Thorax (1965), 20, 149.

Oxidative enzymes of the interalveolarseptum of the rat1

W. S. TYLER2 AND A. G. E. PEARSEFrom the Department of Pathology, Postgraduate Medical School, London, W.12

Though the interalveolar septum is the targettissue for many deleterious agents, and one ofits constituent cell-types is thought to be thesource of a surfactant essential to normalpulmonary function (Klaus, Reiss, Tooley, Piel,and Clements, 1962), little is known concerningthe metabolic potential of this tissue. Histochemi-cal methods, though mainly qualitative, permitprecise localization of chemical activities amongvarious components of an organ. This type ofinformation is not obtainable by the classical bio-chemical methods of extraction, fractionation ortissue incubation. The object of this study is toreveal the metabolic potential of the cells of theinteralveolar septum.The few histochemical studies that have been

reported have concerned only a few of the histo-chemically demonstrable oxidative enzymes(Pliess and Suhr, 1959; Sorokin, Padykula, andHerman, 1959; Sorokin, 1961). The small num-ber of studies appears to be due in part to thedifficulty in preparing sections of lung suitablefor dehydrogenase histochemistry. The authorshave devised a simple method for preparing thincryostat sections of distended lung which aresuitable for histochemical procedures.

MATERIALS AND METHODS

Lungs from 10 female white rats weighing 150 to250 g. were studied. Six of the rats were euthanizedby the inhalation of coal gas. The other four receivedNembutal intraperitoneally. The thorax was openedand the lungs were allowed to collapse. The tracheawas isolated in the cervical region, cannulated, and4% gelatin (Bacteriological grade from G. T. Gurr)at 37' C. was injected until the lungs approximatelyfilled the thorax. The gelatin in the lungs was con-gealed by cooling in running cold water for five to10 minutes. Small blocks of gelatin-infiltrated lung

5Supported in part by U.S.P.H.S. Special Fellowship 1-F3-HE-20317-01 and U.S.P.H.S. IHE-061012 On Sabbatical leave from the Department of Anatomy, Universityof California, Davis, Calif., U.S.A.

were quenched in liquid oxygen. Sections 8, and 20Athick were cut using a microtome cryostat maintainedat - 15' C., picked up on coverslips at room tempera-ture, and allowed to thaw and dry at room tempera-ture. All sections were incubated within three hoursof being cut.The histochemical methods were those given by

Pearse (1960). The monotetrazolium-cobalt chelationmethod using 3(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTI) was employed to demon-strate nicotinamide-adenine dinucleotide and nico-tinamide-adenine dinucleotide phosphate diaphorases(NAD-D and NADP-D), succinic dehydrogenase(SDH), a-glycerophosphate dehydrogenase (a-GPDH)using menaphthone as an intermediate hydrogencarrier, lactate dehydrogenase (LDH), malate dehydro-genase (MDH), glucose-6-phosphate dehydrogenase(G6PDH), isocitrate dehydrogenase (ICDH), andP-hydroxybutyrate dehydrogenase (18-OHBDH). Cyto-chrome oxidase (CytO) was demonstrated usinga-naphthol and 4-amino-N,N-dimethylnaphthylamine(ADN). Monoamine oxidase (MAO) was localizedusing 2,2',5,5'-tetra-p-nitrophenyl-3,3'-(3,3'-dimethoxy,4,4'-biphenylene)-ditetrazolium chloride (Tetra-Nitro-BT). Ubiquinones (Ubq.) were identified by themethod of Tranzer and Pearse (1963).

Sections for diaphorases and dehydrogenases wereincubated 15 to 60 minutes at 370 C. Those for CytOand MAO were incubated 60 and 90 minutes. Chloro-form-extracted 2% methyl green was employed as anuclear counterstain for all the preparations exceptthose for CytO.The possible influence of the gelatin on the oxida-

tive enzymes was studied by comparing reactions ininjected and uninjected lungs. The lungs from two ofthe rats, one euthanized by coal gas and the otherby Nembutal, were not injected with gelatin. Smallblocks of the collapsed lung were quenched withliquid oxygen and cut using the microtome cryostat.Only fragmentary sections of the collapsed lung couldbe cut, but the sections obtained were incubated inthe same solutions and at the same time as gelatin-infiltrated lung sections.

