19-nor-DOC biosynthesis in the isolated perfused rat kidney

19-nor-deoxycorticosterone (19-nor-DOC) is a potent salt retaining and hypertensinogenic mineralocorticoid that is excreted in the urine. While the precursor of 19-nor-DOC, 19-oxo-DOC, is produced by the adrenal cortex, conversion to 19-nor-DOC does not occur in the adrenal gland. We have examined the hypothesis that 19-nor-DOC is synthesized from precursors in the kidney. 19-oxo-DOC was added to the perfusate of isolated rat kidney preparations (n = 5) at a concentration of 10 μM. During 1 h of perfusion following addition of 19-oxo-DOC, 71 ± 6% of the precursor was converted to 19-oic-DOC, an immediate precursor of 19-nor-DOC, and 8.3 ± 1.8% was converted to 19-nor-DOC. This represents the first definitive evidence that 19-nor-DOC is produced in the kidney from adrenal precursors.     

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.     

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).     

ADPase activity of isolated perfused rat lung.

More than 90% of 3H-ADP was metabolized following bolus injection into rat isolated perfused lungs. The major metabolite was inosine, with lesser amounts of adenosine and AMP. The mean pulmonary transit time for the radioactivity associated with ADP and its metabolites was the same as that for the vascular marker 14C-dextran, indicating that ADP is metabolized by enzymes in the pulmonary vessel walls. The metabolism of 3H-ADP was apparently unaffected by the simultaneous injection of prostacyclin or by continuous infusion of indomethacin or aspirin. 3H-ADP was similarly metabolized by the lung following continuous infusion, although relatively higher amounts of adenosine were observed. The metabolism of ADP by the lung represents biological inactivation since over 95% of the platelet-aggregatory activity of ADP was lost on passage through the lung.     

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.     

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.     

Effects of terbutaline on sodium transport in isolated perfused rat lung

We have previously presented evidence that cultured alveolar epithelial cell monolayers actively transport sodium from medium to substratum, and that this process can be stimulated by beta-agonists. In this study the isolated perfused rat lung was utilized to investigate sodium transport across intact mammalian alveolar epithelium. Radioisotopic tracer(s) (22Na and/or [14C]sucrose) were instilled into the airways of isolated Ringer-perfused rat lungs. The appearance of isotope(s) in the recirculated perfusate was measured and a permeability-surface area product was calculated. Pharmacological agent(s) (terbutaline and/or propranolol) were present in the instillate or were added to the perfusate during the experiments. Terbutaline alone, whether in the instillate or perfusate, caused a significant increase in 22Na flux. This increase was prevented by the presence of propranolol. [14C]sucrose fluxes were unaffected by the presence of terbutaline. These data are consistent with the presence of an active component of sodium transport across intact mammalian alveolar epithelium that leads to removal of sodium from the alveolar space.     

Selenium deficiency potentiates paraquat-induced lipid peroxidation in isolated perfused rat lung

Glutathione peroxidase (GSHPx), a seleno-enzyme, reduces lipid hydroperoxides while producing oxidized glutathione (GSSG), which can efflux from cells. To study the role of GSHPx in antioxidant defense, isolated lungs from selenium-deficient rats were perfused for 2 h with or without 1 mM paraquat. Perfusate GSSG was measured as an index of GSHPx activity, and malondialdehyde (MDA) as an index of lipid peroxidation. Selenium deficiency decreased lung GSHPx activity 75-80%. During perfusion control lungs showed GSSG efflux of 8.5 +/- 4.5 nmol/h and with paraquat 49.1 +/- 12.1 nmol/h. Selenium-deficient lungs with or without paraquat showed GSSG efflux of 16.4 +/- 5.3 and 13.7 +/- 8.9 nmol/h, respectively. MDA efflux occurred only in paraquat-perfused selenium-deficient lungs (7.8 +/- 2.7 nmol/h). Lung homogenates from this group had lower GSH + GSSG than the other three groups. These results indicate an inverse correlation between GSSG efflux and MDA accumulation from paraquat-perfused lungs and suggest that increased turnover of the GSHPx reaction protects paraquat-perfused lungs from lipid peroxidation.     

