Elevated D-glucose induces insulin insensitivity in human umbilical endothelial cells isolated from gestational diabetic pregnancies.

1. The effects of human insulin and elevated D-glucose on L-arginine transport and synthesis of nitric oxide (NO) and prostacyclin (PGI2) have been investigated in human umbilical vein endothelial cells isolated from gestational diabetic pregnancies. 2. The increase in the Vmax for L-arginine transport (9.0 +/- 1.1) pmol (micrograms protein)-1 min-1) in diabetic endothelial cells cultured in 5 mM D-glucose was unaffected following 24 h exposure to 25 mM D-glucose. 3. Gestational diabetes-induced increases in basal intracellular cGMP and L-citrulline levels (inhibitable by L-NAME) and [Ca2+], were unaffected by elevated D-glucose. In contrast, PGI2 release was inhibited in diabetic cells exposed to either 5 or 25 mM D-glucose. 4. Elevated D-glucose attenuated histamine (10 microM, 5 min)-stimulated accumulation of cGMP and L-citrulline in endothelial cells isolated from gestational diabetic pregnancies. 5. The membrane hyperpolarization (-79 +/- 0.9 mV) sustained in diabetic endothelial cells in culture was insensitive to elevated D-glucose. 6. Elevated D-glucose abolished the stimulatory effect of human insulin (1 nM, 8 h) on L-[3H]leucine incorporation in diabetic endothelial cells cultured in 5 mM D-glucose. 7. Human insulin reduced the elevated rates of L-arginine transport and cGMP accumulation in diabetic cells cultured in 5 mM D-glucose but failed to reduce increased rates of transport or NO production in cells exposed to 25 mM D-glucose or cycloheximide. 8. Our findings demonstrate that hyperglycaemia impairs the actions of human insulin on umbilical vein endothelial cells isolated from gestational diabetic pregnancies. Changes in insulin sensitivity and/or its signalling cascade may be affected by hyperglycaemia associated with gestational diabetes, resulting in insulin resistance in endothelial cells derived from the fetal vasculature.

Activation of A2-purinoceptors by adenosine stimulates L-arginine transport (system y+) and nitric oxide synthesis in human fetal endothelial cells.

1. Human umbilical vein endothelial cells were challenged acutely with adenosine and its analogues to examine whether adenosine modulates L-arginine transport (system y+) and synthesis of nitric oxide (NO) and prostacyclin (PGI2). 2. L-Arginine transport was stimulated by adenosine (10 microM, 2 min) and the A2-receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680; 100 nM), but not by the A1-receptor agonist N6-cyclopentyladenosine (CPA). 3. Activation of L-arginine transport was inhibited by the A2-receptor antagonists ZM-241385 and 3,7-dimethyl-1-propargylxanthine (DMPX), but unaffected by the A1-receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine and 8-phenyltheophylline or the adenosine transport inhibitor nitrobenzylthioinosine. 4. Adenosine and CGS-21680 evoked a rapid membrane hyperpolarization. 5. Adenosine and CGS-21680 induced increases in intracellular cGMP levels, whereas release of PGI2 was unaffected. NG-nitro-L-arginine methyl ester (an NO synthase inhibitor) and the A2-receptor antagonists ZM-241385 and DMPX prevented increases in cGMP accumulation. 6. Our findings provide the first evidence that activation of human fetal endothelial cell A2-purinoceptors, but not A1-purinoceptors, leads to a membrane hyperpolarization and stimulation of basal rates of L-arginine transport and NO biosynthesis.

Hypoxanthine enters human vascular endothelial cells (ECV 304) via the nitrobenzylthioinosine-insensitive equilibrative nucleoside transporter.

