Postnatal development of hepatocellular apolipoprotein B assembly and secretion in the rat.

In this study, we explored the paradox that in suckling rats the serum concentration of LDL is high although the liver secretes only minimal quantities of VLDL, the presumed precursor of LDL. Freshly isolated hepatocytes and hepatocytes in primary culture obtained from adult (90 days old) and suckling (17 days old) rats were used to investigate the synthesis and secretion of apolipoprotein B (apoB) and lipids as well as the density profile of secreted apoB-containing lipoproteins. Furthermore, the effects of dexamethasone and oleate on apoB biogenesis were investigated in primary cultures of hepatocytes from adult and suckling rats. Hepatocytes from suckling rats were unable to assemble mature VLDL but secreted apoB as primordial lipoprotein particles in the LDL-HDL density range. Intracellular degradation of apoB was also reduced in hepatocytes from suckling rats compared with that in hepatocytes from adults. The immaturity in VLDL assembly and apoB degradation of hepatocytes from suckling rats could be overcome by treating the cultures with dexamethasone plus oleate or dexamethasone alone. The lower microsomal triacylglycerol transfer protein (MTP) mRNA concentrations in hepatocytes from suckling rats in comparison with hepatocytes from adult rats were not reflected in lower MTP activity levels. Furthermore, dexamethasone plus oleate treatment had no effect on MTP activity although VLDL assembly and secretion were clearly stimulated. We conclude that, during the suckling period of the rat, serum LDL is directly produced by the liver. This is a result of impaired hepatic VLDL assembly, which is a consequence of low triglyceride synthesis and an inefficient mobilization of bulk lipids in the second step of VLDL assembly.

Glycosylphosphatidylinositol-specific phospholipase D in blood serum: is the liver the only source of the enzyme?

In cases of systemic inflammatory response syndrome, sepsis, and septic shock, the activity of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) in serum amounts to 20 to 25% of the activity found in a healthy control group. The activity of serum GPI-PLD is positively correlated with inflammatory markers and counts of monocytes and stab cells (bands) and negatively correlated with polymorphonuclear neutrophils and lymphocytes in severe diseases. This indicates a yet unknown involvement of the inflammatory system in GPI-PLD liberation and suggests that the liver is not the only source of the plasma enzyme. Plasma was shown to contain an effective inhibitor of GPI-PLD which is soluble in organic solvents. Its concentration in capillary plasma is 20-fold higher than in venous plasma. To find possible other sources of plasma GPI-PLD besides the liver, the GPI-degrading activity was measured in different organs of the rat. Product formation was analysed using [125I]TID-labeled GPI-AP.

Dexamethasone enhanced by insulin, but not by thyroid hormones stimulates apolipoprotein B mRNA editing in cultured rat hepatocytes depending on the developmental stage.

The increase of hepatic apolipoprotein B (apoB) mRNA editing during rat development was not affected by hypothyroidism. Furthermore, the addition of 3,3',5'-triiodothyronine (T3) to cultured hepatocytes taken from fetal, neonatal and adult rats had no effect on apoB mRNA editing. In contrast, dexamethasone markedly stimulated apoB mRNA editing in hepatocytes taken from neonates. This effect was enhanced by the addition of insulin. For the first time our data provide evidence that glucocorticoids together with insulin are important for the regulation of apoB mRNA editing during postnatal development, whereas thyroid hormones are not critical for this process.

Luminol-and lucigenin-amplified chemiluminescence with rat liver microsomes. Kinetics and influence of ascorbic acid, glutathione, dimethylsulfoxide, N-t-butyl-a-phenyl-nitrone, copper-ions and a copper complex, catalase, superoxide dismutase, hexobarbital and aniline.

