Comparison of leucine, serine and glycine transport across the ovine placenta

To estimate the transport rate of maternal glycine across the placenta [1-13C]glycine and L-[1-13]serine were infused intravenously in pregnant sheep using both continuous and bolus infusions. Each tracer was infused together with L-[1-13C]leucine, to enable a comparison with the placental transport of an essential amino acid. At steady state, fetal plasma leucine enrichment was 40 per cent of maternal enrichment, indicating that approximately 60 per cent of the entry rate of leucine into fetal plasma is derived from protein breakdown in the placenta and fetus. Fetal plasma glycine enrichment was 11 per cent of maternal and there was no detectable fetal serine enrichment. The direct flux of maternal leucine into the fetal circulation was approximately 3.0 (bolus experiments) to 3.6 (continuous infusion experiments) mumol/min (kg fetus) and greater than the estimated 1.4 mumol/min (kg fetus) direct flux of maternal glycine, despite the fact that the net umbilical uptake of glycine exceeds that of leucine. This supports the conclusion that placental glycine production is a quantitatively important contribution to fetal glycine uptake via the umbilical circulation. The fetal glycine supply from the placenta is provided by a relatively small direct maternal glycine transplacental flux and a larger contribution derived from serine utilization within the placenta for glycine production.     

Pathways of Rb+ influx and their relation to intracellular [Na+] in the perfused rat heart. A 87Rb and 23Na NMR study

The aims of this study were to characterize the routes of influx of the K+ congener, Rb+, into cardiac cells in the perfused rat heart and to evaluate their links to the intracellular Na+ concentration ([Na+]i) using 87Rb and 23Na nuclear magnetic resonance (NMR) spectroscopy. The rate constant for Rb+ equilibration in the extracellular space was 8.5 times higher than that for the intracellular space. The sensitivity of the rate of Rb+ accumulation in the intracellular space of the perfused rat heart to the inhibitors of the K+ and Na+ transport systems has been analyzed. The Rb+ influx rates were measured in both beating and arrested hearts: both procaine (5 mmol/L) and lidocaine (1 mmol/L) halved the Rb+ influx rate. In procaine-arrested hearts, the Na+,K(+)-ATPase inhibitor ouabain (0.6 mmol/L) decreased Rb+ influx by 76 +/- 24% relative to that observed in untreated but arrested hearts. Rb+ uptake was insensitive to the K+ channel blocker 4-aminopyridine (1 mmol/L). The inhibitor of Na+/K+/2 Cl- cotransport bumetanide (30 mumol/L) decreased Rb+ uptake only slightly (by 9 +/- 8%). Rb+ uptake was dependent on [Na+]i: it increased by 58 +/- 34% when [Na+]i was increased with the Na+ ionophore monensin (1 mumol/L) and decreased by 48 +/- 9% when [Na+]i was decreased by the Na+ channel blockers procaine and lidocaine. Dimethylamiloride (15 to 20 mumol/L), an inhibitor of the Na+/H+ exchanger, slightly reduced [Na+]i and Rb+ entry into the cardiomyocytes (by 15 +/- 5%). 31P NMR spectroscopy was used to monitor the energetic state and intracellular pH (pHi) in a parallel series of hearts. Treatment of the hearts with lidocaine, 4-aminopyridine, dimethylamiloride, or bumetanide for 15 to 20 minutes at the same concentrations as used for the Rb+ and Na+ experiments did not markedly affect the levels of the phosphate metabolites or pHi. These data show that under normal physiological conditions, Rb+ influx occurs mainly through Na+,K(+)-ATPase; the contribution of the Na+/K+/2 Cl- cotransporter and K+ channels to Rb+ influx is small. The correlation between Rb+ influx and [Na+bdi during infusion of drugs that affect [Na+]i indicates that, in rat hearts at 37 degrees C, Rb+ influx can serve as a measure of Na+ influx. We estimate that, at normothermia, at least 50% of the Na+ entry into beating cardiac cells is provided by the Na+ channels, with only minor contributions (< 15%) from the Na+/K+/2 Cl- cotransporter and the Na+/H+ exchanger.     

