 nmol x min-1 x mg protein-1, respectively; P < 0.05). These differences were not observed for a higher lactate concentration (50 mM). These results suggest that the level of physical activity plays a role in the regulation of sarcolemmal lactate transport activity implicated in the exchanges of lactate between producing and utilizing cells, organs, and tissues, which are major ways of carbohydrate energy distribution in humans and others species.     

Human erythrocyte anion permeabilities measured under conditions of net charge transfer

1. The permeability of the human erythrocyte to anions has been measured under conditions of net charge transfer: for Cl(-) and HCO(3) (-) ions, at 37 degrees C, this permeability is 5 orders of magnitude too small to account for the rate of the electroneutral anion exchange which is responsible for the chloride, or Hamburger, shift.2. The method is an indirect one in which the ionophore, valinomycin, is used to increase the erythrocyte K(+) permeability: in the absence of permeant cation externally, the rate of the resulting K(+) efflux may be limited by the slowness of the accompanying anion efflux, allowing the true anion permeability to be estimated.3. The average Cl(-) permeability estimated in ACD-stored erythrocytes (seven experiments) and erythrocytes from fresh blood (two experiments) was 2.1 x 10(-8) cm/sec at 37 degrees C and pH 7.4: this may also be expressed as a Cl(-) conductance of about 1.0 x 10(-5) Omega(-1) cm(-2). The apparent activation energy for net efflux of Cl(-) was found to be 3.9 kJ/mole (16.4 kcal/mole).4. In fresh cells, the ratios of Cl(-), HCO(3) (-), Br(-) and I(-) permeabilities (or conductances) were 1:0.8:1.5:5. The three halide ions follow Eisenman's Sequence I, representing a binding site of low field strength.     

Urea transporter UT3 functions as an efficient water channel. Direct evidence for a common water/urea pathway

A family of molecular urea transporters (UTs) has been identified whose members appear to have an exceptionally high transport turnover rate. To test the hypothesis that urea transport involves passage through an aqueous channel, osmotic water permeability was measured in Xenopus oocytes expressing UTs. The UT3 class of urea transporters functioned as efficient water channels. Quantitative measurement of single channel water permeability (pf) using epitope-tagged rat UTs gave pf (in cm3/s x 10(-14)) of 0.14 +/- 0.11 (UT2) and 1.4 +/- 0.2 (UT3), compared with 6.0 and 2.3 for water channels AQP1 and AQP3, respectively. Relative single channel urea permeabilities (purea) were 1.0 (UT2), 0.44 (UT3), and 0.0 (AQP1). UT3-mediated water and urea transport were weakly temperature-dependent (activation energy <4 kcal/mol), inhibited > 75% by the urea transport inhibitor 1,3-dimethylthiourea, but not inhibited by the water transport inhibitor HgCl2. To test for a common water/urea pore, the urea reflection coefficient (sigmaurea) was measured by independent induced osmosis and solvent drag methods. In UT3-expressing oocytes, the time course of oocyte volume in response to different urea gradients (induced osmosis) gave sigmaurea approximately 0.3 for the UT3 pathway, in agreement with sigmaurea determined by the increase in uptake of [14C]urea during osmotic gradient-induced oocyte swelling (solvent drag). In oocytes of comparable water and urea permeability coexpressing AQP1 (permeable to water, not urea) and UT2 (permeable to urea, not water), sigmaurea = 1. These results indicate that UT3 functions as a urea/water channel utilizing a common aqueous pathway. The water transporting function and low urea reflection coefficient of UT3 in vasa recta may be important for the formation of a concentrated urine by countercurrent exchange in the kidney.     

Glucocorticoid-mediated inhibition of interleukin-2 receptor alpha and -beta subunit expression by human T cells

