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We investigated the specific uptake (reference: [H3]-L-glucose) as a measure of membrane transfer of [C14]-labeled L-ascorbic acid (AA), L-dehydroascorbic acid (DHA) and diketogulonic acid using the single injection, double tracer dilution method in the artificially perfused lobe of the near-term human placenta. The uptake of DHA (40-60%) on both the fetal and the maternal side was 3-6 times higher than the uptake of AA, whereas an uptake of diketogulonic acid was not detected. AA transport was slightly higher on the maternal side. Uptake of DHA was suppressed by phloretin and cytocholasin B, whereas AA transfer was not affected. Low sodium concentrations inhibited the uptake of DHA. D-glucose (> 20 mmol/l) inhibited the DHA uptake, and DHA inhibited D-glucose uptake but not L-alanine uptake. The Km value (self inhibition) for DHA was 6-14 mmol/l. Vitamin C enters the trophoblast predominantly as L-dehydroascorbic acid. Its transfer through the microvillous and basal membrane might use the glucose transporter, however, a specific sodium-dependent pathway is not ruled out. Our transfer data suggest an intracellular pool of vitamin C which fills up with increasing plasma DHA-concentrations.

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The development of a new technique to investigate maternal-fetal transfer across the near term guinea-pig yolk sac placenta by in-situ perfusion of the yolk sac vessels is described. The maternal-fetal transfer of labeled water, D- and L-glucose, O-methyl-D-glucose (oMDG), D- and L-alanine, D- and L-aspartate, L-lactate and alpha-amino-isobutyric acid (AIBA) was investigated after injection of these substances into the maternal circulation. After 15 min of perfusion at 0.5 ml/min the water clearance was 132 +/- 12 microliters/min (SEM, n = 30). The clearances for D- or L-glucose were less than 1.2 microliters/min. The activity of label in the venous yolk sac perfusate of all other substances was not different from background activity when 14C-label was used. The clearance of 3H-L-alanine approached the clearance value of water. The total uptake (as defined for single-injection double tracer dilution experiments) from the perfusate of D-glucose, oMDG, alanine and aspartate in comparison to L-glucose was also studied. Mean D-glucose uptake was 11.2 +/- 1.9 percent (n = 8), it was significantly reduced to 4.9 +/- 2 percent (n = 5) by cytochalasin B (1 X 10(-4) mmol/l), and by increasing concentrations of D-glucose (1 to 20 mmol/l, n = 4). The uptake of oMDG was 8.8 +/- 1.5 percent (n = 8). L-alanine uptake was 25 +/- 3.4 percent, D-alanine uptake was 8.3 +/- 1.5 percent (n = 12). Both uptake values were decreased significantly by 10 mmol/l L-alanine, but unaffected by [Na+] (less than 15 mequ/l). There was no uptake of AIBA. The uptakes of L-aspartate were 34.9 +/- 3.7 percent and of D-aspartate 40.4 +/- 4.8 percent (n = 11). Both uptake values were significantly and reversibly reduced by 1 mmol/l L-aspartate and D-aspartate, and by low [Na+] (less than 15 mequ/l). It is concluded that water can move by diffusion from maternal circulation into the yolk sac capillaries in considerable amounts whereas the contribution of the yolk sac placenta to fetal nutrition with D-glucose, L-alanine and L-aspartate is negligible. The membranes of yolk sac cells contain specific transport systems for D-glucose, D-/L-alanine and D-/L-aspartate transfer. The function of the vitelline placenta in the near-term guinea-pig is comparable more to the gut than to the chorio-allantoic placenta.

