The interaction of endothelin receptor responses in the isolated perfused rat lung

To gain more insight into the complex pulmonary interactions of endothelins (ET), we studied airway and vascular responses to endothelins in isolated perfused rat lungs in the presence of the novel ET(B)-receptor antagonist BQ788. In particular we focused on airway responses and on prostacyclin release. The effectiveness of BQ788 in our system was shown by its ability to concentration-dependently prevent vasoconstriction (IC50 0.1 microM), bronchoconstriction (IC50 0.1 microM) and prostacyclin production (IC50 < 0.1 microM) induced by the ET(B)-receptor agonist IRL1620 (1 nmol). Airway responses to ET-1: ET-1-induced bronchoconstriction was aggravated by BQ123 (1 or 8 microM), while BQ788 pretreatment (1 or 8 microM) showed no significant effect. Simultaneous treatment with 8 microM BQ123 and BQ788 attenuated the ET-1-induced bronchoconstriction. Vascular responses to ET-1: ET-1 (1 nmol)-induced vasoconstriction was potentiated by BQ788 (1 or 8 microM), but attenuated by the ET(A)-receptor antagonist BQ123 (1 microM). In the presence of BQ788 diminished amounts of the stable prostacyclin metabolite 6-keto-PGF1alpha were detected in the perfusate. Simultaneous treatment with 8 microM BQ123 and BQ788 completely prevented the ET-1-induced vasoconstriction. Conclusions: Both ET(A)- and ET(B)-receptors contribute to ET-1-induced vasoconstriction and bronchoconstriction. The ET-1-induced vasoconstriction is attenuated by stimulation of ET(B)-receptors, a response that is partly mediated by prostacyclin. Due to the mutual interactions between ET(A)- and ET(B)-receptors, simultaneous inhibition of both receptors is required to prevent the deleterious effects of ET-1 on lung functions.     

Hydroperoxide-induced chemiluminescence of the perfused lung

Light-emission of the perfused lung is induced by t-butyl hydroperoxide, giving chemiluminescence yields that oscillate between 800 and 1500 counts/s depending on the site and position of the lung. The response of the perfused lung to infusion with different hydroperoxides gives a pattern similar to that observed with the liver microsomal fraction; ethyl hydroperoxide shows a much higher chemiluminescence yield than the tertiary (t-butyl and cumene)hydroperoxides. Alveolar oedema affected the light-emission of the perfused lung depending on the time at which oedema developed, decreasing light emission on infusion of hydroperoxide in the oedematous lung and increasing it when oedema appeared after the maximal chemiluminescence yield was already achieved. Paraquat, administered in vivo, augmented light-emission by approximately 2-fold. The effect of paraquat was a time-dependent process. Lung chemiluminescence, compared with liver chemiluminescence, needed higher hydroperoxide concentration to induce light-emission.     

Lipid synthesis by perfused lung

An isolated lung ventilated with pulses of negative pressure and perfused through the pulmonary vasculature was utilized for the study of 3-sn-phosphatidylcholine synthesis. The perfusion fluid consisted of a Krebs-Ringer phosphate buffer with 6% bovine serum albumin, pH 7.4, and the appropriate substrate. The simultaneous incorporation of (1-14C) palmitate and (2-3H) glycerol and the simultaneous incorporation of (CH3-14C) choline and (CH3-3H) methionine were examined. From these experiments it is concluded: 1) lung tissue incorporates (2-3H) glycerol into 3-sn-phosphatidylcholine to a greater extent than any other lipid examined; 2) both choline and methionine contribute to the synthesis of 3-sn-phosphatidylcholine, and 50-70% of the label in its nitrogen base is derived from choline and 30-50% from methionine; and 3) a high PO2 appears to reduce the synthesis of 3-sn-phosphatidylcholine.     

