Hg during helium ventilation (95 percent He:5 percent CO,). Even at these low values for alveolar Po,, the mitochondrial space was not completely reduced. This indicates some mitochondrial oxidative activity when alveolar Po, was in the 0.5-1.0 mrn Hg range.
These studies have demonstrated substrate oxidation and respiratory control with isolated lung mitochondria. Measurements of surface fluorescence and tissue redox couples show intracellular reduction of pyridine nucleotides in response to oxidative inhibitors. These studies indicate in situ activity of lung mitochondria and demonstrate the involvement of these organelles in mainte nance of lung intracellular redox state. An additional feature of lung mitochondria is the relatively rapid rate of a-glycerophosphate oxidation and the CaU requirement for activation of lung mitochondrial aGP dehydrogenase. Inbcellular Ca* may influence the aGP/DHAP ratio and thereby determine the relative availability of these triose phosphates for incorporation into lung phospholipids.
Discussion
Dr. T i e m y : I have seen reports where the Qo, for the mitochondria is actually lower than the Qo, for the whole tissue. I wonder what we would consider to be the normal consumption of oxygen by the mitochondria? Dr. Fisher: In absolute terms, the 0, consumption of lung mitochondria is around 40-50 nrnoles/min/mg protein at 28°C. That is around half the rate normally obtained for liver preparations. Dr. Tiemey: Your lactate-to-pyruvate ratios appear rather high. Dr. Fisher: These are tissue levels and tissue levels are always higher than perfusate levels.
Evidence for Different Forms of Monoamine Oxidase in Perfused Rabbit ~ u n ~ * C . N . Gillis, Ph.D., Jerome A. Rdh, Ph.D. and K . R. Baker, B.A.
Activity of lung mitochondria was studied with a preparation of isolated mitochondria. This was correlated with responses of the isolated perfused lung as determined from surface fluorescence measurements and tissue redox couples. Lung mitochondria demonstrated substrate oxidation, respiratory control, and Ca++ activation of a-glycerophosphate dehydrogenase. In response to oxidative inhibitors, the isolated lung showed increased surface fluorescence and increased tissue ratios of redox couples indicating reduction of pyridine nucleotides. The results demonstrate functional activity of lung mitochondria in situ. ACKNOWLEDGMENTS: Significant contributions to these experiments were made by D.J.P. Bassett, G. A. Huber, L. Furia, B. Stuart, and D. Jamieson. Helpful advice was provided by J. R. Williamson and K. LaNoue. Dr. B. Chance loaned the surface fluorometry equipment. Supported by HL 15013, HL 15061 ( SCOR ) , and the Veterans Administration Research Senice. 1 Reiss OK: Studies in lung metabolism. I. Isolation and properties of subcellular fractions from rabbit lung. J Cell Biol30:4557, 1966 2 Fisher AB, Scarpa A, LaNoue KF, et al: Respiration of rat lung mitochondria and the influence of Caw on substrate utilization. Biochemistry 12:1438-1445, 1973 3 Fisher AB, Huber GA, Bassett D: Oxidation of a-glycerophosphate by mitochondria from lungs of rabbits, sheep, and pigeons. Comp Wysiol Biochem (in press) 4 Chance B, Cohen P, Jobsis F,et al: Intracellular oxidationreduction states in Goo. Science 137:499508,1962 5 Scholz R, Thurman RG, Williamson JR, et al: Flavin and pyridine nucleotide oxidation-reduction changes in perfused rat liver. J Biol Chem 224:2317-2324, 1969 6 Williamson JR, Corkey BE: Assays of intermediates of the citric acid cycle and related compounds by fluorometric enzyme methods. In: Methods in Enzymology Vol XI11 (Lowenstein JM, ed) New York, Academic Press, 1961, 434-490
26S lRH ASPEN LUNG CONFERENCE
S
tudies in a number of laboratories have demonstrated the lungs of several species,'-' including man,5 have the capacity to remove varying amounts of the biogenic amines 5-hydroxytryptamine (5-HT) and norepinephrine (NE) from the pulmonary vascular space. Removal in these cases is equivalent to functional inactivation of these amines, since they are extensively deaminated within the lung to physiologically inactive products by the enzyme, monoamine oxidase (MAO).S14Recently, there has been increasing recognition that MAO, at least in broken cell preparations, exists in several forms that can be differentiated on the basis of their substrate and inhibitor ~pecificities.6.~ The type A form of mitochondrial MA0 which metabolizes 5-HT and NE is selectively inhibited by clorgyline, harmaline and related alkaloids.8 The type B form of mitochondrial MA0 deaminates benzylamine and the endogenous biogenic amine, phenylethylamine, and is inhibited selectively (except in the rabbit) by deprenyl and pargyline. A third form of arnine oxidase found in plasma and the microsomal fraction of several a r t e r i e ~ ~ metabolizes ~~l~ phenylethylamine and benzylamine and is inhibited specifically by semicarbazide. It should be emphasized that the existence of these different forms of amine oxidase in the intact organ has not been demonstrated and therefore their physiologic significance is presently unclear. Since perfused lung is capable of extensively deaminating the biogenic monoamines, it was of interest to use this preparation in an attempt to demonstrate multiple forms of the oxidase in a morphologically and functionally intact organ. 'From the De artment of Anesthesiology, Yale University School of ~ d c i n eNew , Haven. Su ported by Grant no. HL 13315 from the National Heart an$ ~ u n g Institute.