Low-affinity potassium uptake system in the archaeon Methanobacterium thermoautotrophicum: overproduction of a 31-kilodalton membrane protein during growth on low-potassium medium

During growth on low-K+ medium (1 mM K+), Methanobacterium thermoautotrophicum accumulated K+ up to concentration gradients ([K+]intracellular/[K+]extracellular) of 25,000- to 50,000-fold. At these gradients ([K+]extracellular of < 20 microM), growth ceased but could be reinitiated by the addition of K+ or Rb+. During K+ starvation, the levels of a protein with an apparent molecular weight of 31,000 increased about sixfold. The protein was associated with the membrane and could be extracted by detergents. Cell suspensions of M. thermoautotrophicum obtained after K+-limited growth catalyzed the transport of both K+ and Rb+ with apparent Km and Vmax values of 0.13 mM and 140 nmol/min/mg, respectively, for K+ and 3.4 mM and 140 nmol/min/mg, respectively, for Rb+. Rb+ competitively inhibited K+ uptake with an inhibitor constant of about 10 mM. Membranes of K+-starved cells did not exhibit K+-stimulated ATPase activity. Immunoblotting with antisera against Escherichia coli Kdp-ATPase did not reveal any specific cross-reactivity against membrane proteins of K+-starved cells. Cells of M. thermoautotrophicum grown at a high potassium concentration (50 mM) catalyzed K+ and Rb+ transport at similar apparent Km values (0.13 mM for K+ and 3.3 mM for Rb+) but at significantly lower apparent Vmax values (about 60 nmol/min/mg for both K+ and Rb+) compared with K+-starved cells. From these data, it is concluded that the archaeon M. thermoautotrophicum contains a low-affinity K+ uptake system which is overproduced during growth on low-K+ medium.     

Vasopressin reduces taurochenodeoxycholate-induced hepatotoxicity by lowering the hepatocyte taurochenodeoxycholate content

BACKGROUND/AIMS: Vasopressin has been reported to reduce bile flow, but its effects on bile acid secretion and bile acid-related hepatotoxicity are still unclear. We therefore investigated the influence of vasopressin on the hepatotoxicity and biliary excretion of taurochenodeoxycholic acid in primary cultured rat hepatocytes and isolated perfused rat liver models. METHODS/RESULTS: 1) Addition of vasopressin to hepatocyte cultures significantly decreased lactate dehydrogenase release as compared to cultures exposed to 1 mM taurochenodeoxycholic acid alone, and also reduced intracellular taurochenodeoxycholic acid content from 19.3 +/- 2.2 to 13.0 +/- 1.6 nmol/mg protein. After 30 min of preincubation with 1 mM taurochenodeoxycholic acid, rinsing and reculture of hepatocytes in bile acid-free medium resulted in gradual decrease in the intracellular level of the bile acid, and addition of vasopressin (10(-9) M) to the reculture medium accelerated this process. 2) Superimposition of vasopressin (330 pmol/l) for 10 min on taurochenodeoxycholic acid infusion (1.0 mumol/min: 25 mumol/l) caused a rapid increase in bile flow and biliary excretion of taurochenodeoxycholic acid (697 +/- 42 vs 584 +/- 27 nmol/10 min per g liver) from perfused rat livers, and significantly reduced lactate dehydrogenase release. 3) Superimposition of the PKC blocker H-7 (5 mumol/l) on taurochenodeoxycholic acid infusion (1.0 mumol/min: 25 mumol/l) caused a gradual increase in bile flow and biliary excretion of taurochenodeoxycholic acid. Furthermore, an additional infusion of vasopressin (100 pmol/l) for 10 min in the presence of H-7 produced a greater increase in bile flow and biliary excretion of taurochenodeoxycholic acid as compared with H-7 alone (754 +/- 71 vs. 657 +/- 26 nmol/g liver). 4) Continuous infusion of vasopressin (330 pmol/l) significantly increased the late peak (10-50 min) of horseradish peroxidase excretion from perfused livers (from 8.48 +/- 1.02 to 21.7 +/- 6.02 ng/g liver). CONCLUSIONS: These findings suggest that vasopressin exerts a protective effect against taurochenodeoxycholic acid-induced hepatotoxicity by stimulating the secretion of this bile acid via intracellular vesicular transport systems.     

