Pulmonary Pharmacology (1991) 4, 146-150 Q 1991 Longman Group UK Ltd
Isolated Perfused Lungs of Guinea-pig, in Contrast with Rat, Lack an Uptake Process for Noradrenaline L . J . Bryan-Lluka, S . R . O'Donnell Department of Physiology & Pharmacology, University of Queensland, Brisbane, Queensland 4072, Australia SUMMARY. The uptake and metabolism of noradrenaline were compared in isolated perfused lungs of guineapigs and rats . Lungs were perfused with 3 H-(-)-noradrenaline either a) at a concentration of 10 nM for 20 min in experiments to measure the metabolism of the amine or b) at a concentration of 2 nM for 2 min, in the presence or absence of 10 tM cocaine and with MAO and COMT inhibited, in experiments to measure the uptake of noradrenaline . The total formation of metabolites during the 20 min perfusion period was 36 .2 ± 2 .3 pmol g - ' (n = 6) in guinea-pig lungs, and 526 ± 26 pmol g -' (n = 6) in rat lungs (14.5-fold greater) . In guinea-pig lungs, the rate of uptake of noradrenaline was 0 .392 ± 0.044 pmol g - 1 min - ' (n = 3) and was unaffected by cocaine, whereas in rat lungs it was 5 .63 ± 0.03 pmol g -' min -' (n = 5) and was inhibited (88%) by cocaine . It is concluded from these results that the lungs of the guinea-pig lack the specific uptake process that, in rat lungs, allows removal of noradrenaline from the pulmonary circulation .
INTRODUCTION
under conditions in which the metabolizing enzymes were inhibited . Preliminary results of this study were presented to the Xth International Congress of Pharmacology ."
The lungs have an important role in the removal, biosynthesis and release of vasoactive hormones .' In particular, various groups have shown that noradrenaline is removed when it passes through the pulmonary circulation of rats, 2-4 rabbits, 5-8 cats, 9 dogs 9 and man ." Extensive studies on isolated lungs from rats' , ' and rabbits' and in vivo studies in rabbits' , ' have shown that this removal of noradrenaline involves an uptake process for the amine and its subsequent metabolism by monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) . Autoradiographic and fluorescence histochemical evidence has shown that it is the endothelial cells of the capillaries and post-capillary venules that take up noradrenaline in the pulmonary circulation of rats' , " and rabbits ." Removal of noradrenaline has also been reported by Mathe and co-workers to occur in isolated perfused lungs of guinea-pigs . 12-14 However, in preliminary experiments in our laboratory, no evidence of metabolism of noradrenaline in guinea-pig lungs could be obtained . This prompted us to compare the metabolism of noradrenaline in guinea-pig lungs with that in rat lungs in this study . As a result of our findings on the species difference in pulmonary noradrenaline metabolism, we also directly compared the uptake of noradrenaline in guinea-pig and rat lungs,
METHODS Preparation of lungs Adult, male, specific pathogen-free albino guinea-pigs (396 ± 9 g, n = 12) or adult, male, specific pathogenfree Wistar rats (176± 17 g, n = 15) were anaesthetized by intraperitoneal injection of pentobarbitone sodium (guinea-pigs : 75 mg kg - ' ; rats : 60 mg kg - ') . In uptake experiments (see below), the animals were pretreated with 75 mg kg - ' pargyline intraperitoneally 18 h and 2 h prior to the experiment to inhibit MAO . When the animals were in deep anaesthesia, the chest was opened, heparin sodium was administered intracardially (guinea-pigs : 3750U kg - ' ; rats : 250O U kg - ') and the animal was tracheostomized . The pulmonary artery and the left atria were cannulated quickly and the blood was flushed from the pulmonary circulation via the pulmonary artery cannula with Krebs solution at 37°C . The lungs were quickly removed from the thorax, placed in a closed chamber saturated with water vapour at 37°C and ventilated under negative pressure (peak inspiratory pressure of -2 kPa ; frequency 70 min - ') . Metabolism experiments
Address correspondence to : Dr Lesley J . Bryan-Lluka, Department of Physiology & Pharmacology, University of Queensland, Qld 4072, Australia .
