Pentoxifylline ameliorates cerulein-induced pancreatitis in rats: role of glutathione and nitric oxide.

Reactive oxygen radicals, nitric oxide, and cytokines have been implicated in the initiation of pancreatic tissue damage and impairment of the pancreatic microcirculation in acute pancreatitis. Pentoxifylline is a methylxanthine derivative with rheologic and marked anti-inflammatory properties and inhibits the production of proinflammatory cytokines. We have examined whether pentoxifylline ameliorates interstitial edema, inflammatory infiltrate, and glutathione depletion associated with cerulein-induced pancreatitis. Cotreatment of animals with pentoxifylline significantly reduced cerulein-induced pancreatic inflammation and edema and attenuated the depletion of pancreatic glutathione and the increase in serum lipase activity, nitrate, and tumor necrosis factor-alpha levels. Pentoxifylline also prevented both mitochondrial swelling and damage to mitochondrial cristae caused by cerulein. Our findings provide an experimental basis for using pentoxifylline to attenuate inflammatory responses within the pancreas in acute pancreatitis and as an adjuvant in the treatment of acute pancreatitis.

Acute oxidative stress modulates secretion and repetitive Ca2+ spiking in rat exocrine pancreas.

The effects of the oxidant tert-butylhydroperoxide (t-buOOH) on carbachol-stimulated pancreatic secretion in the vascularly perfused rat pancreas have been studied in parallel with [Ca2+]i signalling and amylase output in perifused rat pancreatic acinar cells. Perfusion of the pancreas with t-buOOH (0.1-1 mM) caused a rapid and irreversible inhibition of carbachol-stimulated (3x10-7 M) amylase and fluid secretion. Pre-perfusion of the pancreas with vitamin C and dithiothreitol or a cocktail of GSH and GSH-precursor amino acids provided only marginal protection against the deleterious effects of t-buOOH, even though GSH levels were elevated significantly. In perifused pancreatic acini, repetitive [Ca2+]i spikes evoked by carbachol (3x10-7 M) were sustained for 40 min. t-buOOH (1 mM) acutely increased the amplitude and duration of Ca2+ spikes, then attenuated Ca2+ spiking and subsequently caused a marked and sustained rise in [Ca2+]i. t-buOOH-induced alterations in carbachol-stimulated [Ca2+]i signalling and amylase release in perifused pancreatic acini were prevented by vitamin C. Although vitamin C restored impaired Ca2+ signalling and maintained amylase output in pancreatic acini, it seems likely that oxidative stress inhibits fluid secretion irreversibly in the intact pancreas, resulting in a loss of amylase output. Thus, perturbations in [Ca2+]i signalling may not fully explain the secretory block caused by oxidative stress in acute pancreatitis.

Role of oxidative stress in the pathogenesis of acute pancreatitis.

During the last 10 years, the role of oxidative stress in pancreatitis and the benefits or otherwise of antioxidants has been the subject of numerous research papers. There is general agreement that glutathione and other sulphydryl compounds are depleted while lipid peroxidation is increased in pancreatic tissue during the development of acute pancreatitis. Treatment with antioxidants has been shown to reduce acinar cell injury and oedema in various animal models of pancreatitis, suggesting that the sustained generation of reactive oxygen species depletes cellular antioxidant defences. Evidence for a role for bradykinin and nitric oxide in pancreatitis has been conflicting with some studies suggesting these agents might ameliorate pancreatic dysfunction by enhancing pancreatic blood flow and secretion in response to bradykinin-stimulated generation of nitric oxide from endothelium, while other studies suggest that nitric oxide potentiates pancreatic oxidative stress. Thus, there is clearly a need for well-designed clinical trials to evaluate the protective role of antioxidant therapy in acute pancreatitis.

Insulin stimulates cationic amino acid transport activity in the isolated perfused rat pancreas.