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TIME (min)
FIGURE1. Effect of harmaline on 14C-5-hydmxytryptamine and 14C-phenylethylmine deamination in perfused rabbit lung.
Experiments were carried out using rabbit lung preparations perfused in a manner previously described by Gillis and Iwasawa.12 Left and right lungs were perfused independently at a constant rate of 10 mVmin with Krebs bicarbonate medium for a 20 min equilibration period. Subsequently, both lungs were perfused with solutions of Krebs medium containing either 1"-phenylethylamine (PEA) or 14C-5hydroxytryptamine (5-HT) via the first branches of the pulmonary artery and the effluent from each lung was collected independently. The MA0 inhibitors pargyline, semicarbazide, or harmaline were added to the Krebs medium containing the 1%-labeled mine for perfusion through either the right or left lung. The untreated lung of each pair served as the control. Lungs to be exposed to the amine oxidase inhibitors, were also pretreated by perfusion for 15 minutes with these substrates before perfusion with the 14Cmonoamines and inhibitors were started. Aliquots of the total effluent, or individual fractions of effluent, were passed over columns of Bio Rex-70 cation exchange resin to separate unchanged PEA or 5-HT from their respective acid metabo1ites.13
In lungs perfused with 14C-PEA alone 60 to 80 percent or total radioactivity in the venous effluent was in the form of dearninated products. Table 1 indicates that treatment with 0.01 mM pargyline reduced PEA deamination by approximately 60 percent; increasing pargyline concentration to 1.0 mM failed to increase the Table 1-Effect o f Pargyline and Semicarbaade on Pulmonary Deamination o f Phenylethybmine (0.71
a)
Treatment
% Inhibition ( f SE) of deaminated product formed
Pargyline, 0.01 mM
59.9 f3.6 (n =4)
Pargyline, 1.0 mM
57.8 k2.4 (n-4)
Semicarbazide, 1.0 mM
27.7 k5.9 (n-5)
Pargyline, 0.01 or 1.0 mM Semicarbazide, 1.0 mM
+
>99
CHEST 67: 2, FEBRUARY, 1975 SUPPLEMENT
(n=4)
percent inhibition of PEA deamination (Table 1 ) . In the presence of the plasma amine oxidase inhibitor, semicarbazide, a mean value of 28 percent inhibition of PEA deamination was achieved. Treatment of lungs simultaneously with pargyline at either 0.01 or 1.0 mM concentration and semicarbazide ( 1.0 mM) completely prevented deamination of PEA. These experiments clearly demonstrate the existence of both the mitochondria1 type B and the plasma forms of amine oxidase in the intact perfused lung. The lung preparation used is also capable of extensive deamination of 5-HT, which is a substrate for the type A monoamine 0xidase.~1~ To verify the presence of this form of arnine oxidase in lung, harmaline, a specific type A inhibitor, was perfused in several experiments along with either "C-PEA in one lung or simultaneously with l4C-5-HT in the contralateral lung. As illustrated in Figure 1, which presents the results of a typical experiment, the production of 5-hydroxyindole acetic acid in the presence of harmaline was progressively reduced to about 30 percent of its original value while the acid metabolite of phenylethylamine was produced at its normal rate despite the presence of this inhibitor. It must be emphasized that during harmaline perfusion the uptake of =*-PEA or lC-5-HT into the lung was a t e r e d . Accordingly, it must be concluded that harrnaline inhibited the type A form of MA0 in lung. We believe these data represent definitive evidence for the existence of three f o k s of amine oxidase in an intact perfused organ. Although the location of the N e r e n t forms of enzyme cannot be determined from our studies, previous work from our laboratory1' has demonstrated that following inhibition of MA0 by pargyline, both 5-HT and NE fluorescence is .found predominantly in the capillary endothelium of rabbit lung. Therefore, it is possible that all three forms of the amine oxidase are associated with this cell type. Certainly the fact that the dearninated product of 14C-PEA appears so rapidly in the venous effluent suggests that the enzymes responsible must be located relatively close to the vascular space.