Dibutyryl cAMP inhibits endotoxin-induced increases in pulmonary vascular resistance and fluid filtration coefficient in the perfused rat lung

We investigated the effects of pre-treatment with dibutyryl cAMP (db-cAMP) or cGMP on endotoxin-induced hemodynamic changes and pulmonary vascular permeability in isolated perfused rat lungs. Intraperitoneal injection of Salmonella enteritidis endotoxin (2 mg/kg) caused increases in pulmonary arterial resistance (Ra) after venous reservoir elevation, in pulmonary filtration coefficient (Kf) and in lung wet-to-dry (W/D) weight ratio. Pre-treatment with db-cAMP blocked endotoxin-induced increases in Ra, Kf and W/D weight ratio. Pre-treatment with cGMP attenuated only the increase in Ra caused by endotoxin. Moreover, administration of db-cAMP 2 hours after endotoxin injection attenuated the increase in Ra induced by endotoxin treatment. The increases in Kf and W/D weight ratio caused by endotoxin were not affected by post-treatment with db-cAMP. Since the increases in Ra, Kf and W/D weight ratio caused by endotoxin were blocked by pre-treatment with db-cAMP, agents that increase intracellular cAMP level may be useful to prevent acute pulmonary vascular injury.     

Relaxin induces ovulations in the in-vitro perfused rat ovary

The effects of human recombinant relaxin on ovulation and ovarian steroidogenesis were investigated in vitro using a perfused rat ovary model. Ovaries of equine chorionic gonadotrophin (ECG; 20 IU)-primed Sprague-Dawley rats were perfused for 21 h. Ovarian release of oestradiol and progesterone was measured during the perfusion period and the number of ovulations was estimated by counting the released oocytes at termination of the experiment. Non-treated control ovaries did not ovulate whereas addition of ovine luteinizing hormone (LH; 100 ng/ml) resulted in a mean (+/- SEM) number of ovulations of 3.0 +/- 0.8 from all treated ovaries. Relaxin (10 micrograms/ml) induced mean (+/- SEM) number of ovulations at 2.4 +/- 0.2 in all treated ovaries but did not further increase the ovulation rate when combined with LH (mean +/- SEM 3.2 +/- 0.4). All ovulated oocytes in the groups stimulated by LH showed signs of nuclear maturation (germinal vesicle breakdown) when harvested, in contrast to ovulated oocytes in the relaxin group, which were immature (presence of germinal vesicle). Progesterone and oestradiol release was significantly increased in the LH-stimulated groups but not in the group treated only with relaxin, in comparison to the untreated control group. These results demonstrate that relaxin may have a paracrine role within the ovary and may facilitate ovulation, possibly by promoting connective tissue remodelling of the follicle wall.     

Disposition of the bromosulfophthalein-glutathione conjugate in the isolated perfused rat kidney

Renal elimination of the bromosulfophthalein-glutathione conjugate (BSP-GSH) after its i.v. administration in the rat in vivo is negligible. In our study we wanted to establish whether the high albumin-binding of BSP-GSH constitutes the major restrictive factor toward the urinary excretion of the compound. The renal disposition of BSP-GSH was studied in the isolated rat kidney during perfusions with or without albumin in the perfusate. The urinary clearance of BSP-GSH in the absence of albumin was very low (< 60 microliters/min) as compared to the inulin clearance (approximately 300 microliters/min). This indicates that albumin-binding is not the major reason for the low urinary clearance of BSP-GSH. Addition of albumin to the perfusate further decreased the urinary excretion by 60%. BSP-GSH is metabolized by the kidney into two major metabolites: the cysteinylglycine conjugate and the di-glutathione conjugate. Both metabolites appear in perfusate, which suggests that BSP-GSH undergoes tubular (re-)uptake. The di-glutathione conjugate is further metabolized to the di-cysteinylglycine conjugate. The di-glutathione conjugate and the di-cysteinylglycine conjugate are the major urinary components and the urinary elimination of BSP-GSH may depend on their formation. Inhibition of gamma-glutamyl transpeptidase activity with acivicin largely prevented the degradation to the cysteinylglycine and dicysteinylglycine conjugates of BSP. The total rate of urinary excretion, however, was only slightly lowered by acivicin. Apparently, cleavage of the gamma-glutamyl moiety is not relevant for the total urinary elimination of BSP-GSH.     