For the investigation of luminol (LM)-and lucigenin (LC)-amplified chemiluminescence (CL) in rat liver microsomes using both a liquid-scintillation counter (LKB/Wallac 1219 Rackbeta) and a Berthold luminometer (AutoLumat LB 953) optimal incubation mixtures and conditions and basic kinetics have been established. Whereas calibration curves for both LM- and LC-CL are performed with hydrogenperoxide (LC quantum yield is 6.25 fold higher as that of LM), distinct differences were revealed with microsomes, indicating that different reactive oxygen species (ROS) are determined: Both LM- and LC-CL follow the kinetics of enzymatic reactions in terms of dependence on protein and NADPH or NADH concentration, time course, temperature etc., but with differences. LM-CL does not work without addition of Fe2+, whereas LC-CL does. Both copper ions and copper bound in a complex abolish CL, LC-CL being much more sensitive. Isolated cytochrome P-450 (P450) and NADPH P450 reductase from liver of pheno-barbital treated rats alone proved to be inactive in LM-and LC-CL production, whereas te combination 1:1 without and with addition of lipid was highly active in both LM-and LC-CL. Ascorbic acid and glutathione as scavengers diminish both LM- and LC-CL in concentrations higher then 10(5). Dimethyl-sulfoxide (DMSO) was ineffective in LM-CL up to concentrations of 0.2 M, the very high concentration of 2 M diminished LM-CL only to 1/3. LC-CL was diminished starting at concentrations of 100 mM and at 2 M only 10% of maximum LC-CL was observed. The trap substance N-t-butyl-a-phenylnitrone (BNP) also diminished LC-CL more effectively than LM-CL. Clearcut differences were revealed by the addition of catalase and superoxide dismutase: both enzymes diminished LM-CL only, without any influence on LC-CL. Hexobarbital, a potent uncoupler of P450, enhances LM-CL fivefold, whereas LC-CL is barely influenced. Aniline (without uncoupling capability) decreased both LM-and LC-CL increasingly with increasing concentrations. Therefore the conclusion is drawn that LM-CL measures in liver microsomes predominantly superoxide anion radicals, whereas LC-CL is mainly a measure for microsomal hydroxyl radical formation or of reactive organic radicals. With microsomes of phenobarbital and beta-naphthoflavone treated rats CL was much higher but in principle the same kinetic characteristics could be shown. All results on microsomes were obtained uniformly with the liquid scintillation counter and the Berthold luminometer, the letter being much more effective and more sensitive.

[Diagnosis of intestinal oxalate hyperabsorption in patients with idiopathic recurrent calcium oxalate urinary calculi]. / Diagnostik der enteralen Oxalathyperabsorption bei Patienten mit idiopathischem rezidivierendem Kalziumoxalatharnsteinleiden.

By oral administration of 14C-labelled oxalic acid (2.2 microns Ci; 2 mg), the mean enteral oxalate absorption in 24-h urines (collecting intervals 3, 3, 6, 12 h) of 19 healthy control subjects was determined to be 8.3%. It was 14.6% (alpha less than 1%) in 20 patients with recurrent idiopathic calcium-oxalate lithiasis. The differences in absorption were most marked in the first two 3-h urines. Under the test conditions described, maximum excretion of 14C oxalate had already occurred after 3 h (55%-57%); after 6 h it was about 85%, and after 12 h 95% of the total activity in the 24-h urine. In 68% of the control subjects, the oxalate absorption values were below 10%; in 32% they were less than or equal to 15%. For the group of lithiasis patients, these findings suggest a metabolic disorder of gastrointestinal origin. In 40%, the oxalate absorption rates were clearly above 15%. The test method described is simple to carry out and can be recommended for the assessment of idiopathic calcium-oxalate lithiasis.

Development and regulation of hepatocellular fatty acid synthesis towards term: studies in isolated fetal rat hepatocytes.

De novo fatty acid synthesis was studied in hepatocytes freshly isolated from rat fetuses on day 20 and 22 (term) of gestation by using the 3H2O incorporation method. Within 1 h, about 4 nmoles palmitate equivalents/10(6) cells were synthesized on day 20 of gestation. Towards term this rate decreases to about two thirds of this value. Both on day 20 and 22 of gestation, fetal hepatocellular de novo fatty acid synthesis is stimulated by the addition of lactate and octanoate, whereas a stimulating effect by the addition of pyruvate was found on day 20 of gestation only. These data provide evidence that both cytosolic and mitochondrially derived NADH2 is an adequate source of reducing equivalents required for NADPH2-dependent fatty acid synthesis. Insulin stimulates fetal hepatocellular de novo fatty acid synthesis at term, whereas db-cAMP remains ineffective on both gestational days under study.

[3H]naloxone as an opioid receptor label: analysis of binding site heterogeneity and use for determination of opioid affinities of casomorphin analogues.

The nonselective antagonist [3H]naloxone was used to identify opioid receptors in rat brain membranes. The multiple naloxone binding sites were related to different opioid receptors by means of selective opioid ligands as well as various beta-casomorphin analogues. Analysis of binding site heterogeneity was performed using several computer curve fitting methods. The results indicate that structurally modified casomorphin peptides are able to discriminate between mu 1- and mu 2-binding sites. The affinities to the mu-sites obtained with [3H]naloxone as label are in a good agreement with those from experiments with the mu-selective radioligand [3H]DAGO. The mu 1-site affinities of these casomorphin derivatives are well correlated with their antinociceptive potencies. This finding suggests the mediation of the analgesic activity via the high-affinity mu 1-subtype.