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In isolated guinea-pig placentae the transfer of D-[3H]glucose and L-[14C]glucose was investigated in single injection (SIE) and steady state (SSE, n = 11, placental weight 4.5 +/- 1.14 g) experiments at constant perfusion flow rates (3 ml/min). In SIE, a mixture of D- and L-glucose was injected as a bolus into either the fetal or maternal side of the placenta, uptake curves were obtained and the maximal extraction values Umax were derived. From these the membrane rate constants Kmc and Kfc of either the maternal or fetal side of the trophoblast membrane were calculated. The specific placental transfer rate constants were computed for the maternal-fetal (Kspec,mf) and fetal-maternal (Kspec,fm) direction from the amount of label transferred to the acceptor side in either SIE or in SSE (where labelled D- and L-glucose had been added to the stock solution). The chemical concentration of D-glucose was changed (5, 50 and 100 mmol/l, n = 11) and it was found that all rate constants decreased with increasing D-glucose concentration. At a D-glucose concentration of 5 mmol/l, Kmc was 3.11 +/- 1.59 ml/min (n = 6) and Kfc 2.69 +/- 0.5 ml/min (n = 5), the combined membrane rate constant (which determines the total placental transfer) was estimated to be 1.44 ml/min. This value was not significantly smaller than the mean (both directions) transfer rate constant Kspec in SIE (1.51 +/- 0.89 ml/min) or SSE (1.52 +/- 0.56 ml/min). Thus the results from uptake and transfer experiments are consistent. From the rate constants D-glucose fluxes at different chemical D-glucose concentrations were estimated. Whereas the mean Km values for all fluxes based on the various rate constants were about 30 mmol/l, the maximal flux Vmax was highest at the maternal trophoblast membrane (159.1 +/- 70.2 mumol/min), it was 107.9 +/- 11.6 mumol/min at the fetal side and 67.3 +/- 7.9 mumol/min in SIE or SSE for placental transfer in both directions. It is concluded that D-glucose carriers predominate at the maternal side of the trophoblast, and that maternal-fetal glucose transfer is proportionate to the transplacental glucose concentration difference within the physiological range of glucose concentrations.

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Pulsed wave Doppler sonography was used to determine peak systolic velocity (Vpeak), mean maximum velocity (Vmean) and Pulsatility Index (PI) in the descending aorta of chronically instrumented, unanesthetized fetal sheep (days 120-130 of pregnancy). Measurements were done 1-4 days after surgery using a duplex mechanical sector scanner (4.5 MHz) and offset attached pulsed wave Doppler probe (2.25 MHz) during periods of control, intrauterine cooling and rewarming. Mean values derived from the envelope curve of maximum shift frequencies were (controls): thoracic: Vmean 60.9 +/- 9.1 cm/sec, Vpeak 118.9 +/- 17.8 cm/sec, PI 1.57 +/- 0.24; abdominal: Vmean 49.6 +/- 10.9 cm/sec, Vpeak 98.9 +/- 20.4 cm/sec, PI 1.59 +/- 0.41. Vpeak was positively correlated to arterial pressure (r = 0.23) and PI showed an inverse correlation to heart rate (r = -0.22). During cold stress (reduction of fetal core temperature by 2.7 degrees C) and consecutive rewarming Vpeak and Vmean were positively correlated to fetal core temperature and arterial pressure (r = 0.2-0.26). PI revealed a weak negative dependency from core temperature and heart rate (r = -0.19 and -0.18 respectively).

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Experiments were performed to investigate the mechanisms of bicarbonate/CO2 transport in the isolated guinea pig placenta. The maximal uptake of [14C]-bicarbonate/CO2 (reference [3H]-L-glucose) was about 60 per cent on both the fetal and maternal side, using the method of paired tracer single injection dilution technique. Carbonic anhydrase inhibitors such as acetazolamide (0.1 and 1 mmol/l) and the poorly permeant aminothiadiazole (0.5 mmol/l) reduced bicarbonate/CO2 uptake on both sides by 25-50 per cent. Inhibition was significantly higher on the fetal side. When lactate (5 mmol/l) was added to the acceptor side solution, the uptake of bicarbonate/CO2 increased 10-20 per cent. DIDS (1 mmol/l) decreased bicarbonate/CO2 uptake additionally under the simultaneous influence of acetazolamide. It is concluded that at least two mechanisms for placental bicarbonate transfer exist. A portion of bicarbonate, predominantly on the fetal side and outside the trophoblastic cells, is converted to CO2 which then diffuses into the tissue and crosses the placental barrier. Another part of bicarbonate is transported as ion using an anion exchange system, which exists in both the multivillous and the basal membranes of the syncytium. The first system is important for the excretion of fetal CO2 into the mother.

