Investigations of thermotropic phase changes in peripheral nerve of frog and rat. A spin label study.

The temperature-dependent fluidity of myelin of frog and rat peripheral nerve (Nervus ischiadicus) was studied using the spin label technique. In frog nerve a phase change was detected at 38 degree C. In rat nerve no sharp phase change could be established, and the lipid-depleted frog and rat nerve also showed no transition. From the spectral data, it was concluded that in frog and rat nerve the lipid-protein interactions are different, i.e. species dependent. Ca-2+-depletion of frog nerve caused a loss of transition, while rat nerve remained unaffected. Thus it was indicated that, in frog nerve, Ca-2+ is involved in the phase change. In the total lipid extract of frog nerve a phase change centered at 32 degree C occurred, while the total lipid extract of rat nerve again showed no transition. It is suggested that a connexion exists between our results and investigations on the temperature dependence of an axonal conduction block of nerve.

6-Phosphogluconate/glucose-6-phosphate ratio in rat pancreatic islets during inhibition of insulin release by exogenous insulin.

Inhibition of glucose-stimulated insulin release by exogenous insulin has been demonstrated in pancreatic islets to be associated with a decrease of the NADPH/NADP ratio and the pentose-phosphate cycle activity. Batches of five islets were incubated for 15 and 90 minutes in 1 ml. of KRB buffer with 2 per cent albumin containing 3 mg./ml. glucose and 0, 200, 400, or 800 microU./ml. of rat insulin, and the glucose-6-phosphate (G6P) and 6-phosphogluconate (6PG) contents were determined by enzymatic cycling. In response to a rise in the concentration of insulin, the 6PG/G6P ratio decreased. A close relationship was observed between this decrease of 6PG/G6P ratio and the net insulin release, the absolute rate of glucose oxidation via the pentose phosphate cycle, and the NADPH/NADP ratios measured under similar conditions. The results suggest that exogenous insulin, directly or indirectly, regulates the pentose cycle activity in the pancreatic islets at the G6P dehydrogenase step.

Age-dependence of molecular and functional changes in biological membrane properties.

Some general aspects including results on the possible mechanisms of membrane ageing are reviewed. The liquid-crystalline fluid state of a biological membrane is an essential condition for maintenance of different membrane functions. The liquid-crystalline state of different plasma membranes changes with age of the organism. The degree of unsaturated fatty acids decreases and the content of cholesterol increases during ageing. It could be shown that superoxide radicals originate from minor side-reactions of oxidoreductase enzymes. Ageing increases the amount of superoxide radicals. A small amount of radicals escape quenching by superoxide dismutase. The formation of radicals leads to degradation of membrane lipids. The age-dependent changes in membrane lipid composition influence respiratory activity in rat heart mitochondria of old animals. Rat liver plasma membrane lipids also show a decrease in membrane fluidity which results in a change in transport parameters of cholic acid and thymidine. The change in age-dependent lipid-protein interactions was demonstrated by spin-label measurements in model membranes. The results demonstrated that peroxidative break-down of lipids is an ongoing post-transcriptional process of ageing. The possible role of protective repair mechanisms is discussed.

Effect of essential phospholipids on the properties of ATPases of isolated rat liver plasma membranes of young and old animals.

"Essential" phospholipids (EPL), rich in dilinoleyl-phosphatidylcholine were given orally to young and 780-800 day-old rats. The double-labelled (14C/32P) dilinoleyl-phosphatidylcholine was incorporated in plasma membranes and the amounts incorporated into different organs investigated. Old animals incorporated higher radioactivity than young. The 14C/32P ratio however decreased in comparison with the substance administered. The amount of (Na+-K+)-ATPase in rat liver plasma membranes of old animals increased after pretreatment with EPL. The thermostability of ATPases was increased in plasma membranes of EPL treated old animals. In old animals EPL significantly lowered the cholesterol content. The membrane fluidity increased. The role of EPl in structural and functional properties of aged plasma membranes is discussed.

Effect of local anesthetics on cellular respiration and thymidine transport in hepatocytes from young and old rats.

