Surfactant cholesterol metabolism of the isolated perfused rat lung

The cuticle (0.15 to 0.5 microns thick) of the microscopic free-living nematode Panagrellus silusiae was isolated intact by incubating worms with 1% sodium dodecyl sulfate at 37 degrees C overnight. After shearing and further treatment with detergent, electron microscopy revealed that the cuticular pieces were free of contaminating material and retained their characteristic in situ ultrastructure. From amino acid determinations, the cuticle is collagen-like with high levels of glycine (approximately equal to 31 residue %), proline (approximately equal to 20 residue %) and alanine (approximately equal to 21 residue %) although the hydroxyproline (2.6 residue %) content is low. Half-cystine (approximately equal to 1 residue %) is present in purified cuticles. Treatment with 8 M guanidine hydrochloride-2% beta-mercaptoethanol can solubilize more than 85% of the cuticular preparation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized cuticles from juvenile, adult and old dead worms revealed, at least, 18 discrete components. Estimated molecular weights ranged from about 26 000 (peak 1) to 250 000 (peak 18).     

Use of the isolated perfused rat lung in studies on lung lipid metabolism

A procedure for the use of the isolated perfused rat lung in studies on metabolic regulation has been developed. The procedure, reasonably uncomplicated, yet physiological, maintains the lung so that edema is not observed. The phospholipid content remains normal, and incorporation of [1-(14)C]-palmitate, [2-(14)C]acetate, and [U-(14)C]glucose is linear with time for a minimum of 2 hr. The incorporation of [1-(14)C]-palmitate and [2-(14)C]acetate into the total lung phospholipid fraction and into the phosphatidylcholine and phospatidylethanolamine fractions has been studied. Increasing the concentration of palmitate in the medium from 0.14 to 0.51 mm increased by 60% the incorporation of [1-(14)C]palmitate into the total lung phospholipid fraction at 2 hr. When the palmitate concentration of the medium was 0.14 mm, addition of 0.11 and 0.79 mm oleate to the medium decreased [1-(14)C]palmitate incorporation into the total lung phospholipid fraction at 2 hr by 37 and 49%, respectively. The results suggest that the incorporation of exogenous fatty acids, present in the medium perfusing the lung, into lung phospholipids may depend upon the fatty acid composition of the medium. Known specific acyltransferase activities may be responsible for the ordered incorporation of available fatty acids into lung phospholipids.     

Effect of experimental diabetes and insulin on lipid metabolism in the isolated perfused rat lung.

The isolated perfused rat lung was used as a model to study the possible hormonal regulation of lipid metabolism in the mammalian adult lung. Experimental diabetes, whether induced by alloxan or streptozotocin, decreased the incorporation of [U-14C]glucose into neutral lipids and phospholipids of both the surfactant fraction and the residual fraction of the lung by 60-80%. Glucose incorporation into phosphatidylcholine and phosphatidylglycerol is decreased in experimental diabetes in both the surfactant and residual fractions to a comparable degree. Glucose incorporation is decreased in both the fatty acid and the glycerophosphocholine moieties of phosphatidylcholine isolated from the surfactant and residual fractions. Insulin treatment of normal animals 30 or 15 min prior to perfusion resulted in an approximate doubling of the incorporation of glucose into the phosphatidylcholine and phosphatidylglycerol isolated from the surfactant and residual fractions of the lung. The incorporation of glucose into palmitic acid isolated from phosphatidylcholine was also shown to increase similarly. The results of these investigations indicate that insulin may play a role in regulating the synthesis of the important lipid components of the mammalian pulmonary surfactant complex.     

Prostaglandin removal and metabolism by isolated perfused rat lung

We have investigated the mechanism(s) involved in the removal of prostaglandins (PG) from the pulmonary circulation by the lung. Unidirectional fluxes of PG from the circulation into the lung are measured in an isolated perfused rat lung preparation. Evidence is presented which suggests that a transport system for PG exists in lung tissue. This transport system is responsible for the removal of some PG from the circulation by the lung. PGE₁ and PGF_2α are substrates for this system, whereas PGB₁, PGA₁, and 15-keto-PGF_2α are not. Since PGA₁ is a substrate for the intracellular PG dehydrogenase, the selectivity of the lung's metabolism system for circulating PG is probably due to the selectivity of the transport system for PG. It is shown that the percentage of the pulmonary arterial concentration (C_A) of PGE₁ or PGF_2α that is metabolized on passage through the pulmonary circulation decreases rapidly as C_A increases. When the lungs were perfused with PGE₁ (PGF_2α), the metabolites detected in the venous effluent were 15-keto-PGE₁ (PGF_2α) and 15-keto-13,14-dihydro-PGE₁ (PGF_2α). The time course pattern of the appearance of metabolites in the venous effluent after the initiation of a constant C_A, and the relative concentrations of the metabolites in the venous effluent, were examined as a function of C_A.     

Dipeptide metabolism in the isolated perfused rat kidney

Renal handling of glycyl-proline was studied in the isolated perfused rat kidney. Glycyl-proline disappeared from the perfusate as a function of time. The dipeptide was freely filtered at the glomerulus but only 6% of the filtered load was excreted in the urine as the intact peptide. More than 90% of the filtered dipeptide was reabsorbed as the intact peptide and/or its hydrolytic products. Non-filtration mechanisms were also involved to a significant extent in the clearance of the peptide. Hydrolysis at intratubular, intracellular and peritubular sites all contribute to the disappearance of the dipeptide from the perfusate, though the relative contributions of each mechanism are not known. Significant metabolic conversions, especially the conversion of glycine to serine, were also observed during perfusion.     