Kinetic analysis of dynamic 13C NMR spectra: metabolic flux, regulation, and compartmentation in hearts

Control of oxidative metabolism was studied using 13C NMR spectroscopy to detect rate-limiting steps in 13C labeling of glutamate. 13C NMR spectra were acquired every 1 or 2 min from isolated rabbit hearts perfused with either 2.5 mM [2-13C]acetate or 2.5 mM [2-13C]butyrate with or without KCl arrest. Tricarboxylic acid cycle flux (VTCA) and the exchange rate between alpha-ketoglutarate and glutamate (F1) were determined by least-square fitting of a kinetic model to NMR data. Rates were compared to measured kinetics of the cardiac glutamate-oxaloacetate transaminase (GOT). Despite similar oxygen use, hearts oxidizing butyrate instead of acetate showed delayed incorporation of 13C label into glutamate and lower VTCA, because of the influence of beta-oxidation: butyrate = 7.1 +/- 0.2 mumol/min/g dry wt; acetate = 10.1 +/- 0.2; butyrate + KCl = 1.8 +/- 0.1; acetate + KCl = 3.1 +/- 0.1 (mean +/- SD). F1 ranged from a low of 4.4 +/- 1.0 mumol/min/g (butyrate + KCl) to 9.3 +/- 0.6 (acetate), at least 20-fold slower than GOT flux, and proved to be rate limiting for isotope turnover in the glutamate pool. Therefore, dynamic 13C NMR observations were sensitive not only to TCA cycle flux but also to the interconversion between TCA cycle intermediates and glutamate.     

Hydroperoxide metabolism in rat liver. K+ channel activation, cell volume changes and eicosanoid formation

Addition of t-butylhydroperoxide (0.2 mM) to isolated perfused rat liver led to a net K+ release of 7.2 +/- 0.2 mumol/g within 8 min and a net K+ reuptake of 6.6 +/- 0.4 mumol/g following withdrawal of the hydroperoxide, in line with earlier findings by Sies et al. [Sies, H., Gerstenecker, C., Summer, K. H., Menzel, H. & Flohé, R. (1974) in Glutathione (Flohé, L., Ben?hr, C., Sies, H., Waller, H. D., eds) pp. 261-276, G. Thieme Publ. Stuttgart]. Net K+ release roughly paralleled the amount of GSSG released from the liver under the influence of the hydroperoxide. The t-butylhydroperoxide-induced K+ efflux was inhibited by approximately 70% in the presence of Ba2+ (1 mM), by 30% in Ca(2+)-free perfusions and was decreased by 50-60% when the intracellular Ca2+ stores were simultaneously depleted by repeated additions of phenylephrine. t-Butylhydroperoxide-induced K+ efflux was accompanied by a decrease of the intracellular water space by 58 +/- 14 microliter/g (n = 4), corresponding to a 10% cell shrinkage. The effect of t-butylhydroperoxide on cell volume was inhibited by 70-80% in the presence of Ba2+. In isolated rat hepatocytes treatment with t-butylhydroperoxide led to a slight hyperpolarization of the membrane at concentrations of 100 nM, but marked hyperpolarization occurred at t-butylhydroperoxide concentrations above 10 microM. t-Butylhydroperoxide (0.2 mM) transiently increased the portal-perfusion pressure by 3.3 +/- 0.6 cm H2O (n = 18), due to a slight stimulation of prostaglandin-D2 release under the influence of the hydroperoxide. In the presence of Ba2+ (1 mM), t-butylhydroperoxide increased the perfusion pressure by 12.7 +/- 1.2 cm H2O (n = 9) and produced an approximately tenfold increase of prostaglandin-D2 and thromboxane-B2 release. Under these conditions, glucose output from the liver rose from 0.9 +/- 0.03 to 2.9 +/- 0.7 mumol.g-1.min-1 (n = 4) with a time course roughly resembling that of portal-pressure increase and prostaglandin-D2 overflow. These effects were largely abolished in the presence of ibuprofen or the thromboxane-receptor-antagonist BM 13.177. The t-butylhydroperoxide effects on perfusion pressure, glucose and eicosanoid output were also enhanced in the presence of insulin or during hypotonic exposure; i.e. conditions known to swell hepatocytes, but not during hyperosmotic exposure. The data suggest that t-butylhydroperoxide induces liver-cell shrinkage and hyperpolarization of the plasma membrane due to activation of Ba(2+)-sensitive K+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of dehydrocholate sodium on the biliary reabsorption of sulfobromophtalein sodium from the rat biliary tree after retrograde intrabilary injection