The transport properties of the nucleobase hypoxanthine were examined in the human umbilical vein endothelial cell line ECV 304. Initial rates of hypoxanthine influx were independent of extracellular cations: replacement of Na+ with Li+, Rb+, N-methyl-D-glucamine or choline had no significant effect on hypoxanthine uptake by ECV 304 cells. Kinetic analysis demonstrated the presence of a single saturable system for the transport of hypoxanthine in ECV 304 cells with an apparent K(m) of 320 +/- 10 microM and a Vmax of 5.6 +/- 0.9 pmol/10(6) cells per s. Hypoxanthine uptake was inhibited by the nucleosides adenosine, uridine and thymidine (apparent Ki 41 +/- 6, 240 +/- 27 and 59 +/- 8 microM respectively) and the nucleoside transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and dipyridamole (apparent Ki 2.5 +/- 0.3, 11 +/- 3 and 0.16 +/- 0.006 microM respectively), whereas the nucleobases adenine, guanine and thymine had little effect (50% inhibition at > 1 mM). ECV 304 cells were also shown to transport adenosine via both the NBMPR-sensitive and -insensitive nucleoside carriers. Hypoxanthine specifically inhibited adenosine transport via the NBMPR-insensitive system in a competitive manner (apparent Ki 290 +/- 14 microM). These results indicate that hypoxanthine entry into ECV 304 endothelial cells is mediated by the NBMPR-insensitive nucleoside carrier present in these cells.

Activation of L-arginine transport (system y+) and nitric oxide synthase by elevated glucose and insulin in human endothelial cells.

1. Modulation of L-arginine transport (system y+) and release of nitric oxide (NO) and prostacyclin (PGI2) by elevated glucose and insulin were investigated in human cultured umbilical vein endothelial cells. 2. Elevated glucose induced a time- (6-12 h) and concentration-dependent stimulation of L-arginine transport, which was reversible and associated with a 3-fold increase in intracellular cGMP accumulation (index of NO synthesis) and 75% decrease in PGI2 production. 3. Elevated glucose had no effect on the initial transport rates for L-serine, L-citrulline, L-leucine, L-cystine or 2-deoxyglucose. 4. Resting membrane potential was unaffected by elevated glucose whereas basal intracellular [Ca2+] increased from 65 +/- 5 nM to 136 +/- 16 nM. 5. Insulin induced a protein synthesis-dependent stimulation of L-arginine transport and increased NO and PGI2 production in cells exposed to 5 mM glucose. 6. In cells exposed to high glucose, insulin downregulated elevated rates of L-arginine transport and cGMP accumulation but had no effect on the depressed PGI2 production. 7. Our findings suggest that insulin's normal stimulatory action on human endothelial cell vasodilator pathways may be impaired under conditions of sustained hyperglycaemia.

Diabetes-induced activation of system y+ and nitric oxide synthase in human endothelial cells: association with membrane hyperpolarization.

1. The activity of the human endothelial cell L-arginine transporter (system y+) has been correlated with cGMP production (index of nitric oxide) and prostacyclin (PGI2) release in umbilical vein endothelial cells cultured from normal or gestational diabetic pregnancies. 2. In non-diabetic and diabetic cells, transport of L-arginine was Na+ and pH independent, inhibited by other cationic L-arginine analogues and unaffected by neutral amino acids. 3. Diabetes was associated with an increased Vmax for saturable L-arginine transport (4.6 +/- 0.13 vs. 9.9 +/- 0.5 pmol (microgram protein)-1 min-1, P < 0.01), but had no effect on initial rates of transport for L-serine, L-citrulline, L-leucine or 2-deoxyglucose. 4. In non-diabetic and diabetic cells, elevated K+ resulted in a concentration-dependent inhibition in the initial rates of transport for L-arginine and the membrane potential-sensitive probe tetra[3H]phenylphosphonium (TPP+). 5. When resting membrane potential was measured using the whole-cell patch voltage clamp technique, diabetic cells were hyperpolarized (-78 +/- 0.3 mV) compared with non-diabetic cells (-70 +/- 0.04 mV, P < 0.04). Accumulation of [3H]TPP+ was also increased in diabetic compared with non-diabetic cells. 6. Basal intracellular cGMP levels were elevated 2.5-fold in diabetic cells, and L-NAME (100 microM), an inhibitor of nitric oxide synthase, abolished basal cGMP accumulation in non-diabetic and diabetic cells. 7. Histamine (10 microM) had no effect on L-arginine transport but evoked significant increases in cGMP in non-diabetic and diabetic cells, which were completely inhibited by L-NAME but unaffected by superoxide dismutase. 8. Basal and histamine-stimulated PGI2 release was decreased markedly in diabetic cells. 9. Our findings demonstrate that gestational diabetes is associated with phenotypic changes in fetal endothelial cells, which result in a membrane hyperpolarization, activation of the human endothelial cell L-arginine transporter (system y+), elevation of basal nitric oxide synthesis and decreased PGI2 production.