The lungs were initially perfused via the pulmonary artery at 37°C for 15 min with Krebs solution contain146
Lack of Noradrenaline Uptake in Guinea-pigs
ing 5% bovine serum albumin at 20 ml min -1 for guinea-pigs or 10 ml min - ' for rats . During this period, the perfusate was recirculated via a reservoir equilibrated with 95% 0 2 and 5% C0 2. The lungs were then perfused at 37°C for 20 min with aerated (95% 0 2 and 5% C0 2) Krebs solution containing 10 nM 3H-noradrenaline at the flow rates stated above . During this perfusion with noradrenaline, perfusate from the pulmonary veins obtained via the cannulated left atrium (venous effluent) was collected over intervals of 0-2 min, 2-4 min, 4-6 min, 6-9 min, 9-12 min, 12-15 min, 15-18 .5 min and 18 .519 .5 min . At the end of the 20 min perfusion with noradrenaline, the lungs were blotted, weighed and placed in 0 .4 M perchloric acid containing antioxidants (2 .7 mM Na 2 EDTA and 10 mM Na2SO3) at 4°C . After at least 1 h in this solution, the lungs were homogenized, centrifuged at 12000 g for 20 min and samples of the supernatant were collected . A sample of perfusion solution was collected from the perfusion system after the lungs had been removed (arterial sample). Samples of venous effluent, arterial solution and supernatant from the lung homogenate were a) counted for 3H (liquid scintillation counter) and b) used to separate noradrenaline from its metabolites by column chromatography .'"' This method allowed the separation of noradrenaline, normetanephrine (NMN), dihydroxyphenylethyleneglycol (DOPEG), dihydroxymandelic acid (DOMA) and, in one further fraction, the two 0-methylated deaminated metabolites of noradrenaline (OMDA) . Uptake experiments The lungs were initially perfused at 37°C for 20 min with Krebs solution containing 5% bovine serum albumin, 10 .tM U-0521 to inhibit COMT and, in some experiments, 10 µM cocaine at 20 ml min -1 for guinea-pigs or 10 ml min - ' for rats . The lungs were then perfused at 37°C for 2 min with aerated Krebs solution containing 2 nM 3H-noradrenaline, 100 µM ' 4C-sorbitol, 10 µM U-0521 and, in some experiments, 10 µM cocaine at the same flow rates . At the end of this period, the lungs were treated as described for the metabolism experiments above and an arterial sample was collected also as described above . Samples of arterial solution and supernatant from the lung homogenate were counted for both 3H and 14 C. Viability of lung preparations The viability of the lungs was monitored by measuring the perfusion pressure throughout the experiments (guinea-pigs : 1 .07 ± 0.11 kPa, n=12 ; rats : 1 .29+ 0.07 kPa, n = 15) and by calculating the lung weight as a percentage of the body weight at the end of the experiment (guinea-pigs : 0 .54±0.02%, n=12 ; rats :
147
0.53+0 .01%, n=15) . Data from any preparation where the perfusion pressure was greater than 2 kPa or where the lung weight was greater than 0 .65% of body weight were not included . Drugs and solutions The drugs used were : cocaine hydrochloride (Drug Houses of Australia, Sydney, Australia); 3',4'dihydroxy-2-methylpropiophenone (U-0521 ; Upjohn, Kalamazoo, MI, USA) ; heparin sodium (as vials of 5000 U ml -1 ; Weddel Pharmaceuticals Ltd, London, UK) ; (-)-noradrenaline bitartrate (Sigma Chemical Co, St Louis, MO, USA) ; (-)-[7- 3H]-noradrenaline (New England Nuclear, Boston, MA, USA ; approximately 650 Bq pmol -1 , purified over alumina before use and diluted with unlabelled noradrenaline to the desired specific activity ; the final concentration of labelled noradrenaline was I nM in metabolism experiments and 2 nM in uptake experiments) ; pargyline hydrochloride (Sigma ; administered as a 92 .5 mg ml -1 solution in normal saline containing 154 mM NaCl) ; pentobarbitone sodium (as Nembutal vials of 60 mg ml - '; Bomac Laboratories Pty Ltd, Sydney, Australia) ; D-sorbitol (Sigma) ; D-[14 C]-sorbitol (Amersham International, Amersham, UK ; 12 Bq pmol -1 , diluted with unlabelled sorbitol to the desired specific activity ; the final concentration of labelled sorbitol was 33 nM) . Bovine serum albumin (Cohn Fraction V, 96-99% albumin) was obtained from Sigma . The Krebs solution contained (in mM) : NaCl 118, KC1 4 .7, CaC1 2 .2H20 2.5, KH 2PO4 1 .2, MgS04.7H20 1 .2, NaHCO 3 25 .0, glucose 11 .7, ascorbic acid 0 .57 and Na 2EDTA 0 .040 . Stock solutions of noradrenaline (10 mM) were prepared in 10 mM hydrochloric acid . Stock solutions of cocaine and sorbitol were prepared in water, and those of U-0521 and all dilutions were prepared in Krebs solution . Calculation of results Results were expressed as arithmetic means + s.e . The significance of differences between mean values was assessed by Students t-test . Percentage inhibition of uptake by cocaine was calculated as the difference between the rate of uptake in the absence and presence of cocaine expressed as a percentage of the rate of uptake in the absence of cocaine .
RESULTS Metabolism of 3H-noradrenaline The rates of appearance of NMN, DOPEG, DOMA and OMDA in the venous effluent from guinea-pig lungs perfused with 10 nM 3H-noradrenaline were low and variable and were very much lower than
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those for rat lungs under the same conditions (Fig . 1) . The rates of appearance of DOMA were very low, viz . less than 0 .1 pmol g - ' min t in guinea-pig lungs and less than 0 .25 pmol g - ' min - ' in rat lungs, and hence are not shown in the Figure . In the guinea-pig lungs, the total formation of metabolites in the 20-min perfusion period was 36 .2 ± 2 .3 pmol g - ' (n=6), comprising 23 .6% NMN, 17 .9% DOPEG, 8 .0% DOMA and 50 .5% OMDA, and the accumulation in the lungs of unchanged noradrenaline was 40 .7 ± 3 .0 pmol g -' (n = 6) . In the rat lungs perfused under the same conditions, the total formation of metabolites was 526 ± 26 pmol g- t (n = 6), which was 14 .5-fold greater (t = 18 .8, d . f. 10, P < 0 .001) than that in guinea-pig lungs . The metabolites in the rat lungs comprised 45 .9% NMN, 17 .2% DOPEG, 1 .7% DOMA and 35 .2% OMDA . The accumulation in the rat lungs of unchanged noradrenaline was 32 .0 ± 5 .2 pmol g - ' (n=6), which was not significantly different from that in the guinea-pig lungs (t = 1 .45, d .f. 10, P > 0 .05) . Uptake of 3 H-noradrenaline In view of the very low rates of pulmonary metabolism of noradrenaline in guinea-pigs compared with rats, the uptake of this amine was measured in lungs perfused with 2 nM 3 H-noradrenaline . Experiments were carried out in the absence and presence of the uptake inhibitor, cocaine (10 µM), and MAO and
15
0
10
20
PERFUSION TIME (MIN) Figure-Comparison of the metabolism of noradrenaline in guinea-pig (closed symbols) and rat (open symbols) lungs perfused with 10 nM 'H-noradrenaline . Means ± s .e . of rates of appearance (pmol g - ' min - ') of NMN ( •, 0), DOPEG (A, 0) and OMDA (0, o) are plotted against time of perfusion with noradrenaline (min) for 6 guinea-pigs and 6 rats . Neither MAO nor COMT was inhibited . The very low rates of DOMA appearance (< 0 .1 pmol g - ' min - ' in guinea-pig lungs and < 0 .25 pmol g - ' min - ' in rat lungs) have been omitted for clarity .