Bilary re-excretion of sulfobromophthalein sodium (BSP) (1.2 mumol/rat) after retrograde intrabilary injection is markedly inhibited by dehydrocholate sodium (Decholin) given intravenously as a constant infusion (3.1 mumol/min/kg body weight) or as a bolus injection (24.8 mumol/rat) to rats. Most interestingly Decholin demonstrates the same inhibitory effects on the biliary re-excretion of BSP when it is administered by retrograde intrabiliary injection (24.8 mumol/rat) together with the dye. In contrast to the inhibition of biliary re-excretion of BSP its biliary reabsorption from the biliary tract seems to be rather increased: about 80-85% of 1.2 mumol BSP are reabsorbed in all sets of experiments with Decholin in comparison to about 65% to the control group. Combined with histological data it is suggested that reabosorption of BSP after retrograde intrabiliary injection occurs at the ductular site whereas re-excretion takes place at the cannalicular membrane of the hepatocyte.     

Effects of ischemia on intracellular sodium and phosphates in the in vivo rat liver

Metabolic factors that influence the transition form reversible to irreversible ischemic injury were studied in the rat liver in vivo with 31P-nuclear magnetic resonance (NMR) spectroscopy. Hepatic ischemia for 15, 35, or 65 min was produced by occlusion of the hepatic artery and portal vein in rats. Ischemia caused a rapid decrease in the ATP concentration ([ATP])-to-P(i) concentration ratio and pH within 5 min, but there was little change in these variables detectable by 31P-NMR with longer periods of ischemia. After reperfusion, the [ATP] and P(i) concentration returned toward normal values in livers exposed to 15 or 35 min of ischemia, but 65 min of ischemia were associated with only modest recovery in [ATP], and the [ATP] later decreased. Because the 31P-NMR spectrum was similar after brief compared with prolonged ischemia, it appears that neither ATP depletion, P(i) accumulation, nor acidosis predicts metabolic recovery. Hepatic intracellular NA+ was also measured in separate groups of animals by 23Na-NMR in the presence of a shift agent, thulium (III) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis (methylene-phosphonate) (TmDOTP5-), and by atomic absorption spectroscopy. Under baseline conditions, the concentration of intracellular Na+ was 15.2 mM by atomic absorption spectroscopy and 16.5 mM by 23Na-NMR. Although the 31P-NMR spectrum responded very rapidly to the onset of ischemia, intracellular Na+ concentration measured by 23Na-NMR increased gradually but steadily at approximately 1.0 mM/min during early (up to 15 min) ischemia. These observations demonstrate that a rise in intracellular Na+ does occur early ischemia, that TmDOTP5- can be applied in vivo for analysis of intracellular Na+ in the ischemic liver, and that 31P-NMR spectroscopy is very sensitive to early ischemic injury.     

Caffeine and the contractility of single muscle fibres from the barnacle Balanus nubilus

1. In single striated muscle fibres from the barnacle Balanus nubilus tension development following axial injection of caffeine (50 mM in 150 mM-KCl, pH 7.1) was used as an index of releasable Ca. It was shown that fibres incubated in 0 Ca (Na replaced) salines for up to 400 min gave ca. 15% of the control tension response. Inclusion of 1 mM-La in the 0 Ca (Na) saline significantly reversed this decline.2. Estimates of the total fibre calcium assayed under the same experimental conditions indicated a 67% loss of fibre Ca in 0 Ca, and only a 37% loss in La-0 Ca media. No correction was made for the loss of calcium from the extracellular space.3. Experiments with (45)Ca indicated that the efflux of calcium from this preparation was inhibited by 1 mM-La externally and that this effect was still significant even in the presence of 0 Ca (Na) salines. The caffeine-stimulated efflux of Ca was also reduced by ca. 70% in the presence of 1 mM La saline externally.4. The influx and efflux of (14)C caffeine were shown to be rapid, and apparently passive. The diffusion coefficient for caffeine following intracellular injection was 2.4 +/- 0.2 x 10(-6) cm(2) sec(-1) at 18-22 degrees C.5. There was no significant loss of (140)La over a period of 2 hr following axial micro-injection into the fibres.6. In (140)La uptake experiments there was a progressive increase in the La space over 10.5 hr, in contrast to the results with [(3)H]inulin, whose uptake saturated in ca. 1.5 hr. The probability of surface binding and the precipitation of La salts in the extensive extracellular space was suggested as an explanation.7. It is concluded that internal Ca within the sarcoplasmic reticulum, and not cleft or extracellular Ca is the most significant source for these caffeine-induced contractions. Fluxes across the surface membrane can however alter the internal Ca stores over longer periods of time.     