Quantitation and immunolocalization of glucose transporters in the human placenta.

The subcellular distributions of the mammalian passive glucose transporter isoforms GLUT1, GLUT3 and GLUT4, in the human placenta, were investigated using isoform-specific anti-peptide antibodies. On western blots of both basal and brush-border plasma membranes isolated from the syncytiotrophoblast, antibodies specific for GLUT1 labelled a broad band (apparent Mr 55,000) that co-migrated with the human erythrocyte GLUT1 glucose transporter. In contrast, no labelling was detectable when blots were probed with antibodies specific for the GLUT3 or GLUT4 isoforms. Densitometric analysis of blots showed that GLUT1 accounts for approximately 90 and 65 per cent of the D-glucose-sensitive cytochalasin B binding sites present in brush-border and basal membranes, respectively. Confocal immunofluorescence microscopy of fixed placental tissue showed that GLUT1 is abundant at both maternal- and fetal-facing surfaces of the syncytiotrophoblast whereas it was undetectable at the fetal capillary endothelium. In parallel experiments, no staining by antibodies against either the GLUT3 or the GLUT4 isoforms was detected in placental tissue. These results indicate that GLUT1 is the major isoform responsible for glucose transfer from mother to fetus. The absence of GLUT4 is consistent with the lack of insulin-sensitive glucose transport across the placenta.

Immunolocalisation of nucleoside transporters in human placental trophoblast and endothelial cells: evidence for multiple transporter isoforms.

Polyclonal antibodies raised against the human erythrocyte nucleoside transporter were used to investigate the distribution of the nucleoside transporters in the placenta. Immunoblots of brush-border membranes isolated from the human syncytiotrophoblast revealed a cross-reactive species that co-migrated with the erythrocyte nucleoside transporter as a broad band of apparent M(r) 55,000. In contrast, no labelling was detected in basal membranes containing a similar number of equilibrative nucleoside transporters as assessed by nitrobenzylthioinosine (NBMPR)-binding. The absence of cross-reactive epitopes in basal membranes and their presence in brush-border membranes was confirmed by confocal immunofluorescence microscopy. These results suggest that at least two isoforms of the NBMPR-sensitive nucleoside transporter are present in the human placenta. The lumenal surfaces of fetal capillaries, small placental vessels and umbilical vein were also strongly labelled by the antibody, a finding that suggests that the high fetal-placental adenosine uptake previously reported is due to endothelial transporters.

Transport and metabolism of adenosine in diabetic human placenta.

Pregnancy complicated by diabetes is a relatively frequent event and may result in fetal embriopathy. However, little is known regarding whether placental transport functions are altered. In this study, we have compared the activity of the nitrobenzylthioinosine (NBMPR)-sensitive adenosine transporter and adenosine metabolism in human placental brush-border- and basal-membrane vesicles from placentas of normal and diabetic pregnancies. Neither [3H]NBMPR binding, a marker of the facilitative-diffusion nucleoside transporter in the human placenta, nor adenosine metabolism exhibited a significant difference in either the brush-border- or the basal-membrane vesicles between the normal and diabetic group, except for an increased affinity in [3H]NBMPR binding at the basal side in diabetic placenta. This result contrasts with an earlier finding using the same group of patients that adenosine transport is downregulated in umbilical vein endothelial cells from diabetic pregnancies. It is concluded that adenosine transport is modulated selectively in different tissues in diabetic pregnancies.