Effect of catecholamines on the metabolic fate of nonesterified fatty acids in isolated hepatocytes from newborn rats.

Isolated hepatocytes from newborn rats are able to produce ketone bodies from added medium-chain and long-chain fatty acids. Carnitine enhances the rate of ketone body synthesis from palmitate as well as from caprinoate. The 3-OHB/AcAc ratios indicate a highly reduced state of the mitochondrial redox carriers in the presence of both fatty acids and carnitine. Ketogenesis from palmitate accounts for about 90% of the total beta-oxidation. At recovery of 95% of the radioactivity two thirds of totally fatty acid uptake are channeled into esterification, whereas the remainder is oxidized. alpha- and beta-agonists stimulate glycogen degradation and glucose release and reduce net lactate production in hepatocytes from newborn rats. The (1-14C)-palmitate uptake is not altered by alpha- and beta-agonists. Phenylephrine significantly enhances 14CO2 production from (1-14C)-palmitate. Neither of the agonists affects the rate of esterification or of ketone body production with palmitate as substrate. Isoproterenol, however, stimulates ketogenesis from caprinoate even in the presence of optimal carnitine concentrations.

A simple method for the isolation of highly enriched hormone-sensitive parenchymal liver cells from fetal rats.

A well reproducible method for preparing isolated parenchymal cells from fetal rat liver on the 22nd day of gestation by tryptic digestion is described including criteria for assessing their viability. The content of parenchymal liver cells was higher than 90% as could be demonstrated by light microscopic analysis of thin sections of the pelleted hepatocytes. Electron microscopic examination of the freshly prepared suspension revealed a well preserved ultrastructure of the hepatocytes. The metabolic competence of the isolated fetal hepatocytes was tested by alpha- and beta-adrenergic stimulation of glycogenolysis and glycogenolytic glucose release.

Effect of glucagon, phenylephrine, and isoproterenol on glycogenolysis and glucose release from fetal rat hepatocytes in suspension.

Fetal hepatocytes isolated at day 22 of gestation by trypsin digestion of rat liver retain their responsiveness to hormones. Glucagon significantly stimulates glycogenolysis and glucose release to 115 and 124%, respectively. The effect of alpha- and beta-agonists is more pronounced enhancing glycogen breakdown and glucose release to 133 and 147% (L-phenylephrine) and 183 and 202% (isoproterenol), respectively. The isolated fetal hepatocytes obtained by trypsin digestion are a useful tool for studying the hormonal control of liver metabolism during the perinatal stage without limitations met otherwise with extrahepatic factors and the effect of altered blood flow.

[Tracer kinetic studies on maternal-fetal amino acid transport in Wistar rats]. / Tracerkinetische Untersuchungen zum materno-fetalen Aminosäure-transport bei Wistarratten.

The experiments were designed to determine the amount of free amino acids which passes through the placenta from the maternal pool in plasma to the fetus. On day 19 of gestation we determined the growth rate of the rat fetus to be about 39 microgram amino nitrogen/minute/litter. Uniformly labelled [14 C]-L-leucine was used in our experiments. The specific radioactivity was measured in the maternal plasma amino acids and in fetal amino acids and proteins from zero to sixty minutes after pulse labelling. By means of compartmental analysis we calculated the transfer rate of amino nitrogen from the mother to the fetus to be 89 microgram/min/litter. The rate of amino nitrogen acccumulation in fetal carcass proteins was 39 microgram amino nitrogen/min/litter. The importance of our results in fetal metabolism is discussed.

A new method for producing a chronic E. coli pyelonephritis in rabbits.

Chronic pyelonephritis, especially that caused by the most common invader E. coli, corresponding satisfactorily to the pathophysiology of the human disease is difficult to reproduce. We have developed a pattern of infection with which we succeeded reliably in producing a chronic E. coli pyelonephritis. This was achieved by inserting a plastic catheter into the renal pelvis. A suspension of E. coli was injected via the catheter into the renal pelvis of 32 rabbits. This pattern of infection takes a chronic progressive course and, in all cases, results in typical macroscopic and histological changes in the kidney. The bacterial excretion in the urine remains unchanged over a period of months. With the experimental procedure described, the pathogenesis and pathophysiology of chronic E. coli pyelonephritis can be clearly studied. Drugs currently used on patients can be tested for their effectiveness in long-term application.