Comparative pharmacokinetics of four cholinesterase inhibitors in rats

Pharmacokinetics of a very short-acting, a short-acting and two long-acting cholinesterase (ChE) inhibitors, edrophonium, neostigmine, pyridostigmine and ambenonium, respectively, were compared to elucidate the major determinant of their pharmacokinetics. No dose-dependency in pharmacokinetic behavior was observed within the range of 2-10 mumol/kg for edrophonium, 0.5-2 mumol/kg for pyridostigmine, 0.1-0.5 mumol/kg for neostigmine and 0.3-3 mumol/kg for ambenonium, respectively. Neostigmine has the shortest elimination half-life, and edrophonium, pyridostigmine and ambenonium follow in that. Four ChE inhibitors have similar Vdss values within the range of 0.3-0.7 l/kg, which is similar to the muscle/plasma concentration ratio of these drugs. The liver or kidney to plasma concentration ratio of all ChE inhibitors at 20min after i.v. administration ranged from 5 to 15. Small distribution volumes estimated from the plasma concentration profiles may reflect the distribution to muscle and to the extracellular space of other organs/tissues, while the rapid disappearance of ChE inhibitors from plasma may reflect the concentrative uptake to the liver and kidney.     

Liver and spleen enhancement after intravenous injection of carboxydextran magnetite: effect of dose, delay of imaging, and field strength in an ex vivo model

It has been predicted that liver and spleen enhancement after administration of superparamagnetic contrast agents may be different, depending on the strength of the main magnetic field. With the use of an ex vivo model, we investigated at 0.3, 0.5, and 1.5 T the effects on liver and spleen signal intensity of 5, 15, and 45 mumol/kg body weight of dextran magnetite (SHU 555A) in 54 rats. Nine rats served as controls. At different time delays since injection, the animals were killed, and after perfusion with saline, the liver, brain, and spleen were fixed in formalin. The specimens were embedded in an agar gel matrix and imaged with inversion recovery T1-weighted, proton density spin echo, and T2*-weighted gradient recalled echo (GRE) sequences. At each magnetic field strength, peak liver and spleen signal loss increased with increasing dose of the contrast medium. Signal loss was significantly more conspicuous after a dose of 15 than 5 mumol/kg body weight, but not after a dose of 45 compared with 15 mumol/kg. No signal change was observed in the brain. GRE images showed higher enhancement than proton density-weighted spin echo and inversion recovery images but were noisier. The enhancement showed a plateau between 30 min and 24 hours. Only the signal decrease of the liver after a low dose of contrast medium on GRE images was significantly higher (p < 0.01) at 1.5 than at 0.5 and 0.3 T. Other differences in respect to the field strength were less significant (p < 0.05) or nonsignificant. Differences in the spleen enhancement were nonsignificant. SHU 555A at a dose of 15 mumol/kg is an efficient intracellular contrast agent for liver and spleen at low, mid, and high field strength. Proton density spin echo images are probably the sequence of choice to exploit SHU 555A contrast effects and a wide time window for imaging after its intravenous injection does exist.     