Adenosine transport in cultured human umbilical vein endothelial cells is reduced in diabetes.

Adenosine transport in cultured human umbilical vein endothelial cells (HUVEC) was characterized and shown to be mediated by a single facilitated diffusion mechanism. Initial rates of adenosine influx at 22 degrees C were saturable [apparent Michaelis constant, 69 +/- 10 microM; maximum velocity (Vmax), 600 +/- 70 pmol.10(6) cells-1.s-1] and inhibited by nitrobenzylthioinosine (NBMPR). Formycin B had an unusually high affinity [inhibitory constant (Ki), 18 +/- 4.3 microM], whereas inosine had a low affinity (Ki, 440 +/- 68 microM) and nucleobases were without effect on adenosine influx. The number of transporters (1.2 x 10(6) sites/cell) was estimated by NBMPR equilibrium binding (apparent dissociation constant, 0.11 +/- 0.01 nM; maximum binding, 2.0 +/- 0.15 pmol/10(6) cells). In addition, we compared these endothelial cells with those obtained from cords from pregnancies complicated by diabetes (HUVEC-D), since embriopathy may occur in these conditions. HUVEC-D exhibited a 2.3-fold reduction in both the Vmax for adenosine influx and the maximum number of NBMPR binding sites (260 +/- 40 pmol.10(6) cells-1.s-1 and 0.86 +/- 0.08 pmol/10(6) cells, respectively). However, the turnover number for each nucleoside transporter in normal and diabetic HUVEC was similar (approximately 300 adenosine molecules/s). Adenosine metabolism at 10 microM in HUVEC-D was modified compared with normal cells. Intracellular phosphorylation (> 90%) was the predominant pathway in normal HUVEC, whereas in HUVEC-D, substantial levels of adenine and adenosine were detected. The present results demonstrate therefore the downregulation of the NBMPR-sensitive nucleoside transporter and changes in adenosine metabolism in HUVEC from diabetic pregnancies.

Adenosine transport and nitrobenzylthioinosine binding in human placental membrane vesicles from brush-border and basal sides of the trophoblast.

The nucleoside transport activity of human placental syncytiotrophoblast brush-border and basal membrane vesicles was compared. Adenosine and uridine were taken up into an osmotically active space. Adenosine was rapidly metabolized to inosine, metabolism was blocked by preincubating vesicles with 2'-deoxycoformycin, and subsequent adenosine uptake studies were performed in the presence of 2'-deoxycoformycin. Adenosine influx by brush-border membrane vesicles was fitted to a two-component system consisting of a saturable system with apparent Michaelis-Menten kinetics (apparent Km approx. 150 microM) and a linear component. Adenosine uptake by the saturable system was blocked by nitrobenzylthioinosine (NBMPR), dilazep, dipyridamole and other nucleosides. Inhibition by NBMPR was associated with high-affinity binding of NBMPR to the brush-border membrane vesicles (apparent Kd 0.98 +/- 0.21 nM). Binding of NBMPR to these sites was blocked by adenosine, inosine, uridine, thymidine, dilazep and dipyridamole, and the respective apparent Ki values were 0.23 +/- 0.012, 0.36 +/- 0.035, 0.78 +/- 0.1, 0.70 +/- 0.12 (mM), and 0.12 and 4.2 +/- 1.4 (nM). In contrast, adenosine influx by basal membrane vesicles was low (less than 10% of the rate observed with brush-border membrane vesicles under similar conditions), and hence no quantitative studies of adenosine uptake could be performed with these vesicles. Nevertheless, high-affinity NBMPR binding sites were demonstrated in basal membrane vesicles with similar properties to those in brush-border membrane vesicles (apparent Kd 1.05 +/- 0.13 nM and apparent Ki values for adenosine, inosine, uridine, thymidine, dilazep and dipyridamole of 0.14 +/- 0.045, 0.54 +/- 0.046, 1.26 +/- 0.20, 1.09 +/- 0.18 mM and 0.14 and 3.7 +/- 0.5 nM, respectively). Exposure of both membrane vesicles to UV light in the presence of [3H]NBMPR resulted in covalent labeling of a membrane protein(s) with a broad apparent Mr on SDS gel electropherograms of 77,000-45,000, similar to that previously reported for many other tissues, including human erythrocytes. We conclude that the maternal (brush-border) and fetal (basal) surfaces of the human placental syncytiotrophoblast possess broad-specificity, facilitated-diffusion, NBMPR-sensitive nucleoside transporters.