The effects of exogenous insulin, glucagon and streptozotocin-diabetes on influx (15 s) of L-lysine via a cationic amino acid transporter resembling system y+ were investigated in the isolated perfused rat pancreas. In non-diabetic pancreata, transport of L-lysine was saturable with an apparent Km of 2.11 +/- 0.29 mM and Vmax of 2.21 +/- 0.20 mumol min-1 g-1 (n = 6). Bovine insulin (100 mu u ml-1) increased the maximal transport rate (Vmax = 3.49 +/- 0.30 mumol min-1 g-1, n = 4, P < 0.05) for L-lysine 1.6-fold without altering the Km. L-Lysine transport was not elevated significantly in diabetic pancreata, although insulin (100 mu u ml-1) enhanced transport to values measured in non-diabetic preparations. Human glucagon (1.5 x 10(-9) M) had no stimulatory effect on L-lysine transport. These findings provide the first evidence that exogenous insulin stimulates cationic amino acid transport activity in the exocrine pancreatic epithelium. Activation of the cationic pancreatic amino acid transporter may provide a mechanism to enhance the supply of L-arginine and thus sustain nitric oxide-mediated pancreatic secretion in response to islet hormones and secretagogues.

Induction of the antioxidant stress proteins heme oxygenase-1 and MSP23 by stress agents and oxidised LDL in cultured vascular smooth muscle cells.

Enhanced expression of the antioxidant stress proteins heme oxygenase-1 (HO-1) and macrophage stress protein (MSP23) by oxidative stress agents and oxidatively modified low density lipoproteins (LDL) was investigated in cultured porcine aortic smooth muscle cells. Treatment of smooth muscle cells with glucose oxidase, CdCl2 or diethylmaleate resulted in a time-dependent (6-48 h) induction of HO-1 and MSP23 expression. Exposure of cells to 100 micrograms protein/ml highly oxidised LDL increased the expression of HO-1 and MSP23 within 24 h, and the induction was dependent on the degree of LDL oxidation. The induction of HO-1 and MSP23 may thus play an important cytoprotective role against oxidative stress in atherogenesis.

A role for gamma-glutamyl transpeptidase and the amino acid transport system xc- in cystine transport by a human pancreatic duct cell line.

1. The roles of the gamma-glutamyl cycle and the anionic amino acid transport system xc- in mediating L-cystine uptake were investigated in cultured human pancreatic duct PaTu 8902 cells. This cell line exhibits morphological features of normal pancreatic duct cells and expresses gamma-glutamyl transpeptidase (gamma-GT, EC 2.3.2.2), an enzyme involved in the metabolism and regulation of intracellular glutathione (GSH). 2. Uptake of L-cystine (10 microM) was linear for up to 10 min, temperature dependent, Na+ independent, saturable (Michaelis-Menten constant (Km), 86 +/- 25 microM; maximal velocity (Vmax), 109 +/- 33 nmol (mg protein)-1 h-1) and reduced by 80-90% by a 50-fold excess concentration of L-glutamate and L-homocysteic acid, but not L-aspartate. These transport properties resemble those described for system xc-, which exchanges cystine for intracellular glutamate. 3. Acivicin, a known inhibitor of gamma-GT, decreased gamma-GT activity from 2.58 +/- 0.96 to 0.97 +/- 0.11 mU (mg protein)-1 and decreased the initial rates of L-cystine and L-glutamine uptake by 41-55%. Anthglutin (1-gamma-L-glutamyl-2-(2-carboxyphenylhyl)hydrazine), a structurally different inhibitor of gamma-GT, also caused a concentration-dependent (0.01-1 mM) decrease in gamma-GT activity and L-cystine uptake. 4. Neither acivicin nor anthglutin inhibited the uptake of L-glutamate, a poor substrate for gamma-GT. 5. In the presence of a 500-fold excess concentration of glutamate, which should abolish entry of cystine via system xc-, the remaining fraction of cystine transport was inhibited by 50% by acivicin, suggesting that transport is, in part, dependent on the activity of gamma-GT. 6. Cystine transport was also 60-80% inhibited by a series of gamma-glutamyl amino acids (5 mM) including gamma-glutamyl-glutamate, gamma-glutamyl-glutamine and gamma-glutamyl-glycine. alpha-Dipeptides inhibited cystine transport by only 6-22%. 7. These findings demonstrate that in human pancreatic duct PaTu 8902 cells, cystine uptake is mediated by system xc- (50-60%) and the gamma-glutamyl cycle. Our results provide the first evidence linking gamma-GT with cystine transport in human epithelial cells and are of relevance in view of the importance of cystine as a sulphur amino acid source for GSH synthesis in cells exposed to oxidative stress.