Obesity and high-density lipoprotein cholesterol in black and white 9- and 10-year-old girls: The National Heart, Lung, and Blood Institute Growth and Health Study

Acetaldehyde, the first metabolite of ethanol oxidation, has been proposed as a major initiating factor in ethanol-induced liver injury. The aims of this study were to examine whether acetaldehyde is absorbable from the digestive tract and whether, when delivered chronically in drinking water, it is capable of inducing liver injury in rats. Acetaldehyde concentrations in the rat portal and peripheral blood were measured by head space gas chromatography after intragastric (5 ml) and intracolonic (3 ml) administration of 20 mM acetaldehyde solution. In the hepatotoxicity study, rats were exposed to acetaldehyde (20 and 120 mM) delivered in drinking water for 11 weeks and histopathological changes in the liver were morphometrically assessed. Peak blood acetaldehyde levels were found at 5 min after acetaldehyde infusion and were 235 +/- 11 microM (mean +/- SE) after intragastric and 344 +/- 83 microM after intracolonic infusion of 20 mM acetaldehyde solution. The exposure of rats to 120 mM acetaldehyde solution for 11 weeks resulted in the development of fatty liver and inflammatory changes. Morphometric analysis showed significantly more fat accumulation in rats receiving 120 mM acetaldehyde solution (85 +/- 2 per cent of hepatocytes occupied by fat) than in rats receiving 20 mM acetaldehyde solution (38 +/- 11 per cent) or in controls (36 +/- 10 per cent). The dose of extrahepatic acetaldehyde (500 mg/kg per day) producing liver injury corresponds to only around 3 per cent of that derived from hepatic ethanol oxidation in animals receiving an ethanol-containing totally liquid diet (15 g/kg per day). These results indicate that acetaldehyde delivered via the digestive tract can reach the liver by the portal circulation and that acetaldehyde of extrahepatic origin appears to be more hepatotoxic than acetaldehyde formed during ethanol oxidation within the liver.     

Phosphonates as 31P-NMR markers of extra- and intracellular space and pH in perfused rat liver

We evaluated phosphonates (Po) as markers of the extra- and intracellular space in perfused rat liver. (i) In- and outwash behaviour of phenylphosphonate (PhePo), 3-amino-propylphosphonate (NProPo) and methyl phosphonate (MePo) was compared with that of creatine phosphate (CrP), a marker of the extracellular space, and of dimethyl methylphosphonate (MePoMe2), a marker of the total water-accessible space. In- and outwash of CrP was accurately predicted by the time constant (approximately 12 s) for the in- and outwash of inulin, a standard marker of the extracellular space. MePoMe2 rapidly distributed over the total liver volume (about three times the CrP accessible space). PhePo, NProPo and MePo washed rapidly into the extracellular space with CrP, and then steadily spilled over into the MePoMe2-accessible space. Upon outwash, Po signals rapidly declined in phase with that of CrP. Residual Po (PhePo > NProPo approximately equal to MePo) reflected the amount internalized during prolonged (60 min) inwash. Proportional amounts of residual Po were found in extracts of livers harvested after outwash of perfusate and extracellular markers. Consistent with exclusion from the cells, CrP went undetected in these extracts. (ii) The resonance frequency of residual PhePo after outwash of the extracellular fraction corresponded with the pH reported by cytosolic P1 and responded to transient changes of the intracellular pH, induced by perfusion with and withdrawal of 20 mM NH4Cl. (iii) MePoMe2 homogeneously distributed over perfusate, parenchyma and bile, consistent with unrestricted permeability. Other Po were transported transcellularly and excreted in bile. CrP was virtually excluded from the bile, attesting to a minimal role for 'bulk-phase pinocytotic' transcellular transport, or for 'paracellular' leakage. In summary, charged Po can be used as extracellular markers in liver, provided experimental conditions are adjusted to minimize their internalization. Some Po (e.g. PhePo) can reach intracellular concentrations which suffice for the compound to act as a reporter molecule of the cytosolic pH.     

Decrease of oxygen consumption in the dog liver during temporary arterial occlusion

The effects of occlusion of the hepatic artery on total and regional splanchnic oxygen consumption were studied in lightly anaesthetized dogs. Mean whole body oxygen uptake (+/- S.D.) was 4.72 +/- 0.55 ml/kg b.w. min-1, mean liver oxygen uptake (+/- S.D.) 1.18 +/- 0.42 ml/kg b.w. min-1 and mean oxygen uptake of the portally-drained tissues (+/- S.D.) was 0.80 +/- 0.54 ml/kg b.w. min-1 during the control period. The hepatic artery contributed 45 +/- 24% of the total liver oxygen uptake. The duration of occlusion was 45 min. Mean liver oxygen uptake was found to decrease to 64% of control values. The extraction of oxygen from the portal blood increased slightly. Mean whole body oxygen uptake and mean oxygen uptake of the portally-drained tissues were unchanged. 45 min after release of the hepatic artery occlusion, liver oxygen consumption had returned to control values. It is concluded that oxygen uptake in the liver is correlated to oxygen tension.     