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Fetal sheep (n = 13) were chronically instrumented to measure temperatures in the maternal femoral artery (MAT), the amniotic fluid (AFT), the fetal brown adipose tissue (BFT) and the fetal arterial blood (DAT). Cooling loops were inserted into the amniotic cavity. In 4 fetuses osmotic minipumps delivering triiodothyronine (T3) were implanted subcutaneously. One to seven days after surgery the following results were obtained: 1) During control DAT was 0.59 +/- 0.2 degrees C (SD), BFT 0.60 +/- 0.24 degrees C and AFT 0.38 +/- 0.31 degrees C higher than MAT. T3 levels in treated fetuses were 3.4 +/- 1.5 micrograms/l. 2) Infusion of norepinephrine (NE) (5.2 +/- 0.9 micrograms/min per kg fetal body weight) with phentolamine (26.1 +/- 4.3 micrograms/min per kg) into a fetal vein did not change temperatures. 3) During cooling (-53 +/- 15 W) MAT decreased 0.45 +/- 0.3 degrees C, DAT 1.9 +/- 0.39 degrees C, BFT 1.61 +/- 0.52 degrees C and AFT 4.2 +/- 1.8 degrees C. 4) The amniotic fluid was cooled until steady state temperatures were achieved. Then propranolol (26.1 +/- 4.3 micrograms/min per kg) or suxamethonium (3 +/- 1 mg/kg) were introduced into the fetal vein. No consistent and significant changes of temperatures could be detected. It is concluded that 1) lowering the fetal core temperature by 1.6 - 1.9 degrees C and its ambient temperature (AFT) by 4.2 degrees C does not induce shivering or non-shivering thermogenesis suppressible by pharmacologic agents, 2) thermogenesis in fetal brown adipose tissue cannot be induced by NE (with or without supplemention of T3).(ABSTRACT TRUNCATED AT 250 WORDS)

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An active transport for amino acids exists in maternofetal direction through the hemochorial placenta of man and guinea pig. With respect to the trophoblastic barrier some properties of the transplacental transport at both sides of this tissue are investigated. The results are compared to the methods of the transport of amino acids at intestine and kidney. The maternal and the fetal (basic) membrane can be investigated separately and one after another at one placenta by the technique of the bolus injections. The results were as follows: The 3H-L-alanine uptake is significantly higher by maternal injection than by fetal injection. Opposite to this the backflux is higher out of the trophoblast into the perfusion medium of the donor side at fetal injections. Out of this transplacental transport is higher in materno-fetal direction than in the opposite direction (p less than or equal to 0.05). A sodium dependence of the 3H-L-alanine transport is found at the maternal and fetal side. Nevertheless, the withdrawal of sodium ions inhibits more strongly at the maternal than at the fetal side. The sodium-dependent transport system predominates at the maternal membrane of the trophoblasts. Phloretin only inhibits significantly the uptake (maximal) and the transport of 3H-L-alanine at the fetal side (at the basic membrane). That means that the sodium independent by phloretin inhibited transport system exists mostly, if not totally, at the fetal membrane. Ouabain reduces uptake (maximal and total) and transport of 3H-L-alanine only significantly at the maternal side (p less than or equal to 0.05). Therefore the active sodium transport must be mostly localized at the maternal membrane. The flux of L-alanine diminishes with increasing L-alanine concentrations at the fetal and maternal side. The determinations of the transport capacity (Jmax) and the transport affinity (Km) show that Jmax is significantly higher at the maternal side and Km at the fetal side (p less than or equal to 0.05). By means of these results a model can be described to characterise the transport of amino acid through the placenta.

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The clearance curves of xenon-133 were registered with an external detector situated above the isolated, dually perfused guinea-pig placenta. The curves were analyzed with a computer program that calculated the parameters of a sum of exponentials. Fetal and maternal flow rates were varied systematically. It was attempted to relate subdivisions of the preparation and their flow rates to the results of washout analysis. No unequivocal correspondence between anatomical and calculated compartments could be detected, but mean calculated and real relative perfusion rates correlated reasonably well. It is concluded that the compartment analysis of xenon clearance curves is not advantageous to other methods.