We have shown previously that vanadium ions (vanadate and vanadyl) inhibit autophosphorylation of histidine but not that of serine in ATP citrate lyase (ACL). Here we report the results concerning the effect of monovanadate (+ oligomers), decavanadate as well as vanadyl on the activity of ACL of the rat liver. Susceptibility of ACL to inhibition by vanadate was rather low. Vanadate at concentration 10(-4) mol/l inhibited ACL by only 10% and at 10(-3) mol/l concentration monovanadate inhibited ACL by 37%. Decavanadate had comparable potency to inhibit ACL. So was vanadyl which produced 20%, 32% and 66% inhibition at 10(-4) mol/l, 10(-3) mol/l and 10(-2) mol/l concentrations, respectively. From the kinetic data it appears that inhibition by mono- and deca-vanadate of ACL with respect to both ATP and citrate was of competitive nature. Vanadyl inhibited ACL noncompetitively with respect to these substrates. However, all three species of vanadium ions inhibited ACL noncompetitively with respect to CoA. Endogenous (auto)phosphorylation of the ACL histidine as well as its response to vanadate depended on the presence of he substrate (citrate + CoA). The kinetic characteristics of vanadium ions action of ACL was compared with that previously demonstrated for vanadium inhibition of succinyl-CoA synthetase. Plausibility of our hypothesis that inhibition of histidyl phosphorylation at the catalytic site may be a common mechanism by which vanadium ions suppress the activity of the histidyl containing enzymes catalyzing the phosphoryl transfer is discussed.     

The uptake of 3-deoxy-3-fluoro-D-glucose by synaptosomes from rat brain cortex

D-[3-3H]-3-deoxy-3-fluoroglucose was synthesized chemically and shown to be transported into rat brain synaptosomes by a saturable process with a Km 6.2 x 10(-4) M and a Vmax 2.8 nmole x mg protein-1. After an initial, rapid period of transport, further uptake of the fluorosugar is restricted by the rate of its phosphorylation. Both D-glucose and cytochalasin B are competitive inhibitors of 3-deoxy-3-fluoro-D-glucose transport with Ki values of 93 micron and 6.0 x 10(-7) M, respectively. Phloretin, N-ethylmaleimide and p-chloromercuribenzoate also inhibit the fluorosugar uptake, whereas ouabain and changes in K+, Na+, Mg2+ and Ca2+ ions have only a small effect. The recorded 3-deoxy-3-fluoro-D-glucose influx is slightly reduced by potassium cyanide, antimycin A, 2,4-dinitrophenol, and rotenone. The uptake reduction caused by these four reagents is relieved by the addition of exogenous ATP. The possible influence of hexokinse activity on the uptake process is discussed.     

Deletion polymorphism in the angiotensin-converting enzyme gene as a thrombophilic risk factor after hip arthroplasty

Recent work has suggested a possible role for nitric oxide (NO) in the development of hepatic encephalopathy (HE). In this study, we examined the effect of ammonia and manganese, factors implicated in the pathogenesis of HE, on the transport of arginine (a precursor of NO) into primary cultures of astrocytes. Treatment with 5 mM ammonia for 1-4 days produced a maximal (53%) increase in L-arginine uptake at 3 days when compared to untreated cells. Kinetic analysis following 4-day treatment with 5 mM ammonia revealed an 82% increase in the Vmax and a 61% increase in the Km value. Similar analysis with 100 microM manganese showed a 101% increase in Vmax and a 131% increase in the Km value. These results suggest that both manganese and ammonia alter L-arginine uptake by modifying the transporter for arginine. A decrease of 32% in the non-saturable component of L-arginine transport was also observed following treatment with ammonia. When cultures were treated separately with 5 mM ammonia and 100 microM manganese for 2 days, the uptake of L-arginine increased by 41% and 57%, respectively. Combined exposure led to no further increase in uptake. Our results suggest that ammonia and manganese may contribute to the pathogenesis of HE by influencing arginine transport and thus possibly NO synthesis in astrocytes.     