Quantitative analysis of metabolism of hepatic triglyceride in ethanol-treated rats

An acute intraperitoneal injection of ethanol (0.7 or 2.1g/kg body wt.) causes the reversible, dose-dependent accumulation of hepatic triglyceride in rats. By using a pulse of [14C]palmitate injected into a tail vein, it was found that ethanol (2.1g/kg)had no effect on the flux of unesterified fatty acid of serum (4.3mumol/min per 100g body wt.). However, either dose increased the fraction of the total flux going to liver from 0.16 to0.27 as rapidly as could be measured (30s), and it remained elevated until all ethanol had been cleared from the blood. The fraction of the total radioactivity in lipids of liver that was in triglyceride increased linearly for 1 h from 30 to 50% and there was a simultaneous decrease in phospholipid from 60 to 40%. The rate of synthesis of hepatic triglyceride derived directly from unesterified fatty acid of serum was calculated by using the flux rate of unesterified fatty acid in serum, the fractional hepatic uptake of this flux, and the percentage of liver fatty acid esterified to triglyceride. This contribution is related to the total synthetic rate of hepatic triglyceride (rate of accumulation+rate of release) to determine quantitatively how much of the developing fatty liver is attributable to increased uptake of unesterfied fatty acid of serum. At the higher dose of ethanol, about half of the accumulating triglyceride is derived from this source, whereas with the lower dose of ethanol it can account for all of the build-up.     

A two-dimensional Kolmorogov-Smirnov test for binned data

Exercise leads to marked increases in muscle insulin sensitivity and glucose effectiveness. Oral glucose tolerance immediately after exercise is generally not improved. The hypothesis tested by these experiments is that after exercise the increased muscle glucose uptake during an intestinal glucose load is counterbalanced by an increase in the efficiency with which glucose enters the circulation and that this occurs due to an increase in intestinal glucose absorption or decrease in hepatic glucose disposal. For this purpose, sampling (artery and portal, hepatic, and femoral veins) and infusion (vena cava, duodenum) catheters and Doppler flow probes (portal vein, hepatic artery, external iliac artery) were implanted 17 d before study. Overnightfasted dogs were studied after 150 min of moderate treadmill exercise or an equal duration rest period. Glucose ([14C]glucose labeled) was infused in the duodenum at 8 mg/kg x min for 150 min beginning 30 min after exercise or rest periods. Values, depending on the specific variable, are the mean +/- SE for six to eight dogs. Measurements are from the last 60 min of the intraduodenal glucose infusion. In response to intraduodenal glucose, arterial plasma glucose rose more in exercised (103 +/- 4 to 154 +/- 6 mg/dl) compared with rested (104 +/- 2 to 139 +/- 3 mg/dl) dogs. The greater increase in glucose occurred even though net limb glucose uptake was elevated after exercise (35 +/- 5 vs. 20 +/- 2 mg/min) as net splanchnic glucose output (5.1 +/- 0.8 vs. 2.1 +/- 0.6 mg/kg x min) and systemic appearance of intraduodenal glucose (8.1 +/- 0.6 vs. 6.3 +/- 0.7 mg/kg x min) were also increased due to a higher net gut glucose output (6.1 +/- 0.7 vs. 3.6 +/- 0.9 mg/kg x min). Adaptations at the muscle led to increased net glycogen deposition after exercise [1.4 +/- 0.3 vs. 0.5 +/- 0.1 mg/(gram of tissue x 150 min)], while no such increase in glycogen storage was seen in liver [3.9 +/- 1.0 vs. 4.1 +/- 1.1 mg/(gram of tissue x 150 min) in exercised and sedentary animals, respectively]. These experiments show that the increase in the ability of previously working muscle to store glycogen is not solely a result of changes at the muscle itself, but is also a result of changes in the splanchnic bed that increase the efficiency with which oral glucose is made available in the systemic circulation.     