N-oxidation of imipramine by isolated perfused rat and rabbit lung

Pulmonary uptake and metabolism of imipramine (IMP) was investigated in isolated perfused rat (IPrL) and rabbit (IPRL) lung preparations. Perfusate containing ¹⁴C-IMP (1.2 μmole/g lung) was recirculated through the pulmonary artery in artificially ventilated lungs. The radioactivity in the perfusate declined rapidly and about 80% of the dose was taken up by the lungs within 10 minutes in both IPrL and IPRL preparations. A steady-state was apparently reached thereafter in the IPRL, while a portion of the radiolabel effluxed into the perfusate of the IPrLs, thus reducing the net lung content to 54% of added IMP by 60 minutes. After 60 minutes perfusion, metabolites of IMP accounted for the major radioactivity (80%) in the perfusate, while the lung contained mainly (83%) the unchanged parent compound. The principal metabolite was identified as IMP-N-oxide (IMP-NO) which was found in the perfusate after 5 minutes of perfusion. Only 3% of the added IMP was metabolized by IPRL in 60 minutes. SKF-525A, an inhibitor of cytochrome P-450-mediated monooxygenase system, did not inhibit but enhanced the metabolism of IMP by IPrL to IMP-NO. IMP was principally metabolized to IMP-NO by incubations of 9,000 g supernatant fractions of rat lungs to a significantly higher extent than similar rabbit lung preparations. Including SKF-525A significantly accelerated the metabolism of IMP to IMP-NO in accordance with the perfusion experiments. These results suggest that in contradiction to publishedd reports, IMP is appreciably metabolized by the rat lung $\text{via}$ N-oxidation by non-cytochrome P-450 pathway and the metabolite formed in the lung is released into the circulation indicating its low affinity for the lung tissue.     

The inhibition of oxalate biosynthesis in isolated perfused rat liver by DL-phenyllactate and n-heptanoate

Glycolate oxidase was isolated and partially purified from human and rat liver. The enzyme preparation readily catalyzed the oxidation of glycolate, glyoxylate, lactate, hydroxyisocaproate and α-hydroxybutyrate. The oxidation of glycolate and glyoxylate by glycolate oxidase was completely inhibited by 0.02 m dl-phenyllactate or n-heptanoate. The oxidation of glyoxylate by lactic dehydrogenase or xanthine oxidase was not inhibited by 0.067 m dl-phenyllactate or n-heptanoate. The conversion of [U-¹⁴C] glyoxylate to [¹⁴C] oxalate by isolated perfused rat liver was completely inhibited by dl-phenyllactate and n-heptanoate confirming the major contribution of glycolate oxidase in oxalate synthesis. Since the inhibition of oxalate was 100%, lactic dehydrogenase and xanthine oxidase do not contribute to oxalate biosynthesis in isolated perfused rat liver. dl-Phenyllactate also inhibited [¹⁴C] oxalate synthesis from [1-¹⁴C] glycolate, [U-¹⁴C] ethylene glycol, [U-¹⁴C] glycine, [3-¹⁴C] serine, and [U-¹⁴C] ethanolamine in isolated perfused rat liver. Oxalate synthesis from ethylene glycol was inhibited by dl-phenyllactate in the intact male rat confirming the role of glycolate oxidase in oxalate synthesis in vivo and indicating the feasibility of regulating oxalate metabolism in primary hyperoxaluria, ethylene glycol poisoning, and kidney stone formation by enzyme inhibitors.     

Effect of CO 2 concentration on phospholipid metabolism in the isolated perfused rat lung

Studies have been carried out on the incorporation of [U-(14)C]glucose, [2-(14)C]pyruvate, [2-(14)C]acetate, and [1-(14)C]-palmitate into the phospholipids of the isolated perfused rat lung in the presence of either 6 or 45 mm total CO(2) concentration in the perfusion medium. Incorporation of [U-(14)C]glucose into total phospholipid and into the phosphatidylcholine fraction was increased 19-53% over the 2-hr perfusion period in lungs perfused with medium containing 45 as compared with 6 mm CO(2). The incorporation of [2-(14)C]acetate, [2-(14)C]-pyruvate, and [1-(14)C]palmitate was not affected by the change in medium CO(2) concentration. Increased incorporation of [U-(14)C]glucose combined with a shift toward greater incorporation into the fatty acids of the phosphatidylcholine fraction produced a maximum increase of 90% in [U-(14)C]glucose incorporation into the fatty acids of phosphatidylcholine after 2 hr of perfusion in the presence of medium containing 45 mm CO(2) as compared with 6 mm CO(2). The increase in medium CO(2) concentration produced as much as a 150% increase in [U-(14)C]glucose incorporation into palmitate derived from the phosphatidylcholine fraction. The results provide evidence that glucose functions as an important precursor of palmitate in the phosphatidylcholine fraction of lung phospholipids and that the CO(2) concentration of the perfusion medium affects the incorporation of glucose into palmitate.