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In previous studies our group has shown that unilateral complete ureteral ligation is followed by flow reduction, which clearly precedes renal atrophy and contributes to hydronephrotic renal cortical damage by ischemia. Long-term followup studies in dogs have demonstrated that increased hydronephrotic vascular resistance could be eliminated by infusion of 2-benzyl-2-imidazole, an inhibitor of thromboxane A2 synthesis. This was shown after 1 and 4 weeks of complete renal obstruction, and there was no such effect on the vascular resistance of the contralateral, unobstructed kidney. Flow reduction and vascular resistance were not influenced by the same inhibition of prostaglandin synthesis after 8 weeks of ureteral occlusion, although renal perfusion still responded to a nonspecific vasodilator, such as dopamine. Thus, active preglomerular vasoconstriction, influenced by imidazole, is present only when renal atrophy develops. Irreversible parenchymal loss, judged by renal cortical thickness, begins after 1 to 2 weeks and is complete 6 to 8 weeks after ureteral ligation. Once renal atrophy is established (that is, after 8 weeks of ureteral occlusion), flow reduction represents loss of renal parenchyma, and not active vasoconstriction. The specificity of the possible thromboxane A2 reaction in flow reduction is shown by its absence in the kidney that has been obstructed for 5 to 8 hours when postglomerular vasoconstriction is the cause of flow reduction (indicated by high renal pelvic pressure and intrarenal pressure). If we accept that imidazole selectively inhibits thromboxane A2 synthesis, we reach 2 conclusions that are clinically relevant: 1) thromboxane A2-mediated active vasoconstriction is 1 factor in the pathophysiology of hydronephrotic atrophy, and 2) the presence of thromboxane A2-mediated active vasoconstriction indicates when hydronephrotic atrophy develops and (more importantly) when it is still reversible, with respect to renal function. These findings can be used as a physiologic basis of a clinical test to predict reversibility of hydronephrotic damage.

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Thirty-two isolated cotyledons from human term placentae were perfused artificially with a tissue culture medium on the maternal and the fetal side. The transfer and uptake of labelled L-lactate and D-lactate (test substance) relative to L-glucose (reference substance, extracellular marker) were investigated in steady-state experiments (n = 9) and in a single passage paired tracer dilution technique (n = 23). The L-lactate transfer exceeded the L-glucose transfer two to three times. The L-lactate uptake into the trophoblast--both from the maternal and the fetal side--was more than three times that of D-lactate. L-lactate transfer and uptake were inhibited by phloretin. The L-lactate transfer showed a saturation kinetic at increasing chemical concentrations of lactate. It is concluded that lactate carriers exist both in the maternal and fetal side of the trophoblast.

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The fetal side of the isolated guinea-pig chorio-allantoic placenta has been perfused with a fluid containing horseradish peroxidase. Electron micrographs show that protein is transferred across the fetal capillary endothelium via micropinocytosis as well as by transport through intercellular clefts. Under normal hydrostatic pressure no pinocytosis or other signs of fetomaternal protein transfer across the trophoblast have been observed. Instead, macrophages ingest the proteins that have reached the interstitial space between fetal endothelium and trophoblast. When the fetal venous pressure is raised up to 20 mmHg about 30 to 50 per cent of the arterial perfusion fluid, including proteins, is shifted into the maternal lacunae. In the beginning of this process slender tubules, and later on bag-like channels, appear that cross the trophoblast and probably connect lacunae and interstitium. The inner surface of the channels and the maternal lacunar surface are labelled by peroxidase. This indicates a fetomaternal transfer of proteins conveyed by the fluid shift. Protein transfer and fluid shift return to their previous levels when the fetal venous pressure is reduced. The size and number of channels are reduced but their structure and the reversibility of the events refute the possibility that they are of traumatic origin. It cannot be decided whether the channels are pre-existent but unrecognizable, or de-novo formations.

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Intrarenal blood flow distribution was measured in normal and hypoperfused dog kidneys by xenon-133 washout measurement in one kidney vary slightly in contrast to those done in different kidneys. In normal kidneys the ratio of compartment I (CI) flow to whole renal flow -- i.e., the CI fraction--as measured by the xenon-133 washout curves is almost identical with the distribution of radioactive microspheres in the outer three-fourths of renal cortex (= SI). In hypoperfused dog kidneys, both techniques demonstrate redistribution of blood flow due to reduction predominantly in the CI fraction and the SI zone. The CI fraction is smaller than the microsphere distribution in zone SI in 3 of 5 hypoperfused kidneys. This is explained on the basis of increased cortical flow inhomogeneity in the hypoperfused kidneys.

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Changes in cortical blood flow were measured simultaneously with radioactive microspheres and the xenon-133 washout method in 6 dogs after 24 h of unilateral complete ureteral obstruction and in 6 dogs after 90 min of unilateral ischemia and compared with the blood flow in the contralateral kidney. Predominant reduction in cortical blood flow after 24 h of ureteral obstruction is accompanied by severe differences in the perfusion pattern of different areas in the renal cortex, thus that some areas have almost no detectable blood flow. This finding supports the thesis of renal ischemia causing at least cortical hydronephrotic atrophy.