1 Vane JR: Release and fate of vasoactive hormones in the circulation. Br J Pharmacol35:209-242,1969 2 Gillis CN: Metabolism of vasoactive hormones by lung. Anesthesiol39:626-632, 1973 3 Hughes J, Gillis CN, Bloom F'E: The uptake and disposition of norepinephrine in perfused rat lung. J Pharmacol Exp Ther 169:237-248, 1969 4 Alabaster VA, Bakhle YS: Removal of 5-hydroxytryptamine in the pulmonary circulation of rat isolated lungs. Br J Pharmacol40:468-482,1970 5 Cillis CN, Greene NM, Cronau LH et al: Pulmonary extraction of 5-hydroxytryptamine and norepinephrine before and after cardiopulmonary bypass in man. Circ Res 30:666-674, 1972 6 Johnston JP: Some observations upon a new inhibitor of monoamine oxidase in brain tissue. Biochem Pharmacol 17:1285-1297, 1968 7 McCauley R, Racker E: Separation of two monoamine oxidases from bovine brain. Mol Cell Biochem. 1:73-81,
17TH ASPEN LUNG CONFERENCE 27s
14 Iwasawa Y, Gillis CN, Aghajanian G: Hypothemic inhibition of 5-hydroxytryptamine and epinephrine uptake by lung: cellular location of mines after uptake. J Warmacol Exp Ther 186:498507,1973
1973 Squires RF: Multiple forms of monoamine oxidase in intact mitochondria as characterized by selective inhibitors and thermal stability: a comparison of eight mammalian species. In Adv. Biochem. Psychopharmacol. (E Costa and M Sander, eds) Vol5, New York, Raven Press, 1972, 355-370 9 Yang H-YT, Neff NM: p-Phenylethylamine: a specffic substrate for type B monoarnine oxidase of brain. J Pharmacol Exp Ther 187:365-371, 1973 10 Rucker RB, Gottlick-Rieman W: Properties of rabbit aorta mine oxidase. Proc Soc Exp Biol Med 139:286-289, 1972 11 Coquil JF, Gordis C, Mack G, et al: Monoamine oxidase in rat arteries: evidence for differentforms and selective localization. Br J Pharmacol48:590-599, 1973 12 Gillis CN, Iwasawa Y: Technique for measurement of norepinephrine and 5-hydroxytryptamk uptake by rabbit lung. J Appl Physiol33:404-408, 1972 13 Roth JA, Gillis CN: Inhibition of lung, liver and brain monoamine oxidase by imipramine and desipramine. Biochem Pharmacol23:1138-1140,1974
Dr. Said: You have been able to separate the two processes that must be involved in the removal by the lung of biogenio amines; namely, the uptake itself and then the enzymatic degradation. It appears that even if you inhibit the enzymes responsible for the inactivation of the various biogenic amines that the lung continues to extract these atnines because you see them concentrated in the endothelial cells and you find in your radioisotope experiments also that they are taken up quite independently of the enzymatic d b d a t i o n . Dr. GiUis: Yes, I think that is true. The rate-limiting process in the overall removal, if one can consider the transport and subsequent metabolism as constituting removal, is the transport into the site of metabolisxp.
Crosslinking and Carbohydrate Studies on the Hydroxylated Glycopeptides which Accumulate in Alveoli and Lamellar ~odies"
proteinosis lavage material was reduced wi* NaH8B, in the presence of 1 percent SDS.' After hydrolysis and ion exchange chromatography, the profile of tritium incorporation demonstrated the apparent presence of hvdroxvnorleucine (HNL), the reduced form of the c~ossli& precursor b-amid adipic semi-aldehyde, and
M. A. PUSSYO,M.D.; S . N . Bhattachryya, Ph.D.;
S. Sahu,Ph.D.;andW.S.Lynn,M.D.
Discussion
I
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wo hydroxylated glycopeptides (apparent MW 62,000 and 36,000) accumulate in alveoli of patients with alveolar proteinosisl and can also be isolated in small (1-3 mg) quantities from normal rabbit and dog lungs.2 The peptides contain 1.5 percent hydroxyproline, 0 percent hydroxylysine, 15 percent glycine, 6.4 percent proline, 2.5 percent galactose, 1 percent mannose, 0.7 percent fucose, 0.6 percent glucosamine, and 1.2 percent sialic acid. Cyanogen bromide cleavage studiesSof the 36,000 MW peptide revealed three major components of apparent MW on SDS gels of 18,000, 12,000 (which both m t a i n e d 1.3 percent OHPRQ and 16 percent glycine) and 6000, which contained 1.2 percent OHPRO and 11.7 percent glycine and is probably heterogeneous. The two heavy fragments contained all the carbohydrate. These CNBr fragments &us appear to d 8 e r from the known CNBr fragments of collagpn. We recently have performed the following studies to attempt to clarify the nature and origin of these glycopeptides : 1 ) Crosslinking Studies: In collaboration with Drs. G. Mechanic and D.S. Jackson at the University of worth Carolina, a 20 mg sample of saline-washed alveolar *From the Departments of Biochemistry and Medicine, Duke
University Medical Center, Durham.
28s 17TH ASPEN LUNG CONFERENCE
FIGURE 1. (Upper) Pattern of tritium incorporation of crude alveolar proteinosis washout material, showing apparent crosslinks. (Locoer) After delipidation, the peaks ahistent with cross-links have either disappeared or are present in questionable amounts.
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