The effect of local anesthetics (procaine, nicotinoyl-procaine, tetracaine, and dibucaine) on thymidine uptake and cellular respiration was investigated in hepatocytes from 3- and 24-month-old rats. All local anesthetics inhibited the "high-affinity" as well as the "low-affinity" thymidine transport system in a non-competitive manner immediately upon addition. Nicotinoyl-procaine, tetracaine, and dibucaine showed a similar inhibition profile with an inhibition of 10--15% at 50 mumol/l, or of more than 60% at 1 mmol/l. The less-lipophilic procaine showed a distinctly lower inhibition (10% at 1 mmol/l). The inhibitory effect was reversible and not dependent on Ca2+. All local anesthetics exerted identical effects in hepatocytes from young and old rats. Nicotinoyl-procaine and tetracaine inhibited cellular respiration in young and old rats up to a maximum of 50%. Procaine did not reduce O2 consumption below 1 mmol/l. This inhibition appeared also immediately upon addition, was not reversible, and not dependent on Ca2+. It is concluded that local anesthetics impair quite different biological processes like thymidine transport and cellular respiration in hepatocytes from young and old animals even at concentrations below 100 mumol/l. The magnitude of inhibition was correlated to the lipid solubility of the local anesthetics. The mechanism seemed to be different to the anesthetic action and it is supposed that it is a direct hydrophobic interaction with membrane proteins. Thus the local anesthetic-induced increase in membrane fluidity could not improve the age-dependent impairment of thymidine transport. The reduction of respiration is considered to be due to reduced O2 diffusion. This inhibition is in striking contrast to previously observed stimulatory effects which in part lead to the use of local anesthetics in geriatrics.

Responses of mitochondrial superoxide dismutase, catalase and glutathione peroxidase activities to aging.

The previous observation (Eur. J. Biochem., 82 (1978) 563--567) that age-dependent accumulation of lipid peroxides follows as a consequence of increased radical formation in mitochondria has prompted an examination of the response of a set of protective enzymes to the above situation. Levels of mitochondrial catalase activity as well as selenium-dependent glutathione peroxidase activity were found to be increased with age, while superoxide dismutase activity remained unchanged. No selenium-independent glutathione peroxidase activity could be detected either in preparations from young 3-month-old controls or in preparations from 2-year-old rats. Both the relatively high and unchanged levels of reduced glutathione and kinetic considerations suggest that glutathione peroxidase is preferentially involved in lipid peroxide metabolism, while catalase predominantly metabolizes mitochondrial H2O2.

Altered hepatobiliary transport of taurocholic acid in aged rats.

Hepatobiliary transport of taurocholic acid was studied in adult (3 months) and old (2 years) rats using an isolated perfused rat liver technique in order to determine the effect of age on hepatic uptake and secretion of bile acids simultaneously. The results were analyzed using a steady-state compartmental model to estimate the uptake and secretion of taurocholic acid. Hepatic secretion was decreased to a greater extent than the uptake in old rats. These changes in transport activities were associated with increases in perfusate and liver bile acid pool sizes. These results can explain the decrease in total pool size and synthesis rate of bile acids observed previously in old rats using in vivo studies. It has been suggested that the age-dependent decrease in bile acid transport capacity of the liver is secondary to the altered lipid composition of the liver plasma membranes of old rats.

Age-dependent physiochemical and biochemical studies of human red cell membranes.

Erythrocyte ghosts of male persons of two age groups, younger than thirty and older than seventy years, were analyzed to investigate the imfluence of age on lipid composition, the physical state of the lipid phase, Na+K+-ATPase activity and sialic acid content. The phospholipid content in red cell membranes of old donors is significantly lower than in young ones. Cholesterol and fatty acid compositions shows no difference between the two donor groups. The membrane fluidity in liposomes prepared from total lipids of old donor decreased. No significant difference in lipid composition and membrane fluidity reflected by the spin labels was observed between blood group A and O. The activity of Na+K+-ATPase of the young donors with blood group A is significantly higher than those of old donors. The results also demonstrate a decrease of sialic acid content of red cells of old donors.

Effects of concanavalin A on the isolated perfused rat liver.

In the isolated perfused liver, Concanavalin A provoked a significant decrease of flow rate within 2 to 4 min. which was dose-dependent and could be partly inhibited by specific antagonists. Furthermore it was found that the lectin led to a decline of the respiration, an increase of the lactate/pyruvate ratio and a release of the transaminases into the medium. It was suggested that Concanavalin A displaced endothelial cells in the liver capillaries, which occluded the vessels and decreased the flow rate. The decreased respiration was considered to be secondary to this effect.

Interaction of fusidates with bile acid uptake by isolated rat hepatocytes.

The interaction of fisidic acid and two of its conjugates with carrier-mediated uptake of bile acids was investigated in isolated rat hepatocytes. All three fusidates inhibited the uptake of both cholate and taurocholate competitively suggesting a direct interaction of fusidates with bile acid carrier. The inhibition constants for all three fusidates for the inhibition of cholate uptake were significantly different from the respective inhibition constants for the inhibition of taurocholate uptake. This would indicate that both cholate and taurocholate are transported by more than one carrier into hepatocytes. The results may also indicate that taurine conjugated bile acids may be transported preferentially by one transport system while unconjugated bile acids may be preferentially transported by another transport system.

A compartmental model for hepatic transport of taurocholic acid in isolated perfused rat liver.