Properties of a cell volume-sensitive potassium conductance in isolated guinea-pig and rat hepatocytes.

1. Whole-cell voltage clamp and intracellular recording techniques were used to study the increase in K+ conductance that accompanies swelling in isolated guinea-pig and rat hepatocytes in short-term culture at 37 degrees C. 2. Swelling was induced (i) by the application of pressure (15 cmH2O) to the shank of the patch pipette, (ii) by exposing the cells to hypotonic solutions and (iii) as a consequence of leakage of electrolyte from an intracellular microelectrode. 3. Applying pressure to the patch pipette caused a large outward current (approximately 600 pA) to develop in guinea-pig hepatocytes voltage clamped to 0 mV. This current reversed direction at -86 mV, close to the reversal potential for K+, EK (-93 mV), and is attributable to the activation of a K+ conductance. 4. Spectral analysis of current noise during this response suggested a single-channel conductance of 7 pS, though this may well be an underestimate. The power spectrum could be fitted by the sum of two Lorentzian components, with half-power frequencies of 7 and 152 Hz. Seventy per cent of the variance was associated with the lower frequency component. 5. The steady-state current-voltage relationship for guinea-pig hepatocytes, as determined by whole-cell recording, was linear over the range -70 to +40 mV both before and during the increase in K+ conductance induced by swelling. 6. Confirming earlier work, intracellular recording using microelectrodes filled with 1 M-potassium citrate sometimes resulted in a slow hyperpolarization and a large rise in input conductance. These changes are also attributable to an increase in K+ conductance as the cell swelled because of leakage from the electrode. 7. Application of hypotonic external solutions during intracellular recording caused hyperpolarization and an increase in conductance. Conversely, hypertonic solution evoked depolarization and a fall in conductance in partly swollen cells. 8. The volume-activated K+ conductance was reversibly blocked by cetiedil, which caused half-maximal inhibition at 2.3 microM. Bepridil, quinine and barium were also effective, with IC50s (concentrations giving 50% maximal inhibition) of 2.7, 12 and 67 microM respectively. 9. Much greater concentrations of cetiedil and bepridil (IC50 approximately 1 mM and 77 microM respectively) were required to inhibit the loss of K+ which follows the application of angiotensin II (100 nM) to guinea-pig hepatocytes, and which occurs via Ca(2+)-activated K+ channels. Our evidence suggests that the activation of K+ channels by cell swelling is Ca2+ independent.(ABSTRACT TRUNCATED AT 400 WORDS)

Influx and incorporation into protein of L-phenylalanine in the perfused rat pancreas: effects of amino acid deprivation and carbachol.