Blood-tissue transport of substrates in the heart: studies by single circulation tracer dilution.

Passive capillary permeability is usually not the rate limiting step to movement of small lipid-insoluble molecules from plasma into the heart. At physiological concentrations the rate of carrier-mediated transport and or receptor binding of blood-borne substrates is often measurable in a single circulation through the coronary bed. Here I review some studies on uptake measured by the single-circulation multiple tracer dilution technique. The method consists of a sudden intra-arterial injection of a mixture of tracers followed by sequential rapid sampling of the venous outflow. Generally the tracers include a molecule confined to the intravascular space (e.g. albumin), another confined to the extracellular space and a third test tracer. The data analysis can give information about: a) capillary permeability-surface product (PS), b) endothelial cell membrane transport, c) the size of the interstitial space, d) transport across the sarcolemma, e) uptake by nerve endings, f) binding to receptors, g) rapid metabolic transformations. Studies were performed in the isolated perfused heart (dog and rabbit). Early observations on rapid tissue uptake of potassium and ouabain have recently been extended. Kinetics of unidirectional trans-sarcolemmal influx of lactate, a metabolic substrate for the myocardium and the neuronal uptake of amines, were investigated as well as the myocardium and the neuronal uptake of amines, were investigated as well as the effects of specific inhibitors for these transport mechanisms. Uptake was also measured for glucose, the amino acids L-phenylalanine, L-alanine and taurine; and for acetate and palmitate. Recent literature which proposes an important role for uptake and metabolism of adenosine by the endothelium is discussed. These studies are of interest for clinical applications such as positron emission tomography (PET) which makes use of short-life isotopes.

Effect of insulin, prostaglandin E1 and uptake inhibitors on glucose transport in the perfused guinea-pig placenta.

The effects of insulin, prostaglandin E1 (PGE1) and uptake inhibitors on unidirectional D-glucose influx at brush border (maternal) and basal (fetal) sides of the guinea-pig syncytotrophoblast were investigated in the intact, perfused guinea-pig placenta by rapid, paired-tracer dilution. Experiments were performed in either an in situ preparation artificially perfused through the umbilical vessels (intact maternal circulation) or in the fully isolated dually-perfused placenta in which both interfaces were studied simultaneously. Kinetic characterization of unidirectional D-glucose influx gave apparent Km values (mean +/- SEM) at maternal and fetal sides of 70 +/- 6 and 87 +/- 16 mM respectively; corresponding Vmax values were 53 +/- 3 and 82 +/- 6 mumol min-1g-1. At the fetal side (singly-perfused placenta) cytochalasin B (50 microM), ethylidene-D-glucose (100 mM) and PGE1 (1 microM) partially inhibited D-glucose uptake whereas cortisol (50 microM) and progesterone (100 microM) had no effect. Abolition of the sodium gradient across the fetal interface did not modulate the kinetics of influx. In the presence of 150 mu units ml-1 insulin (dually-perfused placenta), unidirectional uptake into the trophoblast and transplacental D-[3H]glucose transfer were unaltered. In contrast, prostaglandin E1 (1 microM) markedly reduced the Km and Vmax for D-glucose at both interfaces and the inhibitory effect was reflected in a reduction in specific transplacental D-glucose transfer. Further experiments showed that the isolated placenta releases prostaglandins (PGE; PGF2 alpha) into both circulations. Bilateral insulin perfusion did not affect either lactate release by the placenta or rapid metabolism of D-[14C]glucose to [3H]lactate (usually less than 10% effluent [14C]lactate in 5 min). An asymmetric degradation of exogenous insulin was observed in the dually-perfused placenta: uterine venous samples contained 24 +/- 7 microunits ml-1 immunoreactive insulin when compared to the arterial concentration (151 +/- 3 microU ml-1 perfusate) while no change was measureable in the fetal circulation within the same time period (152 +/- 5 microU ml-1). This asymmetry was confirmed in experiments employing [125I]insulin. These results demonstrate that glucose transport in the intact guinea-pig placenta occurs by a sodium-independent, cytochalasin B-inhibitable system which is insulin-insensitive. Prostaglandin E1 appeared to be a potent transport inhibitor which suggests that prostaglandins may be involved in the 'down' regulation of placental glucose transport in vivo.