Sections of rat kidney and liver were incubated inthe same solutions as the lung sections to determinewhether or not the histochemical procedures werereacting properly. Histochemical controls consistedof sections of lung and other tissues which were

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incubated in media lacking substrate, co-enzyme orboth.The rela,tive intensity of the overall reaction was

estimated on sections incubated for 60 minutes andinspected without magnification and at x25. Overallreactions were graded as strong, moderate or weak.The intensity of the reaction in the various cells wasestimaited at x400 to x 1000 and recorded as+ + + + for very strong to ± for very weakreactions.The identification anid nomenclature of the cells of

the interalveolar septum has been extensively reviewedby Bertalanffy (1964), and his nomenclature was usedin this study. Thus the cells were identified asalveolar cells, pulmonary epithelium or pulmonaryendoithelium. Free cells in the alveolar lumen wereconsidered to be pulmonary macrophages and werenot a primary object of this study as they are not partof the interalveolar septum.

RESULTS

The relative intensities of the reactions arepresented in the Table. Overall reactions ofcontrol tissues were generally stronger than thoseof lung. Strong overall reactions in lung wereobserved for NAD-D, NADP-D, and Ubq.Overall reactions in lung for MDH, LDH,a-GPDH and CytO were judged to be moderateand those for f3-OHBDH, ICDH, G6PDH, SDH,and MAO were estimated to be weak.

Typically the cells of the interalveolar septumhad weaker reactions than those observed in otherlung tissues, especially bronchial epithelium.Differences in the reaction of cells forming theblood-air pathway were observed only for CytO,and it was always difficult to differentiate thereactions of pulmonary epithelium from those ofpulmonary endothelium. Generally the reactionswere more intense in the alveolar cells than in thepulmonary epithelium or endothelium (Fig. 1). The

FIG. 1. NADP-D activity in rat interalveolar septa. Thereaction is much stronger in the alveolar cells than in thepulmonary epithelium orpulmonary endothelium. Incubated30 min. 8,u. x 370.

exceptions were the reactions for G6PDH, ICDH,and MAO, which appeared to have a more nearlyuniform intensity in all three of these cell types(Fig. 2). The final reaction product in alveolar

TABLEHISTOCHEMICAL REACTIONS OF INTERALVEOLAR SEPTUM OF RAT FOR OXIDATIVE ENZYMES

Enzyme Control Overall Alveolar Pulmonary PulmonaryTissues Lung Cell Epithelium Endothelium

Nicotinamide-adenine dinucleotide diaphorase (NAD-D) Strong Strong ++ + + ++ +-+Nicotinamide-adenine dinucleotide phosphate diaphorase(NADP-D) Strong Strong - ± + + 4-

Ubiquinones (Ubq.) ...Strong Strong + + + + 4Lactate dehydrogenase (LDH) Strong Moderate + + + +a-Glycerophosphate dehydrogenase (a-GPDH) Moderate Moderate + +Malate dehydrogenase (MDH) .Strong Moderate + + +± +Isocitrate dehydrogenase (ICDH).Moderate Weak + 4Succinic dehydrogenase (SDH) ..Moderate Weak 0 0 0Glucose-6-phosphate dehydrogenase (G6PDH). Moderate Weak + 4±f6-Hydroxybutyrate dehydrogenase (#-OHBDI-f) Moderate Weak 0 0 0Cytochrome oxidase (CytO) Strong Weak --+ + +--+ +--±-Monoamine oxidase (MAO) Strong Weak + + +

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Oxidative enzymes of the interalveolar septum of the rat

DISCUSSION

;.u.X B~

FIG. 2. ICDH activity in rat interalveolarreaction is nearly uniform in all cell type.reaction product appears in the form of coarin the alveolar cells than in the epithelial o0cells. Incubated 60 min. 20p. x 370.

cells was usually in the form of coarsethan that in the other cells. The rea/3-OHBDH and SDH were essentially nthe cells of the interalveolar septum, buin other pulmonary tissues were estimor + +. Free cells in the alveolar 1

reactions similar to those of the alveolaThe reactions in control tissues

expected on the basis of previous eSections incubated in media lacking suco-enzyme were negative.No evidence of inhibition or transl

enzymes due to the injection of ge]observed. Reactions in the frag:uninjected lungs had similar intensitiein injected lungs, but it was very <determine cell types in the collapsed,lungs. No differences due to the neuthanasia were detected.