COMT were inhibited to prevent metabolism of noradrenaline after uptake . The rate of uptake of noradrenaline in guinea-pig lungs was very low (0 .392±0 .044 pmol g - ' min - ', n=3) and was not affected by cocaine (0 .249+ 0 .041 pmol g - ' min', n=3 ; t = 2 .37, d .f. 4, P>0 .05) . In rat lungs under the same conditions, the rate of uptake of noradrenaline was 5 .63±0 .39 pmol g - ' min -' (n = 5), 14 .4-fold greater than in the guineapig lungs, and was inhibited (88%) by cocaine (0 .684±0 .098 pmol g - ' min - ', n=4 ; t=11 .0, d .f. 7, P 0 .05) lungs .
DISCUSSION In the present study, perfusion of guinea-pig lungs with 10 nM noradrenaline resulted in very little metabolism of noradrenaline by MAO or COMT in the pulmonary circulation, in contrast with rat lungs . Although there were some differences between guineapig and rat lungs in the percentages of the different metabolites formed (viz . O-methylated deaminated metabolites comprised a greater percentage of metabolites in guinea-pig than rat lungs), the major difference was that the total formation of metabolites was approximately 14-fold less for lungs from guinea-pigs than from rats . Hence, there was metabolism by MAO and/or COMT of only 2% of the total noradrenaline perfused through the guinea-pig lungs in the 20-min perfusion period and accumulation in the lungs of a further 2% as unchanged noradrenaline . In contrast, in rat lungs, 26% of the total noradrenaline perfused through the lungs was metabolized and a further 2% was accumulated unchanged . We have concluded that the difference between the two species can be explained by the lack, in guinea-pig lungs, of the noradrenaline uptake process that is present in rat lungs . The reasons for this conclusion are that a) the rate of uptake of noradrenaline was approximately 14-fold lower in guinea-pig compared with rat lungs, thus accounting for the difference in the formation of metabolites described above and b) the uptake that did occur in guinea-pig lungs was not inhibited by a concentration of cocaine that caused almost complete inhibition of uptake in the rat . The low rate of uptake of noradrenaline in rat lungs in the present study when cocaine was present to inhibit Uptake, (0 .684 pmol g - ' min - ' with 2 nM noradrenaline) could be attributed to entry of noradrenaline into the cells by passive diffusion . We have based this conclusion on unpublished results which show that corticosterone (an Uptake, inhibitor) or higher concentrations of cocaine cause no further
Lack of Noradrenaline Uptake in Guinea-pigs
reduction in the rate of uptake of noradrenaline in rat lungs . Also, there is already evidence for low rates of diffusion of noradrenaline into various cells, including myocardial cells in rat heart 18 and pulmonary endothelial cells in rabbit lungs . , " Furthermore, the very low rate of uptake of noradrenaline in guineapig lungs in this study, even in the absence of cocaine (0 .392 pmol g-' min' with 2 nM noradrenaline), can also be attributed to passive diffusion, in that the rates are even lower than those thought to represent diffusional entry in the rat lungs (see above) . The results obtained in guinea-pig lungs in this study differ from those previously reported by Mathe and co-workers," - " who found that about 41% of noradrenaline perfused through the lungs was metabolized and a further 1 % was accumulated unchanged. 12 The small differences between the experimental conditions in their study and ours (e .g . noradrenaline concentration and perfusion time) cannot explain the major differences in the results . No data are available to exclude the possibility that the lungs in the study of Mathe and Volicer 12 developed oedema during the perfusion period . However, oedema would not be expected to increase the entry of noradrenaline into the lung tissue, based on data from rat lungs that appeared to have developed oedema ." Mathe and co-workers concluded that the removal of noradrenaline from the perfusate by guinea-pig lungs occurred predominantly by Uptake, . 12 However, this conclusion appears not to be justified since classical Uptake 2 inhibitors had only very small inhibitory effects on the uptake and metabolism of noradrenaline in guinea-pig lungs in their studies ." It also appeared that Uptake, was not the major mechanism for the noradrenaline removal that they found in guinea-pig lungs . 12 In rat and rabbit lungs, the uptake process for noradrenaline has been considered by most workers to have some properties of both Uptake, and Uptake 2. 1 However, we have recently provided evidence that the uptake of noradrenaline in rat lungs cannot be distinguished from Uptake,, 20 which is normally attributed only to noradrenergic neurones. Our data in the present study indicate that this process is lacking in guineapig lungs. In conclusion, we can find no evidence for substantial rates of removal of noradrenaline from the perfusate of guinea-pig lungs such as occurs in rat lungs . We have concluded that this is because the lungs of guinea-pigs lack the specific uptake process that transports noradrenaline in rat lungs . On this basis, the guinea-pig is not a useful species for studies on the effects of disease conditions or drug treatments on the pulmonary uptake and/or metabolism of catecholamines . Interestingly, this is not the only report of a guinea-pig uptake process being different to that in other species . We have previously shown that myocardial cells in guinea-pig atria" lack the Up-