Lysine and alanine transport in the perfused guinea-pig placenta.

The characteristics of L-lysine transport were investigated at brush-border (maternal) and basal (fetal) sides of the syncytiotrophoblast in the term guinea-pig placenta artificially perfused either through the umbilical vessels in situ or through both circulations simultaneously. Cellular uptake, efflux and transplacental transfer were determined using a single-circulation paired-tracer dilution technique. Unidirectional L-[3H]lysine uptake (%) (perfusate lysine 50 microM) was high on maternal (M = 87 +/- 1) and fetal (F = 73 +/- 2) sides. L-[3H]Lysine efflux back into the ipsilateral circulation was asymmetrical (F/M ratio = 2.3) and transplacental flux occurred in favour of the fetal circulation. Unidirectional lysine influx kinetics (0.05-8.00 mM) gave Km values of 1.75 +/- 0.70 mM and 0.90 +/- 0.25 mM at maternal and fetal sides, respectively; corresponding Vmax values were 1.95 +/- 0.38 and 0.87 +/- 0.10 mumol.min-1.g-1. At both sides, lysine influx (50 microM) could be inhibited (about 60-80%) by 4 mM L-lysine and L-ornithine and less effectively (about 10-40%) by L-citrulline, L-arginine, D-lysine and L-histidine. At the basal side: (i) lysine influx kinetics were greatly modified in the presence of 10 mM L-alanine (Km = 6.25 +/- 3.27 mM; Vmax = 2.62 +/- 0.94 mumol.min-1.g-1), but unchanged by equimolar L-phenylalanine or L-tryptophan; (ii) in the converse experiments, lysine (10 mM) did not affect the kinetic characteristics for either L-alanine or L-phenylalanine; (iii) L-lysine and L-alanine influx kinetics were not dependent on the sodium gradient; (iv) the inhibition of L-[3H]lysine uptake by 4 mM L-homoserine was partially (60%) Na+-dependent. At the maternal side the kinetic characteristics for alanine influx were highly Na+-dependent, while lysine influx was partially Na+-dependent only at low concentrations (0.05-0.5 mM). Bilateral perfusion with 2,4-dinitrophenol (1 mM) reduced L-[3H]lysine uptake into the trophoblast and abolished transplacental transfer. It is suggested that lysine transport in the guinea-pig placenta is mediated by a specific transport system (y+) for cationic amino-acids. The asymmetry in the degree of sodium-dependency at both trophoblast membranes may in part explain the maternal-to-foetal polarity of placental amino-acid transfer in vivo.

Transport and metabolism of adenosine in the perfused guinea-pig placenta.