These studies revealed that the alveolar cell hasa greater potential for metabolic activity than thepulmonary epithelium or the pulmonary endo-

Z thelium. The latter two cell types have nearlysimilar histochemical reactions. These observa-tions correlate well with electron microscopicstudies (Gillespie, Pangborn, Addison, and Tyler,1964) which revealed more numerous mito-chondria in the alveolar cells than in thepulmonary epithelium or endothelium. Thealveolar cell may require this greater metabolicpotential to produce the osmiophilic inclusionstypical of this cell. These osmiophilic inclusionsare thought by Klaus et al. (1962) to be the sourceof the pulmonary surfactant and our workreported here clearly supports their conclusions.The similarity between the pulmonary epitheliumand the endothelium is supported by the observedsimilarity of their cytoplasm in the electronmicroscopic studies of De Groodt, Lagasse, andSebruyns (1958).Although exact correlations between enzymes

demonstrated by histochemical and biochemicalmethods are not available for all the enzymesystems studied, speculation concerning thepossible functional significance of these observa-tions may contribute to a better understanding of

septa. The structure-function relationships in the lung.s. The final Strong reactions for NAD-D and NADP-D oftenrser granules accompany high activities of NAD and NADPr endothelial linked dehydrogenases. NADP linked dehydro-

genases are frequently associated with bio-synthetic systems. The strong reactions forNADP-D in alveolar cells are specially significant

or granules in view of the possible role of these cells in theLctions for production of pulmonary surfactant. The preciseegative for role of ubiquinones, or coenzymes Q, in theIt reactions electron transport system is not known, but Greeniated as + (1964) has suggested that they are important inLumen had the transfer of electrons across lipid layers fromr cells. his complex II (or complex I) to complex III inwere as mitochondria. CytO is the terminal oxidase of

experience. the cytochrome chain and transfers electrons toibstrate or molecular oxygen. The concentration of this

enzyme is directly correlated with oxygen require-ocation of ment. MAO functions in the destruction orlatin were detoxication of amines including adrenalin andments of serotonin.s to those Reactions for enzymes of the glycolytic scheme,lifficult to LDH and a-GPDH, were moderately strong inuninjected alveolar cells, indicating that this metabolic path-nethod of way is potentially functional in these cells. Though

the reaction for a-GPDH was very weak in

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pulmonary epithelium and endothelium, it isthought that the glycolytic scheme is functionalin these cells. The presence of G6PDH in all thecells of the interalveolar septa indicates that thepentose cycle is potentially functional. This cyclecould provide pentoses for nucleotide derivativesnecessary for phospholipid synthesis. The absenceof demonstrable ,B-OHBDH may indicate thatfatty acid metabolism is much less important tothe cells than the glycolytic scheme or the pentosecycle.The results for the three histochemically

demonstrable dehydrogenases of the tricarboxylicacid (TCA) cycle, MDH, ICDH, and SDH, werepuzzling, and these data are difficult to interpret.Our own observations agree only in part withthose of Sorokin et al. (1959), Sorokin (1961), andPliess and Suhr (1959). In all these studies SDHwas present and had the strongest reaction invascular musculature. The studies of Sorokinet al. (1959) and Sorokin (1961) concerned in vivoand in vitro development of lungs. They did notspecifically state the SDH activity in the cells ofthe interalveolar septum of their post-natal rats,but they did report a decrease in SDH activityof the stroma with increasing age. Their earlierstudies were conducted before the development ofMTT or Nitro-BT, and their later studiesemployed Nitro-BT at pH 7 6. Pliess and Suhr(1959) reported the presence of SDH in alveolarcells. However, they used very long (two to threehours) incubation times with a very easily reducedtetrazolium salt, Nitro-BT. The possibility offalse localizations under the condition of theseearlier studies has been discussed (Pearse, 1960)and must be considered. Using the MIT-cobaltchelation method we were able to demonstratevery weak reactions for SDH in the exchange areaof avian lungs (Tyler and Pearse, 1 964a), but foundno activity in the interalveolar septa of horselungs (Tyler and Pearse, 1964b). In our opinion,additional studies using other methods, probablythose of Lowry, Roberts, Wu, Hixon, andCrawford (1954), are required to determinewhether or not the TCA cycle is functional inthe cells of the interalveolar septa.The method used in this study to prepare cryo-