Kinetics of insulin action in vivo. Identification of rate-limiting steps

To examine the kinetic steps in insulin's in vivo action, we have assessed the temporal relationship between arterial insulin, interstitial insulin, glucose disposal rate (GDR), and insulin receptor kinase (IRK) activity in muscle and between portal insulin, hepatic glucose production (HGP), and IRK activity in liver. Interstitial insulin, as measured by lymph-insulin concentration (muscle only), and IRK activity were used as independent methods to determine the arrival of insulin at its tissue site of action. Euglycemic clamps were conducted in seven mongrel dogs and consisted of an activation phase with a venous insulin infusion (7.2 nmol.kg-1.min-1, 100 min) and a deactivation phase. Liver and muscle biopsies were taken to assess IRK activity. Arterial, portal, and lymph insulin rose to 636 +/- 12, 558 +/- 18, and 402 +/- 24 pmol/l, respectively. GDR increased from 13.9 +/- 0.6 to 41.7 +/- 2.8, and HGP declined from 14.4 +/- 0.6 to 1.1 +/- 0.6 mumol.kg-1.min-1. Muscle and liver IRK activity increased significantly from 5.9 +/- 0.9 to 14.6 +/- 0.6 and 5.5 +/- 0.7 to 23.7 +/- 1.9 fmol P/fmol insulin receptor (IR), respectively. The time to half-maximum response (t1/2a) for stimulation of GDR (19.8 +/- 4.8 min) and suppression of HGP (21.5 +/- 3.7 min) were similar. The t1/2a for stimulation of GDR, muscle IRK, and rise in lymph insulin were not significantly different from one another and were all markedly greater than that for the approach to steady state of arterial insulin (2.3 +/- 1.2 min, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of pH on the uptake of 5-fluorouracil into isolated tumour cells

To investigate the possible dependence of 5-fluorouracil (5FU) uptake in tumours on the intra- (pHi) and extracellular (pHe) pH, a pH gradient (deltapH) was imposed across the plasma membrane of ascites tumour cells in vitro, similar to that known to occur in some solid tumours in vivo, by incubation in media of PHe 5-8. A > or = 2:1 (intracellular/extracellular) accumulation of radiolabelled 5FU occurred after 5 min incubation of the cells with 0.5 mM 5FU at pHe of 5.0, 5.5 or 6.0. 5FU metabolism is slow under these conditions, and 5FU uptake was not affected by longer incubations up to 20 min, nor by the absence of a sodium gradient. pHi was estimated from the distribution of the weak acid, 5.5-dimethyl-2,4-oxazolidione ([14C]DMO) across the cell membrane. There was significant correlation between the intracellular/extracellular 5FU ratio and pHe (from pHe 6-8), deltapH and pHi (P < 0.02). Similar results were obtained with HT29 cells. Incubation with a drug that made plasma membranes permeable to H+ significantly decreased 5FU uptake in Lettre cells. The co-transport of 5FU may occur on a proton symport using the proton motive force of the deltapH.     

Immunizations: current recommendations

BACKGROUND & AIMS: Most studies of intestinal amino acid absorption use methods in which intestinal function is studied immediately after surgical manipulation. The unphysiological experimental conditions present in these studies limit the ability to extrapolate their results to normal physiological conditions. The aim of this study was to determine the rates of proline and leucine absorption under normal physiological conditions. METHODS: Absorption of proline and leucine was measured in long-term catheterized rats using a method of dual infusion of radiolabeled isotopes. RESULTS: The maximum transport velocity and apparent membrane permeability for proline were 16.1 mumol/ min and 0.07 mumol.min-1.mmol/L-1. For leucine, the maximum transport velocity and apparent membrane permeability were 14.9 mumol/min and 0.08 mumol.min-1.mmol/L-1. Surgical bowel manipulation decreased the maximum transport velocities for proline and leucine by > 80%. The adverse effects of surgery were present for 24 hours. CONCLUSIONS: Under normal physiological conditions, most proline and leucine is absorbed by active transport. Measurements of amino acid absorption using methods in which the intestine has been surgically manipulated within the previous 24 hours significantly underestimate proline and leucine absorption and do not reflect absorption under normal physiological conditions.