Peritubular transport of ochratoxin A in rabbit renal proximal tubules

The transport of the nephrotoxic mycotoxin ochratoxin A across the renal peritubular membrane was examined in suspensions of rabbit renal proximal tubules. Ochratoxin A transport across the peritubular membrane was a high-affinity, low-capacity carrier-mediated process with a Jmax value of 0.12 +/- 0.4 nmol/mg of protein/min and a Km value of 1.4 +/- 0.1 microM. The apparent Michaelis constants for inhibition of [3H]para-aminohippurate (PAH) uptake by ochratoxin A inhibition was 1.5 microM, which is similar to the Km value for ochratoxin A uptake in tubule suspensions and suggests that ochratoxin A could be a substrate for the organic anion pathway. The capacity and affinity for peritubular ochratoxin A transport were 40-fold lower and > 100-fold greater, respectively, than those measured for the peritubular uptake of [3H]PAH in tubule suspensions. A concentration of 2.5 mM PAH, which reduced the uptake of [3H]PAH by 90%, reduced ochratoxin A uptake by only 40% to 50%, whereas probenecid concentrations of 0.6 to 2 mM reduced ochratoxin A accumulation in tubule suspensions up to approximately 80% to 90%. This probenecid-sensitive, PAH-insensitive uptake of ochratoxin A suggested that at least one mediated pathway other than the organic anion transporter was involved in the peritubular uptake of this mycotoxin. A 2 mM concentration of the fatty acid octanoate and 1.5 mM concentration of the nonsteroidal anti-inflammatory agent piroxicam were as effective as probenecid in blocking ochratoxin A uptake. The apparent Ki values for inhibition of ochratoxin A uptake by probenecid, piroxicam and octanoate were 30.5 +/- 7.9, 23.2 +/- 10.4 and 81.5 +/- 8.7 microM, respectively. The ability of octanoic acid to inhibit ochratoxin A transport to the same extent as probenecid and a greater extent than PAH suggests that a separate fatty acid transport pathway may be involved in the accumulation of ochratoxin A by suspensions of rabbit renal proximal tubules.     

Purification, physicochemical characterization and biological properties of a lectin from Erythrina velutina forma aurantiaca seeds

A lectin was purified from seeds of Erythrina velutina forma aurantiaca by affinity chromatography on cross-linked guar gum. The lectin is a potent agglutinin for human (minimal concentration of protein able to cause visible agglutination of a 2% erythrocyte suspension varying from 1 to 4 micrograms/ml), rabbit (4 micrograms/ml) and chicken erythrocytes (8 micrograms/ml) but presented low activity against cow (250 micrograms/ml) or sheep (333 micrograms/ml) blood cells. Hemagglutination of human O+ erythrocytes was inhibited by D-lactose (0.2 mM) > D-galactose (0.8 mM) > D-raffinose (2.1 mM). At pH 7.5, chromatography on a Superose 12 HR 10/30 column showed that the lectin was primarily a dimer (56.0 kDa) composed of two identical subunits (31.6 kDa each). A small amount of a tetrameric form was also apparently present. The lectin is a glycoprotein (7.3% carbohydrate), has a pI of 4.5, contains high levels of acidic (Asp and Glu, 64.2 and 51.6 residues/mol, respectively) and hydroxy amino acids (Ser and Thr, 42.9 and 38.5 residues/mol, respectively) but relatively low amounts of sulfur amino acids (Cys and Met, 1.0 and 5.0 residues/mol, respectively) and has an N-terminal sequence of Val-Glu-Thr-Ile/Leu-Pro-Phe-Ser. Its hemagglutinating activity was abolished by heating at 70 degrees C for 10 min. The activation energy (delta G') required for denaturation measured by loss of hemagglutination activity was 24.87 kcal/mol. In rats, the purified lectin (100 micrograms) induced neutrophil migration into the peritoneal cavity (3.7 +/- 0.6 x 10(6) neutrophils/ml) or into the air pouch (2.75 +/- 0.25 x 10(6) neutrophils/ml), 8 and 10 times greater than the negative control, respectively.     

Mechanism of intestinal absorption of ranitidine and ondansetron: transport across Caco-2 cell monolayers

We have investigated the transport of ranitidine and ondansetron across the Caco-2 cell monolayers. The apparent permeability co-efficients (Papp) were unchanged throughout the concentration range studied, indicating a passive diffusion pathway across intestinal mucosa. No metabolism was observed for ranitidine and ondansetron during the incubation with Caco-2 cell monolayers. Papp values for ranitidine and ondansetron (bioavailability of 50 and approximately 100% in humans, respectively) were 1.03 +/- 0.17 x 10(-7) and 1.83 +/- 0.055 x 10(-5) cm/sec, respectively. The Papp value for ranitidine was increased by 15- to 20-fold in a calcium-free medium or in the transport medium containing EDTA, whereas no significant change occurred with ondansetron, indicating that paracellular passive diffusion is not rate determining for ondansetron. Uptake of ondansetron by Caco-2 cell monolayers was 20- and 5-fold higher than that of ranitidine when the uptake study was carried out under sink conditions and at steady state. These results suggest that ranitidine and ondansetron are transported across Caco-2 cell monolayers predominantly via paracellular and transcellular pathways, respectively.     