1 49
take 2 transport process that is present in rat 22 and cat 2 ' atria . Acknowledgements We would like to thank Helen Vuocolo and Wendy Harris for excellent technical assistance and Upjohn Pty Ltd for a gift of U-0521 . The support of the work by a grant from the National Health and Medical Research Council of Australia is also gratefully acknowledged . References I .Gillis C N. Pharmacological aspects of metabolic processes in the pulmonary microcirculation . Ann Rev Pharmacol Toxicol 1986 ; 26: 183-200 . 2 .Alabaster V A, Bakhle Y S . The removal of noradrenaline in the pulmonary circulation of rat isolated lungs . Br J Pharmacol 1973 ; 47 : 325-331 . 3 .Hughes J, Gillis C N, Bloom F E . The uptake and disposition of dl-norepinephrine in perfused rat lung . J Pharmacol Exp Ther 1969; 169: 237-248 . 4 .Nicholas T E, Strum J M, Angelo L S, Junod A F. Site and mechanism of uptake of 3 H-1-norepinephrine by isolated perfused rat lungs . Circ Res 1974; 35 : 670-680 . 5 .Gillis C N, Iwasawa Y . Technique for measurement of norepinephrine and 5-hydroxytryptamine uptake by rabbit lung. J Appl Physiol 1972 ; 33 : 404-408 . 6 .Gillis C N, Roth J A . The fate of biogenic monoamines in perfused rabbit lung . Br J Pharmacol 1977 ; 59 : 585-590 . 7 .Catravas J D, Gillis C N. Pulmonary clearance of [ 14 C]-5-hydroxytryptamine and [ 3 H]norepinephrine in vivo : Effects of pretreatment with imipramine or cocaine. J Pharmacol Exp Ther 1980; 213 : 120-127 . 8 .Catravas J D, Gillis C N. Single-pass removal of [ 14C]5-hydroxytryptamine and [ 3 H]norepinephrine by rabbit lung, in vivo : Kinetics and sites of removal . J Pharmacol Exp Ther 1983 ; 224: 28-33 . 9 .Ginn R, Vane J R . Disappearance of catecholamines from the circulation . Nature 1968 ; 219: 740-742 . 10 .Gillis C N, Greene N M, Cronau L H, Hammond G L . Pulmonary extraction of 5-hydroxytryptamine and norepinephrine before and after cardiopulmonary bypass in man . Circ Res 1972 ; 30 : 666-674. 1 Llwasawa Y, Gillis C N, Aghajanian G . Hypothermic inhibition of 5-hydroxytryptamine and norepinephrine uptake by lung: Cellular location of amines after uptake. J Pharmacol Exp Ther 1973; 186: 498-507 . 12 .Mathe A A, Volicer L . Norepinephrine uptake in guinea pig lung. Effects of anaphylaxis, phenoxybenzamine and cocaine . Int Archs Allergy Appl Immunol 1977 ; 54 : 356-363 . 13 .Mathe A A, Levine B I, Antonucci M J . Uptake of catecholamines in guinea pig lung . Influence of cortisol and anaphylaxis . J Allergy Clin Immunol 1975 ; 55 : 170-179 . 14.Mathe A A, Vachon L, Bookbinder S . Inhibition of catecholamine uptake by steroids in the isolated guinea pig lung . Res Comm Chem Path Pharmacol 1975 ; 11 : 511-514 . 15 .Bryan L J, O'Donnell S R. Perfused lungs of the guinea-pig lack an uptake process for noradrenaline . Abstracts Xth Int Congr Pharmacol 1987 ; P754 . 16.Fiebig E R, Trendelenburg U. The neuronal and extraneuronal uptake and metabolism of 3H-(-)noradrenaline in the perfused rat heart . NaunynSchmiedeberg's Arch Pharmacol 1978 ; 303 : 21-35. 17 .Trendelenburg U, Stefano F J E, Grohmann M . The isotope effect of tritium in 3 H-noradrenaline . NaunynSchmiedeberg's Arch Pharmacol 1983 ; 323 : 128-140.
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18.Fiebig E R, Trendelenburg U . The kinetic constants for the extraneuronal uptake and metabolism of 3 H(-)-noradrenaline in the perfused rat heart. NaunynSchmiedeberg's Arch Pharmacol 1978 ; 303 : 37-45 . 19 .Metting P J, Levin J A, Samuels J T . Compartmental analysis of the efflux of 1-[3 H]norepinephrine from isolated perfused rat lungs. J Appl Physiol 1985 ; 58 : 244-250 . 20 . Bryan L J, O'Donnell S R, Westwood N N . The uptake process for catecholamines in endothelial cells in rat perfused lungs is the same as Uptake, in noradrenergic neurones . Br J Pharmacol 1988 ; 95 : 539P . 21 .Anning E N, Bryan L J, O'Donnell S R. The
extraneuronal accumulation of isoprenaline in trachea and atria of guinea-pig and cat : A fluorescence histochemical study. Br J Pharmacol 1979; 65 : 175-182 . 22 .Bryan L J, Cole J J, O'Donnell S R, Wanstall J C . A study designed to explore the hypothesis that beta-1 adrenoceptors are `innervated' receptors and beta-2 adrenoceptors are `hormonal' receptors . J Pharmacol Exp Ther 1981 ; 216 : 395-400 .