In order to characterize the transport of bile acids through the liver and to study the influence of drugs on these processes, a kinetic model for hepatobiliary transport of taurocholic acid (TC) using the isolated perfused liver was developed. After the system was brought to a steady state by infusing TC at a constant rate, a tracer dose of 14C-TC was injected into the medium. The medium disappearance of 14C-TC followed a first-order kinetic with a single rate constant. The plot of the biliary secretion rate of radioactivity versus time revealed a curve composed of at least three exponential components. From the described results and the present knowledge of hepatobiliary transport of bile acids we proposed a three compartment model, composed of a perfusion medium compartment and two liver compartments. Parameters calculated from the model constants agreed well with model-independent estimations. The influence of bromosulfophthalein (BSP) on the kinetic parameters was studied to compare the result with the known effect of BSP on hepatic transport of taurocholic acid. BSP decreased the constant describing the fractional transfer of taurocholic acid from medium into the liver, which is in agreement with the inhibition of hepatic uptake of taurocholic acid by BSP. Thus a three compartment model may adequately define the hepatobiliary transport of taurocholic acid in the isolated perfused rat liver.

The lack of active bile acid transport in AS-30 D ascites hepatoma cells.

The uptake of cholic acid as well as taurocholic acid into AS-30 D ascites hepatoma cells showed linearity with respect to incubation concentrations. It has been suggested that these processes can be described as simple diffusion. In further experiments it could be shown that ascites hepatoma cells were unable to conjugate cholic acid. These results may have significance in the phalloidin action on hepatocytes.

The interaction of rifamycin SV with hepatic transport of taurocholic acid in the isolated perfused rat liver.

The effect of rifamycin SV on hepatic transport of taurocholic acid was investigated using isolated perfused rat liver technique. In all experiments, the perfused liver was maintained at taurocholic acid steady state by infusing constant amount of taurocholic acid. Infusion of rifamycin SV at various rates decreased biliary secretion of bile acids in a dose-dependent manner. Replacement of rifamycin SV by perfusion medium reversed this effect. To determine the site of action of rifamycin SV, kinetic experiments with 14C-taurocholic acid were undertaken. Rifamycin SV elevated the half-life of the medium disappearance of 14C-taurocholic acid. Furthermore, the antibiotic delayed the biliary appearance of 14C-taurocholic acid. The analysis of the results gave indications that the antibiotic interferred with hepatic uptake as well as biliary secretion of taurocholic acid.

Inhibition of hepatic uptake of bile acids by rifamycins.

The effect of rifamycin SV and rifampicin on hepatic acid uptake was studied using isolated rat hepatocytes in presence and in absence of albumin. The drugs inhibited cholate uptake more than taurocholate uptake and the inhibition was of non-competitive type. In presence of 3% albumin the inhibitory effect of the drugs was more for cholate and less for taurocholate uptake than in absence of albumin. Neither the binding of bile acids nor that of the drugs to albumin was altered by one another. Thus the effect in presence of albumin cannot be explained by the binding of the drugs and bile acids to albumin alone. It is suggested that albumin interacts with hepatic bile acid uptake process and this interaction with cholate uptake is different from that with taurocholate uptake. This additional and different effect of albumin may explain the effect of the drugs in presence of albumin. The results may be of clinical significance in rifamycins treatments.

Therapeutic doses of erythromycin esteolate is not cholestatic in rats in vivo.

The effect of erythromycin esteolate (EE) on bile flow and bile acid secretion was studied in male Wistar rats in vivo. Daily oral treatment with a dose of up to 100 mg/kg for 1 week increased the bile flow and the bile acid secretion. Increasing the days of treatment to 4 weeks with a dose of 20 mg/kg did not alter the measured parameters significantly. Acute intravenous injection of erythromycin lactobionate (50 mg/kg) also increased bile flow and biliary bile acid secretion temporarily. The increase in bile flow may partly be due to the osmotic effect of the drug and its metabolites in bile. Since EE failed to produce cholestasis in the range of therapeutic doses, rats do not seem to be a suitable experimental model for studying EE-cholestasis.

Leucocidin from Pseudomonas aeruginosa and membrane functions.

Leucocidin from Pseudomonas aeruginosa (strain 158) induced loss of potassium from isolated hepatocytes. The (Na+-K+) stimulated ATPase activity of isolated rat liver plasma membranes showed dose-dependent activation up to 56%. Electron-spin-resonance (ESR) measurements gave no indication of toxin-induced changes in membrane fluidity. On isolated guinea pig heart auricles the toxin produced an increase in frequency from 180/min to about 300/min, with arrhythmia and transitory flutter. On isolated nerve-diaphragm preparations the toxin caused a contracture and a decline in twitch tension, with a loss of potassium into the bathing solution. The action potential of the electrically stimulated N. ischiadicus of rat or frog was not affected when leucocidin was added to the bathing solution up to a concentration of 10 micrograms/ml.