Metabolism of 2,3-butanediol stereoisomers in the perfused rat liver

The identification of 2,3-butanediol in sera of alcoholics led to the hypothesis that it may be a specific marker of alcohol abuse. We have investigated the metabolism of the individual isomers of 2,3-butanediol (2R,3R-, 2S,3S-, meso-2,3-butanediol and racemic 2,3-butanediol) in perfused livers from fed rats. Rates of uptake of the isomers decrease in the order (i) 2R,3R-, (ii) meso-, (iii) 2S,3S-2,3-butanediol. We observed interconversion of isomers and oxidation to acetoin with 2R,3R- and meso- but not with 2S,3S-2,3-butanediol. In perfusions conducted in deuterium oxide, interconversion of isomers was accompanied by incorporation of deuterium. Thus, interconversion of isomers occurs via a reversible oxidation to acetoin with incorporation of hydrogen from water. In perfusions with either 2R,3R- or meso-[2-14C]2,3-butanediol, the substrates were converted to labeled acetate, R-3-hydroxybutyrate and CO2, suggesting that 2,3-butanediol is oxidized to acetyl-CoA via acetoin.     

Ultrastructural localization of concanavalin A in the perfused rat heart

The purpose of this investigation was to assess the alteration in serum free fatty acid concentrations during heat stress and dehydration. Each subject was exposed to heat stress in an environment chamber on 2 separate occasions. During the first exposure the subjects remained seated until the core temperature was elevated 1.4degreesC resulting in a mean weight loss of 1.66 kg due to dehydration. The second condition involved water replacement equal to the weight loss of the initial dehydration condition. Blood samples were obtained prior to heat exposure, when the core temperature was elevated 0.7degreesC and 1.4degreesC. They were subsequently analyzed for free fatty acids (FFA), glucose and lactin acid. Heart rates and core temperatures were monitored at 4 min intervals. During the dehydration condition the mean change in serum FFA was 0.9 muEq/ml in contrast to 0.2 muEq/ml for the rehydration condition. Serum levels of glucose increased moderately throughout the exposure (8 mg-%).     

Silybin inhibition of amatoxin uptake in the perfused rat liver

Silybin dihemisuccinate, in a concentration of 0.4 mg/ml, almost completely inhibited the uptake of an amatoxin by the perfused rat liver. Similary, silybin should also interrupt absorption of toxins due to enterohepatic circulation, e.g. in dog and man. This effect may become important in the therapy of human Amanita poisoning.     

The isolated air perfused rat heart

A simple method has been developed for continuous monitoring of metabolic activity of an isolated, perfused rat heart by O2/CO2 respirometer. Since respirometer provides vital data on oxygen consumption and carbon dioxide production of a preserved organ on a continuous basis over a long period of time, it will be possible to use this method to monitor viability of not only isolated heart but also any given donor organ under preservation.     

Vitamin B2 metabolism in the isolated perfused rat liver

We note the existence of a "partially cis-acting" regulatory protein of bacteriophage lambda: the product of the phage Q gene. We suggest that there may be a complete spectrum from "all cis" to "all trans" for such regulatory proteins. This behavior might arise because a DNA-binding protein either acts at a nearby (cis) site soon after synthesis or becomes "lost" for its trans activity on another genome through nonspecific interactions with DNA. Our proposed explanation provides one evolutionary basis for the linkage of genes for regulatory proteins and the sites at which such proteins act; it also suggests a possible rationale for the "metabolic instability" of certain regulatory proteins.     