The rate of protein synthesis in the isolated perfused rat pancreas was measured from the rate of incorporation of L-[3H]phenylalanine into total protein, and was compared with the transport of this amino acid into the epithelium. Unidirectional (15 s) and net (15-30 min) uptake of L-[3H]phenylalanine was measured relative to D-[14C]mannitol (extracellular marker) using a cell loading technique. The fractional rate of protein synthesis in the pancreas was also measured in vivo using a flooding dose technique and found to be 118 +/- 10% day-1 (corresponding to an absolute rate of incorporation of L-Phe into protein of 36.1 +/- 3 nmol min-1 g-1) in overnight fasted rats. Compared with the in vivo rate, the perfused pancreas exhibited a markedly lower rate of protein synthesis which increased significantly when amino acids were added to the perfusate (15.6 +/- 1.9 vs. 22.5 +/- 0.9% day-1 or 4.7 +/- 0.6 vs. 6.9 +/- 0.3 nmol L-Phe min-1 g-1). Carbachol (3 x 10(-7) M) stimulated protein synthesis provided amino acids were also supplied in the perfusate. Protein synthesis rates measured under all conditions in vivo and in vitro were at least an order of magnitude lower than the unidirectional influx (121 +/- 14 nmol min-1 g-1) of L-phenylalanine into the pancreatic epithelium. These results demonstrate that amino acid transport across the basolateral membrane of the epithelium is not rate-limiting for pancreatic protein synthesis.

Pancreatic microvascular permeability in caerulein-induced acute pancreatitis.

Microvascular permeability was studied in the isolated perfused rat pancreas using a rapid multiple indicator-dilution technique. Capillary extractions, permeability-surface area products (PS), and extravascular volumes of distribution (EVV) were determined for 22Na+, 51Cr-labeled EDTA, [57Co]-cyanocobalamin (B12), and 125I-labeled insulin at various perfusion flows. Permeability to albumin was negligible. PS for Na+ and EDTA increased with increasing flow, whereas PS for cyanocobalamin and insulin approached diffusion-limited exchange at flows greater than 3 ml.min-1.g-1. Permeability coefficients for Na+, EDTA, B12, and insulin were 36, 22, 11, and 3.48 x 10(-5) cm/s, respectively, and the permeability ratio for B12/insulin (3.16) indicated restricted diffusion to insulin. In the presence of unlabeled B12 and insulin EVV (0.15-0.19 ml/g) for EDTA, B12 and insulin approximated the interstitial volume. Caerulein-induced pancreatitis or treatment with the synthetic protease inhibitor camostate had no significant effects on permeability. In caerulein-treated rats, EVV for B12 was elevated (0.17 +/- 0.01 vs. 0.28 +/- 0.06; P less than 0.01), reflecting the interstitial edema associated with this model of pancreatitis. Permeability of the rat pancreatic microvasculature is similar to that of other fenestrated tissues, but it is 10- to 20-fold greater than that of continuous capillaries. Contrary to previous assumptions, permeability does not appear to be increased after induction of acute interstitial pancreatitis.

Cis-inhibition and trans-stimulation of cationic amino acid transport in the perfused rat pancreas.

Transport of cationic amino acids in the isolated perfused rat pancreas was studied using dual-isotope dilution techniques. At 50 microM substrate concentration, unidirectional tracer uptakes for L-arginine (56 +/- 1%), L-lysine (49 +/- 2%), and L-ornithine (44 +/- 3%) were followed by rapid tracer efflux. In the presence of Na+, influx of L-arginine [Michaelis constant (Km) = 1.74 +/- 0.15 mM, maximum velocity (Vmax) = 1.97 +/- 0.07 mumol.min-1.g-1] and L-lysine (Km = 2.48 +/- 0.17 mM, Vmax = 2.42 +/- 0.08 mumol.min-1.g-1) was mediated by a common transport system, sensitive to cis-inhibition by L-ornithine, 2,4-L-diaminobutyric acid, D-lysine, and D-arginine. Substrates for system A [alpha-(methylamino)isobutyric acid] and an anionic carrier (L-aspartate) were poor cis-inhibitors of L-arginine entry. Removal of Na+ resulted in a 40% reduction in cationic amino acid influx. After cell loading (20 min), L-[3H]-lysine cleared predominantly from a slowly exchanging pool with a rate constant of 5.97 +/- 0.67 min. An influx/efflux permeability ratio of 14.5 +/- 1.6 was determined, and efflux of L-lysine was trans-stimulated by vascular challenges with cationic or large neutral amino acids. The specificity, relative Na+ independence, and exchange properties of this saturable cationic amino acid transporter in the pancreatic epithelium resemble those reported for system y+ in cultured fibroblasts and hepatocytes.