1. Uptake and metabolism of adenosine were investigated from both maternal (M) and fetal (F) circulations of the isolated, dually perfused guinea-pig placenta by using a single-circulation paired-tracer [( 14C]sucrose as extracellular reference, and [3H]adenosine) dilution technique. 2. Maximal [3H]adenosine uptakes (percentage of dose) from adenosine-free perfusates were 75 +/- 1 and 87 +/- 2% (mean +/- S.E. of mean) at maternal and fetal blood-tissue interfaces respectively. Rapid backflux (percentage of influx) of tritium (labelled adenosine and/or adenosine derivatives) from the placental tissue into the ipsilateral circulation was higher at the fetal (24 +/- 2%) than at the maternal side (11 +/- 2%). 3. Tritium uptakes were reduced to 50 +/- 4 (M) and 60 +/- 6% (F) when the perfusion medium contained 100 microM-unlabelled adenosine; backflux was highly stimulated (44% M and 84% F). Neither uptake nor backflux were affected by inosine, uridine, adenine or hypoxanthine present in the perfusion medium (1 mM). 4. Tissue sequestration of tritium (5-6 min) was approximately 60% of the injected dose when perfusates were adenosine-free and 20% or less in the presence of 100 microM-adenosine. 5. Cellular uptake of [3H]adenosine at both sides of the placenta was markedly reduced by the nucleoside transport inhibitors dipyridamole (DIP, 10 microM) and nitrobenzylthioinosine (NBMPR, 5 microM). 6. Thin-layer chromatographic separation of [3H]inosine, [3H]hypoxanthine and [3H]phosphorylated derivatives in venous effluents following a bolus arterial injection of [3H]adenosine showed a greater fraction of metabolites at the fetal side (about 0.75) than at the maternal side (about 0.50). The percentage of [3H]inosine increased when perfusates contained 100 microM-adenosine and the effect was more marked in the fetal circulation. In the presence of DIP and NBMPR the fractional recovery of 3H-labelled metabolites was greatly reduced. 7. During steady-state perfusion of [3H]adenosine (100 microM) a maintained (5-60 min) tritium uptake of about 55% was observed and all the effluent activity was 3H-labelled metabolites [( 3H]adenosine was only 2.8 +/- 0.2%). Under these conditions high-performance liquid chromatography (HPLC) showed that effluents contained xanthine and urate at 16 +/- 1 and 23 +/- 2 microM respectively. 8. Transplacental transfer (6 min) of tritiated compounds (of which only 10-20% was [3H]adenosine) was often less than that of the extracellular marker [14C]sucrose in both maternal-to-fetal and fetal-to-maternal directions.(ABSTRACT TRUNCATED AT 400 WORDS)

A new form of asymmetry in epithelia: kinetics of apical and basal sulphate transport in human placenta.

Sulphate transport into and out of membrane vesicles prepared from both the brush-border and basal surfaces of the human placental trophoblast has been studied. For both surfaces of this epithelium clear evidence of trans-stimulation of influx is seen but for efflux, acceleration of labelled sulphate movement by addition of external sulphate is seen only in basal and not in brush-border membrane vesicles. This kinetic asymmetry may underlie the previously observed, but unexplained, finding that the concentration of sulphate is greater in the fetal than in the maternal circulation.

Characterization of folate uptake in guinea pig placenta.

Trophoblast uptake of folate and methotrexate (MTX) was investigated in an in situ or dually perfused (maternal and fetal side) guinea pig placenta by using a single-circulation, paired-tracer technique. For [3H]folate, uptake into trophoblast was rapid (s), high (60-80%) and Na+ independent, and exhibited negligible efflux on both poles of placenta. [3H]folate uptake could be inhibited by folate or 5-methyltetrahydrofolate (CH3THF) but not by equimolar (0.1 microM) MTX, folinic acid, aminopterin, trimoprim, or adenine when these compounds were present in perfusate. Inhibitory effect of folate was time dependent, and its complete reversal by folate-free perfusion required up to 20 min. This suggests the presence of a high-affinity folate carrier that exhibits a slow rate of self exchange. A sudden (bolus) increase of 10 microM folate of CH3THF caused a 70-80% inhibition of [3H]folate uptake, whereas folinic acid, MTX, and trimoprim were two- to threefold less effective. [3H]folate uptake was insensitive to DIDS, SITS, nicotine, ethanol, or phenytoin. For [3H]MTX, uptake was high (60-80%) on both sides of trophoblast, however, as distinct from [3H]folate, rapid and complete efflux followed the initial uptake. [3H]MTX uptake was not inhibited by 0.1 microM MTX, but equimolar folate or CH3THF were highly effective (90%) inhibitors; higher concentration (1 microM) of MTX reduced [3H]MTX uptake by 58%. Transplacental transfer of [3H]folate or [3H]MTX in excess of the leak pathway marker in either direction was not observed. Inhibition obtained by highly concentrated substrate bolus injections indicates saturation (less than 2 microM) of membrane folate carrier.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of amino acid influx and efflux at the basolateral plasma membrane of the salivary gland epithelium: effects of parasympathetic nerve stimulation.