Macromolecular permeability across the blood-nerve and blood-brain barriers

The permeability of insulin (Ins), nerve growth factor (NGF), albumin (Alb), transferrin (Trf), and IgG across the blood-nerve barrier (BNB) and blood-brain barrier (BBB) in normal adult rats was quantified by measuring the (permeability coefficient x surface area) product (PS) with the i.v. bolus-injection technique in the cannulated brachial vein and artery using radioiodinated proteins. The PS values of the BNB for IgG and Alb were low: 0.079 +/- 0.029 x 10(-6) and 0.101 +/- 0.088 x 10(-6) ml.g-1.s-1, (mean +/- SD, respectively). The PS values for NGF and Trf were 16.1-fold and 25.5-fold higher than for Alb. The PS for Ins across the BNB was 33.190 +/- 2.053 x 10(-6) ml.g-1.s-1--a remarkable 329-fold increase compared with Alb. The PS values of the BBB for IgG and Alb in different brain regions were all low, from 0.028 +/- 0.017 to 0.151 +/- 0.035 x 10(-6) ml.g-1.s-1 (mean +/- SD). NGF and Trf had comparable PS values from 13- to 32-fold higher than for Alb, except for the brain stem, where the PS for Trf was 66-fold higher than for Alb. The mean PS for Ins across the BBB ranged from 15.78 +/- 5.45 x 10(-6) ml.g-1.s-1 for the cortex to 22.62 +/- 7.50 x 10(-6) ml.g-1.s-1 for the brain stem--again a remarkable 105- to 390-fold increase relative to Alb. Because reliable PS measurements were obtained for all proteins tested, the BBB and BNB cannot be considered impermeable to proteins--a concept that has plagued brain- and nerve-barrier research. The low PS values for IgG and Alb indicate low rates of transfer; however, Alb, in particular, is the major protein of endoneurial and ventricular fluid, which suggests that these PS values may be significant. Ins had the highest PS values, which likely reflect the mechanism of transport across the barriers--that is, receptor-mediated transport. Because NGF and Trf had PS values 13- to 66-fold higher than for Alb, whether this reflects receptor-mediated uptake, adsorptive-mediated transcytosis, or some other mechanism is unclear. That the PS values for NGF and Trf differ from Alb and IgG clearly suggests, however, a different uptake mechanism. Finally, the remarkably high PS values for Ins across the BBB and BNB identify this protein and its putative receptor on capillary endothelial cells as a potential target for drug delivery into the central and peripheral nervous systems.     

Mass spectral fragmentation of 18-norsteroids and 12-oxo-20-hydroxysteroids

The free magnesium concentration in the axoplasm of the giant axon of the squid, Loligo pealei, was estimated by exploting the known sensitivity of the sodium pump to intracellular Mg2+ levels. The Mg-citrate buffer which, when injected into the axon, resulted in no change in sodium efflux was in equilibrium with a Mg2+ level of about 3--4 mM. Optimal [Mg2+] for the sodium pump is somewhat higher. Total magnesium content of axoplasm was 6.7 mmol/kg, and that of hemolymph was 44 mM. The rate coefficient for 28Mg efflux was about 2 X 10(-3) min-u for a 500-mum axon at 22-25degreesC, with a very high temperature coefficient (Q10=4-5). This efflux is inhibited 95% by injection of apyrase and 75% by removal of external sodium, and seems unaffected by membrane potential or potassium ions. Increased intracellular ADP levels do not affect Mg efflux nor its requirement for Na+/o, but extracellularl magnesium ions do. Activation of 28Mg efflux by Na+/o follows hyperbolic kinetics, with Mg2+/o reducing the affinity of the system for Na+/o. Lanthanum and D600 reversibly inhibit Mg efflux. In the absence of both Na+ and Mg2+, but not in their presence, removal of Ca2+ from the seawater vastly increased 28Mg efflux; this efflux was also strongly inhibited by lanthanum. A small (10(-14) mol cm-2) extra Mg efflux accompanies the conduction of an action potential.     

Swelling-stimulated passive potassium transport in camel erythrocytes: inhibitory effects of furosemide and sodium fluoride

The inhibitory effects of furosemide, sodium fluoride, and age on volume-dependent, ouabain-resistant K+ influx were investigated in camel red blood cells. Swelling of young camel erythrocytes hypotonically stimulates ouabain-resistant potassium influx, a response that was lacking in old camel erythrocytes. The swelling-stimulated influx was partially inhibited by 1 mM furosemide and by 10 and 20 mM sodium fluoride. The inhibitory effect of furosemide was significantly increased if rubidium was added to the flux media. There was a significant correlation between potassium influx in normo- and hypotonic media which might indicate that the anion-dependent transport system operates, to some extent, to regulate cell volume.     