Xylitol metabolism in the isolated perfused rat liver

1. Loading the isolated perfused liver from well-fed rats with xylitol (20mm) caused a depletion of adenine nucleotides and P_i and an accumulation of α-glycerophosphate. The ATP content fell to 66% of the control value after 10min and to 32% after 80min. The ADP and AMP contents also fell. After 80min 63% of the total adenine nucleotides and 59% of the P_i had been lost. 2. The α-glycerophosphate content rose from 0.13 to 4.74μmol/g at 10min and reached 8.02μmol/g at 40min. 3. Xylitol was rapidly metabolized, the main products being glucose, lactate and pyruvate. 4. The [lactate]/[pyruvate] ratio in the presence of xylitol rose to 30–40. 5. On perfusion of livers from starved animals the main product of xylitol metabolism was glucose and the mean ratio xylitol removed/glucose formed was 1.29 (corrected for endogenous glucose and lactate production). This is close to the predicted value of 1.2. 6. Evidence is presented indicating that the loss of adenine nucleotides caused by xylitol is not due to the increased ATP consumption but to the accumulation of α-glycerophosphate and depletion of P_i. 7. The loss of adenine nucleotides accounts for the hyperuricaemia which can occur after xylitol infusion in man. 8. The relevance of the findings to the clinical use of xylitol as an energy source is discussed.     

Carbohydrate metabolism in the isolated perfused rat kidney

1. Anaerobic formation of lactate from glucose by isolated perfused rat kidney (411mumol/h per g dry wt.) was three times as fast as in aerobic conditions (138mumol/h per g). 2. In aerobic or in anaerobic conditions, the ratio of lactate production to glucose utilization was about 2. 3. Starvation or acidosis caused a decline of about 30% in the rate of aerobic glycolysis. 4. The rate of formation of glucose from lactate by perfused kidney from a well-fed rat, in the presence of 5mm-acetoacetate (83mumol/h per g dry wt.), was of the same order as the rate of aerobic glycolysis. 5. During perfusion with physiological concentrations of glucose (5mm) and lactate (2mm) there were negligible changes in the concentration of either substrate. 6. Comparison of kidneys perfused with lactate, from well-fed or starved rats, showed no major differences in contents of intermediates of gluconeogenesis. 7. The tissue concentrations of hexose monophosphates and C(3) phosphorylated glycolytic intermediates (except triose phosphate) were decreased in anaerobic conditions. 8. Aerobic metabolism of fructose by perfused kidney was rapid: the rate of glucose formation was 726mumol/h per g dry wt. and of lactate formation 168mumol/h per g (dry wt.). Glycerol and d-glyceraldehyde were also released into the medium. 9. Aerobically, fructose generated high concentrations of glycolytic intermediates. 10. Anaerobic production of lactate from fructose (74mumol/h per g dry wt.) was slower than the aerobic rate. 11. In both anaerobic and aerobic conditions the ratio [lactate]/[pyruvate] in kidney or medium was lower during perfusion with fructose than with glucose. 12. These results are discussed in terms of the regulation of renal carbohydrate metabolism.     

Effect of diabetes on the metabolism of chenodeoxycholic acid in isolated perfused rat liver

The formation of alpha-muricholic acid and beta-muricholic acid from chenodeoxycholic acid was comparatively investigated in livers isolated from normal, streptozotocin-diabetic, and insulin-treated diabetic rats. [24-14C]Chenodeoxycholic acid or [24-14C]alpha-muricholic acid was infused into the perfused livers. There was no difference in biliary excretion of 14C among the different groups of rats after the infusion of each 14C-labelled bile acid. Biliary [14C]bile acids were chromatographed on a thin-layer plate and the distribution of radioactivity on the plate was measured by radioscanning. In the diabetic group, the formation ratio of alpha-muricholic acid and beta-muricholic acid from [24-14C]chenodeoxycholic acid and also that of beta-muricholic acid from [24-14C]alpha-muricholic acid were much smaller than in the normal group. Treatment of the diabetic group with insulin cancelled the difference in the infusion of each [24-14C]bile acid. The results indicate that not only 6 beta-hydroxylation of chenodeoxycholic acid to alpha-muricholic acid but also 7-epimerization of the latter acid to beta-muricholic acid is suppressed in an insulin-deficient state in rats.     

Disappearance of enkephalins in the isolated perfused rat lung

Enkephalin disappearance during a single passage through the isolated, Krebs'-perfused rat lung was examined by superfusion bioassay. The rat colon was used to quantitate enkephalin disappearance since it proved to be sensitive to physiologic concentrations (10^?11M) of met⁵-enkephalin or an analog D-ala²-D-leu⁵-enkephalin. The rat stomach strip was used to assess the release of prostaglandins from the pulmonary vasculature. The rat lung rapidly degraded the enkephalins but released no prostaglandins in the dose-range of 0.1 – 50 ng. Captopril at doses which blocked conversion of angiotensin I to II inhibited the degradation of enkephalins across the lung.