stat sections of lung appears to be very satis-factory. The gelatin did not appear to causeinhibition or translocation of enzymes. Theseobservations are in accord with those of Fishman,Ladue, and Borger (1961), who used gelatin instudies of free cell enzymorphology, and with ourprevious studies of the chicken lung (Tyler andPearse, 1964a). Histochemical methods employing

cryostat sections have many obvious applicationsto lungs from a variety of experimentalprocedures, and we anticipate that these methodswill be very useful in the evaluation of earlypathological changes in pulmonary tissues.The observation that the method of euthanasia

did not influence the results of histochemicalprocedures was not unexpected. However, it wasthought to be an important observation when oneof the agents entered directly through the tissuesunder study. Prolonged exposure to smallerquantities of either of the chemical agents mightproduce entirely different results.

SUMMARY

The distribution and relative intensity of severaloxidative enzymes of the cells of the interalveolarseptum have been investigated histochemically.Alveolar cells generally had stronger reactionsthan had the pulmonary epithelial cells or theendothelial cells. The reactions indicate that theglycolytic scheme and the pentose cycle aremore active pathways of carbohydrate metabolismthan is the tricarboxylic acid cycle. The histo-chemical observations were correlated withprevious electron microscopic studies. Thepossible significance of the histochemical reac-tions of the alveolar cell to the synthesis ofpulmonary surfactant is discussed.

The authors are grateful to Mr. J. D. Bancroft fortechnical assistance with many of the procedures. andto Mr. W. Brackenbury for the photomicrographs.

REFERENCES

Bertalanffy, F. D. (1964). Respiratory tissue: structure, histophysi-ology, cytodynamics. Part I. Review and basic cytomorphology,In Int. Rev. Cytol., 16, 233.

De Groodt, M., Lagasse, A., and Sebruyns, M. (1958). Fine structureof the alveolar wall of the lung. Nature (Lond.), 181, 1066.

Fishman, W. H., Ladue, K. T., and Borger, P. R. F. (i961). Enzymor-phology: A study of free cells. J. Histochem. Cytochem., 9, 424.

Gillespie, J., Pangborn, J., Addison, R., and Tyler, W. S. (1964).Ultrastructure of the pulmonary blood-air barrier of the horse.Anat. Rec., 148, 285.

Green, D. E. (1964). The mitochondrion. Scientific Amterica, 210,No. 1, p. 63.

Klaus, M. H., Reiss, 0. K., Tooley, W. H., Piel, C., and Clenients.J. A. (1962). Alveolar epithelial cell mitochondria as source ofthe surface-active lung lining. Science, 137, 750.

Lowry, 0. H., Roberts, N. R.,Wu, M. L., Hixon, W. S., and Crawford,E. J. (1954). The quantitative histochemistry of brain. I1. Enzymemeasurement. J. biol. Chem., 207, 19.

Pearse, A. G. E. (1960). Histochemistry, Theoretical and Applied,2nd ed. Churchill, London.

Pliess, G., and Suhr, A. (1959). Histochemische Befunde an derRattenlunge nach Cortisonmedikation. Beitr. Path. Anat., 121,406.

Sorokin, S. (1961). A study of development in organ cultures ofmammalian lungs. Develop. Biol., 3, 60.- Padykula, H. A., and Herman, E. (1959). Comparative

histochemical patterns in developing mammalian lungs. Ibid., 1,125.

Tranzer, J.-P., and Pearse, A. G. E. (1963). Cytochemical demonstra-tion of ubiquinones in animal tissues. Nature (Lond.), 199, 1063.

Tyler, W. S., and Pearse, A. G. E. (1964a). Functional and analyticalhistochemistry of the chicken lung lobule with marticular refer-ence to surfactant. Amer. J. Anat. In press.- (1964b). Unpublished observations.

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