Prenatal ultrasonographic diagnosis of the cerebro-costo-mandibular syndrome: case report and review of the literature

Oxethazaine (OXZ), a potent topical anesthetic, was found to induce red blood cell (RBC) lysis in vitro, depending upon concentrations of OXZ, RBC and Ca2+. In a 2% RBC suspension, 100 microM OXZ caused almost complete hemolysis in the presence of 1.3 mM Ca2+ with only a minimal effect in its absence, while higher concentrations of OXZ (400 microM<) produced hemolysis without Ca2+. The hemolysis induced by OXZ plus Ca2+ was preceded by a rapid increase in 45Ca2+ uptake by RBCs, with both the hemolysis and Ca2+ uptake being inhibited by 1 mM CoCl2, NiCl2, and quinine. Together with the Ca2+ influx, rapid influx of Na+ and efflux of K+ occurred, and an increasing external K+/Na+ concentration ratio inhibited both hemolysis and Ca2+ influx. Morphologically, OXZ plus Ca2+ caused rapid transformation to spheroechinocytes, the formation of blebs and the pinching-off of blebs, whereas OXZ alone produced membrane invagination. SDS-PAGE analysis of the ghosts prepared from the RBCs treated with OXZ plus Ca2+ revealed derangement of cytoskeletal components. OXZ is a rare drug that exhibits a Ca2+ ionophore-like action, increasing the Ca2+ permeability of plasma membranes.     

Amiloride-insensitive Na+-H+ exchange: a candidate mediator of erythrocyte Na+-Li+ countertransport

Erythrocyte Na+-Li+ countertransport shows an increased activity in essential hypertension and diabetic nephropathy, but its nature remains unknown. This amiloride-insensitive membrane transport may not be a mode of operation of the amiloride-sensitive NHE1, the only Na+-H+ exchange isoform found in human erythrocytes. Whether an independent, although unknown, amiloride-insensitive isoform mediates Na+-Li+ countertransport is unclear. Na+-H+ exchange activity was measured in acid-loaded erythrocytes. Dimethylamiloride, a specific inhibitor of Na+-H+ exchange and phloretin, a known inhibitor of Na+-Li+ countertransport, gave a reduction in H+-driven Na+ influx (by 31 and 37%, respectively). This effect was additive, and a 66% reduction in H+-driven Na+ influx was found in the presence of both inhibitors. Internal acidification, a stimulus for Na+-H+ exchange, enhanced Na+-Li+ countertransport activity (from 287 +/- 55 to 1213 +/- 165 micromol x Lcell(-1) h(-1), mean +/- SEM, P = 0.003). This transport remained sensitive to phloretin under both conditions. Conversely, external acidification decreased Na+-Li+ countertransport activity (as expected for a Na+-H+ exchanger). Competition between internal H+ and Li+ or Na+ for a common binding site was present. Finally, similar kinetic parameters for external Na+ characterized Na+-Li+ countertransport and the phloretin-sensitive component of H+-driven Na+ influx. These findings suggest that both Na+-Li+ countertransport and the amiloride-insensitive, phloretin-sensitive component of H+-driven Na+ influx can be mediated by a previously unrecognized novel amiloride-insensitive Na+-H+ exchange isoform in human erythrocytes.     

Molecular characterization of the kinetoplastid membrane protein-11 from African trypanosomes