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In the isolated, perfused guinea pig placenta glucose seems to be a major nutrient of energy metabolism, because the excreted amounts of carbon dioxide and lactate can be explained solely by the catabolism of glucose. In steady state experiments hydrogen ions, ammonia, lactate and carbon dioxide are excreted for at least 90 minutes in rather constant amounts. The production of lactate shows no significant dependence on the glucose concentration in the perfusion fluid. The guinea pig placenta seems to cover its energy requirement mainly by means of anaerobic glycolysis. Of the utilized glucose 76% are metabolized anaerobically. The placenta produces significant quantities of lactate, although it is well oxygenated. A reduction of the oxygen tension in the perfusion fluid does not result in an increase of the production of lactate. Of the hydrogen ions excreted nearly 50% are excreted as ammonia. Although the excreted amounts are small compared with the known transfer rates, they have to be taken into consideration, when studying transplacental transfers of these metabolites.

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The isolated and artificially perfused guinea pig placenta produces hydrogen ions, ammonia, lactate and carbon dioxide, which are secreted in significant larger amounts into the maternal than into the fetal circulation. The excretion rates of hydrogen ions, lactate and carbon dioxide increase significantly on both sides when the perfusion flow rates are enhanced. If the maternal perfusion flow is reversed the amount of substances excreted at the maternal side is not different from the amount excreted at the fetal side. A reversal of the fetal perfusion flow does no influence the different excretion rates on the maternal and on the fetal side. This unequal distribution between maternal and fetal side can be explained by the anatomical structure of the placenta. In the interlobium, which has been described as area of metabolism, fetal capillaries are rare. Therefore the metabolites of the placenta itself arrive chiefly at the maternal circulation.

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Maternal and fetal vascular volumes were determined in 13 isolated artificially perfused guinea pig placentas by measuring mean transit times of an intravascular indicator (Evan's blue dye) at constant flow rates. When both maternal and fetal flow rates were 3.1 ml/min, the average maternal volume of the placenta was 1.85 +/- 0.54 (SD) ml, and the mean fetal volume was 0.92 +/- 0.2 (SD) ml. If calculated maternal volumes were corrected for the myometrial vascular volume and for the interlobium volume, the remaining volume attributable largely to the labyrinth averaged approximately 1.2 ml. When flow rates were changed on the fetal or the maternal side between 0 and 6.2 ml/min, vascular volumes also changed. For example, volumes increased directly with flow rates on that side of the placenta in which the flow change was introduced; in most cases, it decreased on the opposite side where the flow rates had not been altered. This intraplacental volume shift may be regarded as the basic event for the sluice flow phenomenon in placentas.

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The transfer of 14C-labelled D- or L-lactate (test substance) relative to 3H-L-glucose (control substance, extracellular marker) into the trophoblast of the isolated guinea pig placenta was determined during an open loop perfusion on both sides. Using a single passage, paired tracer dilution technique, the maximal lactate uptake (Umax) was derived from the venous concentration ratio of lactate to L-glucose. A significant metabolism of L-lactate was not observed. The lactate uptake, which occurred in all placentas studied, was not significantly different on the fetal and maternal side. Within one placenta the L-lactate uptake was always higher than the D-lactate uptake. The uptake of both L- and D-lactate could be inhibited by phloretin. The lactate uptake was inversely correlated with the pH of the perfusate fluid within the range from 6.2-8. A first order saturation kinetic (Hofstee-plot) was used to approximate the relationship between the L-lactate uptake and the chemical L-lactate concentration. We conclude that similar lactate carriers exist in the membranes on both the maternal and the fetal side of the trophoblast.

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We measured mean renal blood flow and intrarenal blood flow distribution in 40 dogs using the xenon-133 washout technique 3 and 7 hr; 1, 2, and 3 weeks; and 4 and 6 months after unilateral complete ligation of the ureter. Mean renal blood flow rapidly decreased before renal atrophy. The data show an early redistribution of intrarenal hemodynamics with a predominant decrease in compartment I flow. These data are discussed with respect to possible ischemic atrophy especially in the renal cortex.

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