Significance of gamma-glutamyltranspeptidase in exocrine pancreatic amino acid transport.

The exocrine pancreas is rich in gamma-glutamyltranspeptidase (GGT, EC 2.3.2.2) and exhibits high rates of amino acid transport and protein synthesis. The role of the gamma-glutamyl cycle in mediating neutral amino acid transport in the isolated perfused rat pancreas was investigated using acivicin, an inhibitor of GGT, and a rapid dual isotope dilution technique. When treatment in vivo with acivicin (50 mg/kg) was followed 1 h later by continuous perfusion of the isolated pancreas with 10 microM acivicin, GGT levels decreased from 53 +/- 3 IU/g to 4.9 +/- 1.5 IU/g. This marked inhibition of GGT activity was not associated with decreased uptake for either L-alanine or L-glutamine, suggesting that the gamma-glutamyl cycle plays a negligible role in amino acid transport across the basolateral membrane of the pancreatic epithelium.

Active potassium absorption in rat distal colon.

1. Active potassium (K+) absorption in rat distal colon was investigated by measuring mucosal-to-serosal (JK, ms) and serosal-to-mucosal (JK, sm) 42K+ fluxes (mu equiv h-1 cm-2) across isolated stripped mucosa under short-circuit conditions in normal and dietary Na-depleted animals. As previously demonstrated, removal of Na+ from both mucosal and serosal solutions bathing the normal colon slightly increased net K+ absorption as a result of inhibition of JK, sm without affecting JK, ms, while in the Na-depleted group net K+ secretion (-0.54 +/- 0.11) was converted to a marked net K+ absorption (1.68 +/- 0.30, P less than 0.001). 2. In both groups of animals in Na(+)-free Ringer solution, JK, ms exhibited saturable and linear components, while JK, sm was a linear function of [K+]. Estimated affinity constants (mM) for saturable net K+ absorption were similar in normal (0.52 +/- 0.12) and Na-depleted (0.67 +/- 0.11) animals; however, there was a greater than 3-fold increase in the saturable flux (Jmax) from 0.54 +/- 0.04 in the normal colon to 1.78 +/- 0.08 mu equiv h-1 cm-2 in Na-depleted animals. 3. Mucosal orthovanadate (100 microM) inhibited JK, ms in both normal (control, 0.66 +/- 0.05 vs. orthovanadate, 0.36 +/- 0.03 mu equiv h-1 cm-2, P less than 0.001) and Na-depleted animals (control 1.20 +/- 0.13 vs. orthovanadate 0.77 +/- 0.07 mu equiv h-1 cm-2, P less than 0.01) without affecting JK, sm or the short-circuit current. In the Na-depleted group mucosal omeprazole or SCH28080 (100 microM), inhibitors of gastric K(+)-H(+)-ATPase, insignificantly or slightly reduced (by 10%) JK, ms respectively; in contrast, mucosal ouabain (1 mM) markedly inhibited JK, ms (control, 1.61 +/- 0.16 vs. ouabain, 0.83 +/- 0.98 mu equiv h-1 cm-2, P less than 0.001). 4. Mucosal Na+ appeared to be a competitor of K+ uptake across the apical membrane. 5. These results indicate that dietary Na-depletion increases electroneutral K+ absorption by increasing its transport capacity and suggest that the mechanism of this active K+ absorption process may involve an apical K(+)-ATPase with properties that are unlike the gastric K(+)-H(+)-ATPase but similar, in part, to Na(+)-K(+)-ATPase.

Characterization of aldosterone-induced potassium secretion in rat distal colon.