Date received: 11 June 1990 Date revised: 16 August 1990 Date accepted : 27 September 1990
Isolated Perfused Lungs of Guinea-pig, in Contrast with Rat, Lack an Uptake Process for Noradrenaline L . J . Bryan-Lluka, S . R . O'Donnell Department of Physiology & Pharmacology, University of Queensland, Brisbane, Queensland 4072, Australia SUMMARY. The uptake and metabolism of noradrenaline were compared in isolated perfused lungs of guineapigs and rats . Lungs were perfused with 3 H-(-)-noradrenaline either a) at a concentration of 10 nM for 20 min in experiments to measure the metabolism of the amine or b) at a concentration of 2 nM for 2 min, in the presence or absence of 10 tM cocaine and with MAO and COMT inhibited, in experiments to measure the uptake of noradrenaline . The total formation of metabolites during the 20 min perfusion period was 36 .2 ± 2 .3 pmol g - ' (n = 6) in guinea-pig lungs, and 526 ± 26 pmol g -' (n = 6) in rat lungs (14.5-fold greater) . In guinea-pig lungs, the rate of uptake of noradrenaline was 0 .392 ± 0.044 pmol g - 1 min - ' (n = 3) and was unaffected by cocaine, whereas in rat lungs it was 5 .63 ± 0.03 pmol g -' min -' (n = 5) and was inhibited (88%) by cocaine . It is concluded from these results that the lungs of the guinea-pig lack the specific uptake process that, in rat lungs, allows removal of noradrenaline from the pulmonary circulation .
INTRODUCTION
under conditions in which the metabolizing enzymes were inhibited . Preliminary results of this study were presented to the Xth International Congress of Pharmacology ."
The lungs have an important role in the removal, biosynthesis and release of vasoactive hormones .' In particular, various groups have shown that noradrenaline is removed when it passes through the pulmonary circulation of rats, 2-4 rabbits, 5-8 cats, 9 dogs 9 and man ." Extensive studies on isolated lungs from rats' , ' and rabbits' and in vivo studies in rabbits' , ' have shown that this removal of noradrenaline involves an uptake process for the amine and its subsequent metabolism by monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) . Autoradiographic and fluorescence histochemical evidence has shown that it is the endothelial cells of the capillaries and post-capillary venules that take up noradrenaline in the pulmonary circulation of rats' , " and rabbits ." Removal of noradrenaline has also been reported by Mathe and co-workers to occur in isolated perfused lungs of guinea-pigs . 12-14 However, in preliminary experiments in our laboratory, no evidence of metabolism of noradrenaline in guinea-pig lungs could be obtained . This prompted us to compare the metabolism of noradrenaline in guinea-pig lungs with that in rat lungs in this study . As a result of our findings on the species difference in pulmonary noradrenaline metabolism, we also directly compared the uptake of noradrenaline in guinea-pig and rat lungs,
METHODS Preparation of lungs Adult, male, specific pathogen-free albino guinea-pigs (396 ± 9 g, n = 12) or adult, male, specific pathogenfree Wistar rats (176± 17 g, n = 15) were anaesthetized by intraperitoneal injection of pentobarbitone sodium (guinea-pigs : 75 mg kg - ' ; rats : 60 mg kg - ') . In uptake experiments (see below), the animals were pretreated with 75 mg kg - ' pargyline intraperitoneally 18 h and 2 h prior to the experiment to inhibit MAO . When the animals were in deep anaesthesia, the chest was opened, heparin sodium was administered intracardially (guinea-pigs : 3750U kg - ' ; rats : 250O U kg - ') and the animal was tracheostomized . The pulmonary artery and the left atria were cannulated quickly and the blood was flushed from the pulmonary circulation via the pulmonary artery cannula with Krebs solution at 37°C . The lungs were quickly removed from the thorax, placed in a closed chamber saturated with water vapour at 37°C and ventilated under negative pressure (peak inspiratory pressure of -2 kPa ; frequency 70 min - ') . Metabolism experiments
Address correspondence to : Dr Lesley J . Bryan-Lluka, Department of Physiology & Pharmacology, University of Queensland, Qld 4072, Australia .