These is debate about the mechanisms mediating adenosine release from neurons. In this study, the release of adenosine evoked by depolarizing cultured cerebellar granule neurons with 50 mM K+ was inhibited by 49 +/- 7% in Ca2+-free medium. The remaining release was blocked by dipyridamole (IC50 = 6.4 x 10(-8) M) and nitrobenzylthioinosine (IC50 = 3.6 x 10(-8) M), inhibitors of adenosine uptake. Ca2+-dependent release was reduced by 78 +/- 9% following a 21-h pretreatment of the cells with pertussis toxin, which ADP-ribosylates Gi/Go G proteins, thereby preventing their dissociation. The nucleoside transporter-mediated component of K+-induced adenosine release also was inhibited by 62 +/- 8% by pertussis toxin and was potentiated by 78 +/- 11% following cholera toxin treatment, which permanently activates Gs. Uptake of [3H]adenosine into cultured cerebellar granule neurons over a 10-min period was not dependent on extracellular Na+ but was reduced by dipyridamole (IC50 = 3.2 x 10(-8) M) and nitrobenzylthioinosine (IC50 = 2.6 x 10(-8) M). Thus, adenosine uptake likely occurs via the same transporter mediating Ca2+-independent adenosine release. Adenosine uptake was potentiated by cholera toxin pretreatment (152 +/- 15% of control), but pertussis toxin had no statistically significant effect. It is possible that Gs, Gi/Go, or free Gbetagamma dimer modulate the equilibrative, inhibitor-sensitive nucleoside carrier to enhance adenosine transport.     

Human neurons express the polyspecific cation transporter hOCT2, which translocates monoamine neurotransmitters, amantadine, and memantine

Recently, we cloned the human cation transporter hOCT2, a member of a new family of polyspecific transporters from kidney, and demonstrated electrogenic uptake of tetraethylammonium, choline, N1-methylnicotinamide, and 1-methyl-4-phenylpyridinium. Using polymerase chain reaction amplification, cDNA sequencing, in situ hybridization, and immunohistochemistry, we now show that hOCT2 message and protein are expressed in neurons of the cerebral cortex and in various subcortical nuclei. In Xenopus laevis oocytes expressing hOCT2, electrogenic transport of norepinephrine, histamine, dopamine, serotonin, and the antiparkinsonian drugs memantine and amantadine was demonstrated by tracer influx, tracer efflux, electrical measurements, or a combination. Apparent Km values of 1.9 +/- 0.6 mM (norepinephrine), 1.3 +/- 0.3 mM (histamine), 0.39 +/- 0.16 mM (dopamine), 80 +/- 20 microM (serotonin), 34 +/- 5 microM (memantine), and 27 +/- 3 microM (amantadine) were estimated. Measurement of trans-effects in depolarized oocytes and human embryonic kidney cells expressing hOCT2 suggests that there were different rates and specificities for cation influx and efflux. The hypothesis is raised that hOCT2 plays a physiological role in the central nervous system by regulating interstitial concentrations of monoamine neurotransmitters that have evaded high affinity uptake mechanisms. We show that amantadine does not interact with the expressed human Na+/Cl- dopamine cotransporter. However, concentrations of amantadine that are effective for the treatment of Parkinson's disease may increase the interstitial concentrations of dopamine and other aminergic neurotransmitters by competitive inhibition of hOCT2.     

Purification and functional reconstitution of soybean nodulin 26. An aquaporin with water and glycerol transport properties

Infection of soybean roots by nitrogen-fixing Bradyrhizobium japonicum leads to expression of plant nodule-specific genes known as nodulins. Nodulin 26, a member of the major intrinsic protein/aquaporin (AQP) channel family, is a major component of the soybean symbiosome membrane (SM) that encloses the rhizobium bacteroid. To investigate the water and solute transport characteristics of nodulin 26, we purified the protein from SMs and reconstituted it into carboxyfluorescein-loaded liposomes for transport studies using stopped-flow spectrofluorimetry. Liposomes containing nodulin 26 exhibited a high osmotic permeability (Pf = 0. 012 +/- 0.0013 cm/s), a value fivefold higher than that obtained with control liposomes. Water flux through nodulin 26 showed a low activation energy (Ea) (4.07 kcal/mol) and was reduced 70% upon addition of 1 mM HgCl2. Reconstituted nodulin 26 exhibited a single-channel conductance of 3.8 +/- 2.5 x 10(-)15 cm3/s (n = 3), a value that is lower than other characterized AQPs. Nodulin 26 proteoliposomes also facilitate glycerol transport, showing a 43-fold higher rate of glycerol flux than control liposomes. This observation was supported by expression experiments in Xenopus oocytes that showed that nodulin 26 facilitated glycerol flux in a manner indistinguishable from the Escherichia coli GlpF glycerol facilitator. Consistent with the results of water transport, glycerol transport was inhibited by HgCl2 and showed a low Ea (4.43 kcal/mol). These results indicate that nodulin 26 is a multifunctional AQP that confers water and glycerol transport to the SM, and likely plays a role in osmoregulation during legume/rhizobia symbioses.