The role of apical and basolateral membranes in aldosterone-induced active potassium (K) secretion in rat distal colon was investigated by measuring mucosal-to-serosal (Jms) and serosal-to-mucosal (Jsm) 42K fluxes (mueq.h-1.cm-2) across isolated stripped mucosa under short-circuit conditions in normal and secondary-hyperaldosterone animals. In normal colons mucosal tetraethylammonium (TEA; 30 mM) or barium (Ba; 5 mM), but not cesium (Cs; 15 mM), reduced Jsm without affecting Jms. In aldosterone animals (a) net K secretion (-0.54 +/- 0.11) was converted to net K absorption (0.63 +/- 0.15) by mucosal TEA, which produced a marked reduction in Jsm (0.82 +/- 0.07) and an increase in Jms (0.35 +/- 0.07). In contrast mucosal Ba resulted in a relatively smaller reduction in JK(sm) without altering JK(ms), whereas mucosal Cs was ineffective; (b) serosal bumetanide or the removal of serosal Na or Cl markedly inhibited JK(sm and abolished net K secretion; and (c) serosal ouabain (1 mM) produced qualitatively similar effects to those of serosal bumetanide. These results demonstrate that (a) normal rat distal colon contains apical TEA- and Ba-sensitive K channels; (b) aldosterone induces TEA-sensitive and Ba-sensitive apical K channels; (c) aldosterone-induced K secretion requires both the Na,K-pump and Na-K-2Cl cotransport for K uptake across the basolateral membrane; and (d) alteration of any of these processes results in inhibition of aldosterone-induced active K secretion simultaneously with stimulation of K absorption.

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)

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.

Transport of folates at maternal and fetal sides of the placenta: lack of inhibition by methotrexate.

Folate (pteroylglutamate) and methotrexate rapid (seconds) uptake by the trophoblast was investigated from either the maternal or fetal circulations of the isolated dually-perfused guinea-pig placenta. Tissue uptake was measured by using a single-circulation paired-tracer (3H-test and 14C-extracellular marker) technique. [3H]Folate uptakes were 80 and 52% (mean) in perfusates without unlabelled folate, on maternal and fetal sides, respectively. There was negligible 3H-tracer backflux into the circulation up to 6 min probably due to metabolic sequestration. [3H]Methotrexate uptakes were about 85 and 22% on maternal and fetal sides, respectively; however these uptakes were followed by rapid and complete backflux of the label. Specific transplacental transfer of [3H]folate or [3H]methotrexate in either direction was not detectable within 5-6 min. At the brush-border side (maternal) uptake of [3H]folate was highly inhibited by 100 nM unlabelled folate or its reduced form, methyltetrahydrofolate (the main form in plasma); however, equimolar methotrexate (an antifolate chemotherapeutic agent) failed to produce any inhibition of folate uptake. Our findings demonstrate that on both sides of the placenta a high-affinity transport system exists for trophoblast uptake of folate compounds. For methotrexate, either a separate transport system may exist or methotrexate may have a very low affinity for the folate system. These results are distinct from the findings reported in mouse L1210 leukemia cells.

Characterization of choline transport at maternal and fetal interfaces of the perfused guinea-pig placenta.