Endotoxin modulates arachidonic acid-induced glycogenolysis in the perfused rat liver

Effects of endotoxin on arachidonic acid (AA)-induced hepatic glycogenolysis were examined in perfused rat liver. In normal rat liver, infusion of AA increased oxygen consumption and glucose production concurrently. In rats injected with lipopolysaccharide (LPS) 6 h before, AA increased glucose production but suppressed oxygen consumption. The changes in LPS-injected rat were abolished by a thromboxane (Tx) A2 receptor antagonist. The release of Tx B2 by AA increased after LPS-injection. These results suggest that priming of hepatic macrophage by endotoxin in vivo enhances Tx synthesis, resulting in modulating hepatic glycogenolysis.     

Stimulation of gluconeogenesis with 1,3-butanediol in the perfused rat liver

An intensified synthesis of glucose is observed in gluconeogenesis from endogenous precursor only for the first 30 min of perfusion. Pyruvate introduction into the medium raises phosphoenolpyruvate carboxykinase and fructose-1,6-diphosphatase activities in the liver and determines maintenance of the glucose formation high rate for 90 min of perfusion. 1,3-butanediol is found to have a stimulating effect on gluconeogenesis from pyruvate. Introduction of 1,3 bytanediol into perfusate decreases the redox state of free NAD-pairs, increases the content of phosphoenolpyruvate, malate. ATP and the phosphoenolpyruvate carboxykinase and fructose-1.6-diphosphatase activity in the perfused liver.     

Comparison of the effects of nicorandil, pinacidil and nitroglycerin on hypoxic and hypercapnic pulmonary vasoconstriction in the isolated perfused lung of rat

Viral genome DNA from four different multiple-enveloped nuclear polyhedrosis virus isolates, obtained from naturally infected larvae of satin moth (Stilpnotia salicis), a pest of poplar tree (Populus) was analysed. Larvae were collected over a period of 11 years, from 1978 to 1989. The genomic DNA restriction patterns pointed to heterogeneity of these wild-type viruses. The differences observed in isolates of several years revealed limited restriction fragment length polymorphism and showed that these viruses contained distinct, but closely related genotypes. The genome size of SsMNPV was established as 128-134 kb, based on HindIII and SacI restriction analysis.     

First-pass metabolism of 5-fluorouracil in the perfused rat small intestine

The first-pass intestinal metabolism of 5-fluorouracil (5-FU) was investigated by single-pass perfusion of the rat small intestine. At the low concentration of 0.06 mg/ml, the fraction of 5-FU absorbed into (i.e., appeared in) the mesenteric venous blood (Fa,b) was about 50% smaller than the fraction absorbed (disappeared) from the intestinal lumen (Fa), indicating the first-pass extraction of 5-FU in the intestinal mucosa. By addition of uracil (6 mg/ml), the Fa of 5-FU was reduced presumably by competition for the pyrimidine carrier at the process of intestinal uptake (entry into the mucosa). The Fa,b was also reduced, but to a lesser extent, resulting in insignificant first-pass extraction. These results suggest that the extraction of 5-FU in the absence of uracil is caused by metabolism and that uracil is a competitor for this pathway. When 5-FU concentration was raised from 0.06 to 0.6 mg/ml in the absence of uracil, the Fa was reduced by about 50%, consistent with the suggestion of the involvement of saturable uptake by the pyrimidine carrier, and thereafter remained unchanged at 6 mg/ml. However, since Fa,b was also reduced by a similar extent, the intestinal availability (FI=Fa,b/Fa) was unchanged at about 0.5, indicating that the intestinal first-pass extraction of 5-FU is independent of concentration with the extraction ratio (difference between unity and FI) of about 0.5 over the wide range of concentration from 0.06 to 6 mg/ml. Thus, the present study demonstrates that the significant first-pass metabolic extraction of 5-FU occurs in the mucosa of the small intestine, supporting our previous suggestion that 5-FU undergoes first-pass metabolism not only in the liver but also in the small intestine after oral administration. Considering that the oral bioavailability of 5-FU in the human (28%) is reportedly comparable with that in the rat (28%), it is likely that intestinal first-pass metabolism may be significant also in the human. Intestinal first-pass metabolism should be taken into account to explore more efficient and controlled oral 5-FU therapy.