The lungs were initially perfused via the pulmonary artery at 37°C for 15 min with Krebs solution contain146
Lack of Noradrenaline Uptake in Guinea-pigs
ing 5% bovine serum albumin at 20 ml min -1 for guinea-pigs or 10 ml min - ' for rats . During this period, the perfusate was recirculated via a reservoir equilibrated with 95% 0 2 and 5% C0 2. The lungs were then perfused at 37°C for 20 min with aerated (95% 0 2 and 5% C0 2) Krebs solution containing 10 nM 3H-noradrenaline at the flow rates stated above . During this perfusion with noradrenaline, perfusate from the pulmonary veins obtained via the cannulated left atrium (venous effluent) was collected over intervals of 0-2 min, 2-4 min, 4-6 min, 6-9 min, 9-12 min, 12-15 min, 15-18 .5 min and 18 .519 .5 min . At the end of the 20 min perfusion with noradrenaline, the lungs were blotted, weighed and placed in 0 .4 M perchloric acid containing antioxidants (2 .7 mM Na 2 EDTA and 10 mM Na2SO3) at 4°C . After at least 1 h in this solution, the lungs were homogenized, centrifuged at 12000 g for 20 min and samples of the supernatant were collected . A sample of perfusion solution was collected from the perfusion system after the lungs had been removed (arterial sample). Samples of venous effluent, arterial solution and supernatant from the lung homogenate were a) counted for 3H (liquid scintillation counter) and b) used to separate noradrenaline from its metabolites by column chromatography .'"' This method allowed the separation of noradrenaline, normetanephrine (NMN), dihydroxyphenylethyleneglycol (DOPEG), dihydroxymandelic acid (DOMA) and, in one further fraction, the two 0-methylated deaminated metabolites of noradrenaline (OMDA) . Uptake experiments The lungs were initially perfused at 37°C for 20 min with Krebs solution containing 5% bovine serum albumin, 10 .tM U-0521 to inhibit COMT and, in some experiments, 10 µM cocaine at 20 ml min -1 for guinea-pigs or 10 ml min - ' for rats . The lungs were then perfused at 37°C for 2 min with aerated Krebs solution containing 2 nM 3H-noradrenaline, 100 µM ' 4C-sorbitol, 10 µM U-0521 and, in some experiments, 10 µM cocaine at the same flow rates . At the end of this period, the lungs were treated as described for the metabolism experiments above and an arterial sample was collected also as described above . Samples of arterial solution and supernatant from the lung homogenate were counted for both 3H and 14 C. Viability of lung preparations The viability of the lungs was monitored by measuring the perfusion pressure throughout the experiments (guinea-pigs : 1 .07 ± 0.11 kPa, n=12 ; rats : 1 .29+ 0.07 kPa, n = 15) and by calculating the lung weight as a percentage of the body weight at the end of the experiment (guinea-pigs : 0 .54±0.02%, n=12 ; rats :
147
0.53+0 .01%, n=15) . Data from any preparation where the perfusion pressure was greater than 2 kPa or where the lung weight was greater than 0 .65% of body weight were not included . Drugs and solutions The drugs used were : cocaine hydrochloride (Drug Houses of Australia, Sydney, Australia); 3',4'dihydroxy-2-methylpropiophenone (U-0521 ; Upjohn, Kalamazoo, MI, USA) ; heparin sodium (as vials of 5000 U ml -1 ; Weddel Pharmaceuticals Ltd, London, UK) ; (-)-noradrenaline bitartrate (Sigma Chemical Co, St Louis, MO, USA) ; (-)-[7- 3H]-noradrenaline (New England Nuclear, Boston, MA, USA ; approximately 650 Bq pmol -1 , purified over alumina before use and diluted with unlabelled noradrenaline to the desired specific activity ; the final concentration of labelled noradrenaline was I nM in metabolism experiments and 2 nM in uptake experiments) ; pargyline hydrochloride (Sigma ; administered as a 92 .5 mg ml -1 solution in normal saline containing 154 mM NaCl) ; pentobarbitone sodium (as Nembutal vials of 60 mg ml - '; Bomac Laboratories Pty Ltd, Sydney, Australia) ; D-sorbitol (Sigma) ; D-[14 C]-sorbitol (Amersham International, Amersham, UK ; 12 Bq pmol -1 , diluted with unlabelled sorbitol to the desired specific activity ; the final concentration of labelled sorbitol was 33 nM) . Bovine serum albumin (Cohn Fraction V, 96-99% albumin) was obtained from Sigma . The Krebs solution contained (in mM) : NaCl 118, KC1 4 .7, CaC1 2 .2H20 2.5, KH 2PO4 1 .2, MgS04.7H20 1 .2, NaHCO 3 25 .0, glucose 11 .7, ascorbic acid 0 .57 and Na 2EDTA 0 .040 . Stock solutions of noradrenaline (10 mM) were prepared in 10 mM hydrochloric acid . Stock solutions of cocaine and sorbitol were prepared in water, and those of U-0521 and all dilutions were prepared in Krebs solution . Calculation of results Results were expressed as arithmetic means + s.e . The significance of differences between mean values was assessed by Students t-test . Percentage inhibition of uptake by cocaine was calculated as the difference between the rate of uptake in the absence and presence of cocaine expressed as a percentage of the rate of uptake in the absence of cocaine .
RESULTS Metabolism of 3H-noradrenaline The rates of appearance of NMN, DOPEG, DOMA and OMDA in the venous effluent from guinea-pig lungs perfused with 10 nM 3H-noradrenaline were low and variable and were very much lower than
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those for rat lungs under the same conditions (Fig . 1) . The rates of appearance of DOMA were very low, viz . less than 0 .1 pmol g - ' min t in guinea-pig lungs and less than 0 .25 pmol g - ' min - ' in rat lungs, and hence are not shown in the Figure . In the guinea-pig lungs, the total formation of metabolites in the 20-min perfusion period was 36 .2 ± 2 .3 pmol g - ' (n=6), comprising 23 .6% NMN, 17 .9% DOPEG, 8 .0% DOMA and 50 .5% OMDA, and the accumulation in the lungs of unchanged noradrenaline was 40 .7 ± 3 .0 pmol g -' (n = 6) . In the rat lungs perfused under the same conditions, the total formation of metabolites was 526 ± 26 pmol g- t (n = 6), which was 14 .5-fold greater (t = 18 .8, d . f. 10, P < 0 .001) than that in guinea-pig lungs . The metabolites in the rat lungs comprised 45 .9% NMN, 17 .2% DOPEG, 1 .7% DOMA and 35 .2% OMDA . The accumulation in the rat lungs of unchanged noradrenaline was 32 .0 ± 5 .2 pmol g - ' (n=6), which was not significantly different from that in the guinea-pig lungs (t = 1 .45, d .f. 10, P > 0 .05) . Uptake of 3 H-noradrenaline In view of the very low rates of pulmonary metabolism of noradrenaline in guinea-pigs compared with rats, the uptake of this amine was measured in lungs perfused with 2 nM 3 H-noradrenaline . Experiments were carried out in the absence and presence of the uptake inhibitor, cocaine (10 µM), and MAO and
15
0
10
20
PERFUSION TIME (MIN) Figure-Comparison of the metabolism of noradrenaline in guinea-pig (closed symbols) and rat (open symbols) lungs perfused with 10 nM 'H-noradrenaline . Means ± s .e . of rates of appearance (pmol g - ' min - ') of NMN ( •, 0), DOPEG (A, 0) and OMDA (0, o) are plotted against time of perfusion with noradrenaline (min) for 6 guinea-pigs and 6 rats . Neither MAO nor COMT was inhibited . The very low rates of DOMA appearance (< 0 .1 pmol g - ' min - ' in guinea-pig lungs and < 0 .25 pmol g - ' min - ' in rat lungs) have been omitted for clarity .