Unidirectional influx and efflux of choline into the syncytiotrophoblast were investigated from both maternal and fetal circulations of the perfused guinea-pig placenta by using a single-circulation paired-tracer (extracellular reference and test substrate) dilution technique. Cellular uptake of [3H]choline at 0.05 mM was (mean percentage +/- S.E. of mean, n = 14 placentae) 51 +/- 2 and 49 +/- 2, on maternal and fetal sides, respectively. Kinetics of unidirectional influx (0.05-4.0 mM-choline) indicated the existence of saturable and non-saturable components on both sides: on maternal and fetal interfaces the Km (mM) values were respectively, 0.12 and 0.13, the Vmax (mumol min-1 g-1) values, 0.08 and 0.07 and the apparent linear transfer constants (min-1 g-1) 0.11 and 0.12. Efflux of [3H]choline from the placenta back into the ipsilateral circulation (backflux) was generally fast (20-60% in 5-6 min) and asymmetric with the fetal: maternal ratio usually above unity. Transplacental specific choline transfer in the dually perfused placenta, when observed, was small (less than 10% of the injected dose) following tracer injections in either direction based on the 5-6 min collection of the contralateral circulation (at 0.05 mM-choline). Placental retention of [3H]choline at the end of the 5-6 min period was about 25% of the injected dose when the tracers were injected from either circulation. Analogues of choline such as hemicholinium-3, thiamine, ethanolamine and N,N-dimethylethanolamine inhibited choline unidirectional influx, whereas betaine and acetate had no effect. The absence of the normal sodium gradient (perfusate sodium was replaced by Tris or by lithium) did not inhibit choline transport. The metabolic inhibitors dinitrophenol (1.0 mM) and potassium cyanide (1.0 mM) were essentially ineffective (up to 40 min perfusion). The sulphydryl reagent N-ethylmaleimide did not appear to inhibit the influx, in comparison with its effect on [3H]choline backflux which was greatly accelerated, resulting in a dramatic reduction in placental net uptake of the label. Our findings show that choline transport into the placenta is a rapid carrier-mediated process occurring at both maternal and fetal sides of the trophoblast, at physiological blood concentrations. This cellular uptake is possibly related to the synthesis of acetylcholine, which is known to occur in human placental tissue. Specific transplacental transfer of choline was a very slow process under the conditions of our experiments and this contrasted with the observed fast and high uptake into the trophoblast.

Asymmetric calcium influx and efflux at maternal and fetal sides of the guinea-pig placenta: kinetics and specificity.

Unidirectional influx of calcium across maternal and fetal sides of the syncytiotrophoblast was investigated in the guinea-pig placenta by using a rapid (less than 30 s) paired-tracer dilution technique. Experiments were performed in an in situ placenta artificially perfused through the umbilical vessels or in an isolated placenta in which both the maternal and fetal circulations were perfused. At equimolar Ca2+ concentrations, unidirectional calcium influx was always significantly lower on the maternal side than on the fetal side. Saturation kinetics were observed: on the fetal side the estimated Km was 1.8 +/- 0.7 mM and Vmax was 1.66 +/- 0.28 mumol/min X g (mean +/- S.E. of mean) and on the maternal side Km ranged from 0.18 to 1.15 mM and Vmax ranged from 0.12 to 0.59 mumol/min X g. When the inhibition of calcium influx was investigated on the fetal side of the trophoblast by using competing cations, the following sequence was observed: Ba2+ greater than Ca2+ congruent to Ni2+ greater than Sr2+ greater than Mg2+ congruent to Li+. Efflux of 45Ca2+ from the trophoblast into the ipsilateral circulation (backflux) was rapid (20-100% in 6 min) and asymmetric since the fetal:maternal ratio was 1.35 +/- 0.11 (mean +/- S.E. of mean) in the presence of 0.1 mM-Ca2+. In the dually perfused placenta, transplacental transfer (6 min) of 45Ca2+ varied over a wide range (0-80%); however, it was similar to that of the extracellular reference tracer, 22Na+, in either maternal-to-fetal or fetal-to-maternal directions. It is suggested that this is a consequence of the 'leakiness' of the dually perfused placenta since the transplacental transfer of 22Na+ and D-[3H]mannitol (or L-[14C]glucose) measured simultaneously were also variable but similar. Transplacental transfer of 45Ca2+ could not be used to characterize specific calcium-transport mechanisms, whereas highly sensitive trophoblast uptake measurements were provided by the single-circulation, paired-tracer technique. Our findings suggest the presence of a specific carrier-mediated transport system for calcium on both maternal and fetal surfaces of the trophoblast. The asymmetries in unidirectional influx into the trophoblast and rapid backflux indicate a mechanism by which the net transfer of calcium from the maternal to the fetal circulation is maintained in favour of the fetus.