Basolateral amino acid transport systems in the salivary epithelium of resting and secreting cat submandibular glands were characterized by means of a rapid paired-tracer dilution technique. Amino acid uptake was measured by comparison of venous tracer concentration profiles for a labeled amino acid and D-mannitol (an extracellular tracer of similar size) following an intra-arterial bolus injection of both radioactive molecules. Unidirectional uptake of 21 amino acids, dopamine, noradrenaline, and serotonin was quantified in non-secreting glands. During 8 Hz parasympathetic nerve stimulation, significant epithelial uptakes were measured for L-[3H] alanine and L-[3H] phenylalanine, but less than 0.2% of the injected amino acid was recovered in the collected saliva. In non-secreting glands, cross-inhibition studies of L-[3H] alanine, L-[3H] phenylalanine, and L-[3H] lysine uptake by unlabeled amino acid competitors and detailed kinetic influx experiments indicated that epithelial uptake was mediated by three distinct parallel transport systems: ASC (short-chain neutral), L (branched-chain and aromatic neutral), and y+ (cationic). Rapid metabolism of alanine was inhibited by aminooxyacetate, and the metabolic uncoupler dinitrophenol selectively accelerated the efflux of transported large neutral amino acids and L-lysine. Concurrent autoradiographic experiments suggest that transport sites for small and large neutral amino acids are localized in the basolateral plasma membrane of acinar, demilunar, and striated ductal cells.

Evidence of saturable uptake mechanisms at maternal and fetal sides of the perfused human placenta by rapid paired-tracer dilution: studies with calcium and choline.

Rapid uptake and efflux of 45Ca2+ and [3H]choline at the maternal and fetal interfaces of the syncytiotrophoblast in the dually-perfused human placenta was investigated by application of the single circulation paired-tracer dilution method (Yudilevich, Eaton, Short & Leichtweiss 1979). Cotyledons were perfused with Krebs-bicarbonate containing dextran (30 g/l; MW = 60-70,000) at 20 and 6 ml/min on maternal and fetal sides, respectively. The paired-tracer (test substrate and extracellular marker) technique consisted of an intra-arterial injection of a tracer bolus, followed by venous sampling over 5-6 min. There was a rapid (sec) uptake of 45Ca2+, followed by backflux (efflux into the ipsilateral circulation) which, over 5-6 min, was 59-100% on the fetal side. It was more variable but generally lower on the maternal interface. At 0.1 mM calcium, 45Ca2+ maximal uptake (Umax) was about 53% on the fetal side but on the maternal side it was variable and averaged 17%. At 2.4 mM calcium fetal side Umax was reduced to 40%. However, on the maternal side the effect was not consistent. Unidirectional influx (nmol/min per g) appeared to be not different on the two sides of the placenta. For [3H]choline (in choline-free perfusates) Umax was about 50% and 30% on fetal and maternal sides, respectively; tracer backflux was variable on the maternal side and averaged 50% on the fetal side. [3H]Choline uptake was highly inhibited by either 1.0 mM choline or the specific competitive inhibitor, hemicholinium-3 (0.1 mM). Specific transplacental transfer of 45Ca2+ (i.e. in excess of the extracellular marker) was not significant in either direction. For [3H]choline there was an apparent small excess (about 4%) preferential towards the fetal circulation. These findings in the human placenta are similar to those demonstrated previously in the guinea-pig placenta which suggested the existence of specific transport systems for choline and calcium on both sides of the syncytiotrophoblast.