COMT were inhibited to prevent metabolism of noradrenaline after uptake . The rate of uptake of noradrenaline in guinea-pig lungs was very low (0 .392±0 .044 pmol g - ' min - ', n=3) and was not affected by cocaine (0 .249+ 0 .041 pmol g - ' min', n=3 ; t = 2 .37, d .f. 4, P>0 .05) . In rat lungs under the same conditions, the rate of uptake of noradrenaline was 5 .63±0 .39 pmol g - ' min -' (n = 5), 14 .4-fold greater than in the guineapig lungs, and was inhibited (88%) by cocaine (0 .684±0 .098 pmol g - ' min - ', n=4 ; t=11 .0, d .f. 7, P 0 .05) lungs .
DISCUSSION In the present study, perfusion of guinea-pig lungs with 10 nM noradrenaline resulted in very little metabolism of noradrenaline by MAO or COMT in the pulmonary circulation, in contrast with rat lungs . Although there were some differences between guineapig and rat lungs in the percentages of the different metabolites formed (viz . O-methylated deaminated metabolites comprised a greater percentage of metabolites in guinea-pig than rat lungs), the major difference was that the total formation of metabolites was approximately 14-fold less for lungs from guinea-pigs than from rats . Hence, there was metabolism by MAO and/or COMT of only 2% of the total noradrenaline perfused through the guinea-pig lungs in the 20-min perfusion period and accumulation in the lungs of a further 2% as unchanged noradrenaline . In contrast, in rat lungs, 26% of the total noradrenaline perfused through the lungs was metabolized and a further 2% was accumulated unchanged . We have concluded that the difference between the two species can be explained by the lack, in guinea-pig lungs, of the noradrenaline uptake process that is present in rat lungs . The reasons for this conclusion are that a) the rate of uptake of noradrenaline was approximately 14-fold lower in guinea-pig compared with rat lungs, thus accounting for the difference in the formation of metabolites described above and b) the uptake that did occur in guinea-pig lungs was not inhibited by a concentration of cocaine that caused almost complete inhibition of uptake in the rat . The low rate of uptake of noradrenaline in rat lungs in the present study when cocaine was present to inhibit Uptake, (0 .684 pmol g - ' min - ' with 2 nM noradrenaline) could be attributed to entry of noradrenaline into the cells by passive diffusion . We have based this conclusion on unpublished results which show that corticosterone (an Uptake, inhibitor) or higher concentrations of cocaine cause no further
Lack of Noradrenaline Uptake in Guinea-pigs
reduction in the rate of uptake of noradrenaline in rat lungs . Also, there is already evidence for low rates of diffusion of noradrenaline into various cells, including myocardial cells in rat heart 18 and pulmonary endothelial cells in rabbit lungs . , " Furthermore, the very low rate of uptake of noradrenaline in guineapig lungs in this study, even in the absence of cocaine (0 .392 pmol g-' min' with 2 nM noradrenaline), can also be attributed to passive diffusion, in that the rates are even lower than those thought to represent diffusional entry in the rat lungs (see above) . The results obtained in guinea-pig lungs in this study differ from those previously reported by Mathe and co-workers," - " who found that about 41% of noradrenaline perfused through the lungs was metabolized and a further 1 % was accumulated unchanged. 12 The small differences between the experimental conditions in their study and ours (e .g . noradrenaline concentration and perfusion time) cannot explain the major differences in the results . No data are available to exclude the possibility that the lungs in the study of Mathe and Volicer 12 developed oedema during the perfusion period . However, oedema would not be expected to increase the entry of noradrenaline into the lung tissue, based on data from rat lungs that appeared to have developed oedema ." Mathe and co-workers concluded that the removal of noradrenaline from the perfusate by guinea-pig lungs occurred predominantly by Uptake, . 12 However, this conclusion appears not to be justified since classical Uptake 2 inhibitors had only very small inhibitory effects on the uptake and metabolism of noradrenaline in guinea-pig lungs in their studies ." It also appeared that Uptake, was not the major mechanism for the noradrenaline removal that they found in guinea-pig lungs . 12 In rat and rabbit lungs, the uptake process for noradrenaline has been considered by most workers to have some properties of both Uptake, and Uptake 2. 1 However, we have recently provided evidence that the uptake of noradrenaline in rat lungs cannot be distinguished from Uptake,, 20 which is normally attributed only to noradrenergic neurones. Our data in the present study indicate that this process is lacking in guineapig lungs. In conclusion, we can find no evidence for substantial rates of removal of noradrenaline from the perfusate of guinea-pig lungs such as occurs in rat lungs . We have concluded that this is because the lungs of guinea-pigs lack the specific uptake process that transports noradrenaline in rat lungs . On this basis, the guinea-pig is not a useful species for studies on the effects of disease conditions or drug treatments on the pulmonary uptake and/or metabolism of catecholamines . Interestingly, this is not the only report of a guinea-pig uptake process being different to that in other species . We have previously shown that myocardial cells in guinea-pig atria" lack the Up-
1 49
take 2 transport process that is present in rat 22 and cat 2 ' atria . Acknowledgements We would like to thank Helen Vuocolo and Wendy Harris for excellent technical assistance and Upjohn Pty Ltd for a gift of U-0521 . The support of the work by a grant from the National Health and Medical Research Council of Australia is also gratefully acknowledged . References I .Gillis C N. Pharmacological aspects of metabolic processes in the pulmonary microcirculation . Ann Rev Pharmacol Toxicol 1986 ; 26: 183-200 . 2 .Alabaster V A, Bakhle Y S . The removal of noradrenaline in the pulmonary circulation of rat isolated lungs . Br J Pharmacol 1973 ; 47 : 325-331 . 3 .Hughes J, Gillis C N, Bloom F E . The uptake and disposition of dl-norepinephrine in perfused rat lung . J Pharmacol Exp Ther 1969; 169: 237-248 . 4 .Nicholas T E, Strum J M, Angelo L S, Junod A F. Site and mechanism of uptake of 3 H-1-norepinephrine by isolated perfused rat lungs . Circ Res 1974; 35 : 670-680 . 5 .Gillis C N, Iwasawa Y . Technique for measurement of norepinephrine and 5-hydroxytryptamine uptake by rabbit lung. J Appl Physiol 1972 ; 33 : 404-408 . 6 .Gillis C N, Roth J A . The fate of biogenic monoamines in perfused rabbit lung . Br J Pharmacol 1977 ; 59 : 585-590 . 7 .Catravas J D, Gillis C N. Pulmonary clearance of [ 14 C]-5-hydroxytryptamine and [ 3 H]norepinephrine in vivo : Effects of pretreatment with imipramine or cocaine. J Pharmacol Exp Ther 1980; 213 : 120-127 . 8 .Catravas J D, Gillis C N. Single-pass removal of [ 14C]5-hydroxytryptamine and [ 3 H]norepinephrine by rabbit lung, in vivo : Kinetics and sites of removal . J Pharmacol Exp Ther 1983 ; 224: 28-33 . 9 .Ginn R, Vane J R . Disappearance of catecholamines from the circulation . Nature 1968 ; 219: 740-742 . 10 .Gillis C N, Greene N M, Cronau L H, Hammond G L . Pulmonary extraction of 5-hydroxytryptamine and norepinephrine before and after cardiopulmonary bypass in man . Circ Res 1972 ; 30 : 666-674. 1 Llwasawa Y, Gillis C N, Aghajanian G . Hypothermic inhibition of 5-hydroxytryptamine and norepinephrine uptake by lung: Cellular location of amines after uptake. J Pharmacol Exp Ther 1973; 186: 498-507 . 12 .Mathe A A, Volicer L . Norepinephrine uptake in guinea pig lung. Effects of anaphylaxis, phenoxybenzamine and cocaine . Int Archs Allergy Appl Immunol 1977 ; 54 : 356-363 . 13 .Mathe A A, Levine B I, Antonucci M J . Uptake of catecholamines in guinea pig lung . Influence of cortisol and anaphylaxis . J Allergy Clin Immunol 1975 ; 55 : 170-179 . 14.Mathe A A, Vachon L, Bookbinder S . Inhibition of catecholamine uptake by steroids in the isolated guinea pig lung . Res Comm Chem Path Pharmacol 1975 ; 11 : 511-514 . 15 .Bryan L J, O'Donnell S R. Perfused lungs of the guinea-pig lack an uptake process for noradrenaline . Abstracts Xth Int Congr Pharmacol 1987 ; P754 . 16.Fiebig E R, Trendelenburg U. The neuronal and extraneuronal uptake and metabolism of 3H-(-)noradrenaline in the perfused rat heart . NaunynSchmiedeberg's Arch Pharmacol 1978 ; 303 : 21-35. 17 .Trendelenburg U, Stefano F J E, Grohmann M . The isotope effect of tritium in 3 H-noradrenaline . NaunynSchmiedeberg's Arch Pharmacol 1983 ; 323 : 128-140.
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18.Fiebig E R, Trendelenburg U . The kinetic constants for the extraneuronal uptake and metabolism of 3 H(-)-noradrenaline in the perfused rat heart. NaunynSchmiedeberg's Arch Pharmacol 1978 ; 303 : 37-45 . 19 .Metting P J, Levin J A, Samuels J T . Compartmental analysis of the efflux of 1-[3 H]norepinephrine from isolated perfused rat lungs. J Appl Physiol 1985 ; 58 : 244-250 . 20 . Bryan L J, O'Donnell S R, Westwood N N . The uptake process for catecholamines in endothelial cells in rat perfused lungs is the same as Uptake, in noradrenergic neurones . Br J Pharmacol 1988 ; 95 : 539P . 21 .Anning E N, Bryan L J, O'Donnell S R. The
extraneuronal accumulation of isoprenaline in trachea and atria of guinea-pig and cat : A fluorescence histochemical study. Br J Pharmacol 1979; 65 : 175-182 . 22 .Bryan L J, Cole J J, O'Donnell S R, Wanstall J C . A study designed to explore the hypothesis that beta-1 adrenoceptors are `innervated' receptors and beta-2 adrenoceptors are `hormonal' receptors . J Pharmacol Exp Ther 1981 ; 216 : 395-400 .
Date received: 11 June 1990 Date revised: 16 August 1990 Date accepted : 27 September 1990
