static pressure-volume curve for th lunes g of the frog...

J. exp. BioL (1978), 76, 149-165 149With 10 figures

Printed in Great Britain

STATIC PRESSURE-VOLUME CURVES FOR THE LUNGOF THE FROG (RANA PIPIENS)

BY G. M. HUGHES AND G. A. VERGARA*

Research Unit for Comparative Animal Respiration,University of Bristol, Woodland Road, Bristol BS8 1UG

(Received 20 January 1978)

SUMMARY

1. Static pressure/volume curves have been determined for isolatedfrog lungs inflated with either air or saline. In both cases a hysteresis waspresent: the pressure required to produce unit change of volume beinggreater during inflation than deflation.

2. The pressure necessary for a given volume change was less for thesaline-filled than the air-filled lungs. The difference between these curvesis due to the surface tension at the air/lung interface.

3. Pressure/volume curves for air-filled lungs in situ were similar to curvesfor isolated lungs. However, a greater pressure was required for the samevolume change during both inflation and deflation.

4. Compliance was calculated from different parts of air pressure/volumecurves and gave values greater than those obtained using similar calculationsfor higher vertebrates.

5. These observations support other evidence for the presence of a sur-factant in the lung lining of frogs in spite of the relatively large diameterof their 'alveoli.' The precise role of such a lining is uncertain and it isconcluded that similar forces may be involved during the inflation and de-flation of lungs of frogs and higher vertebrates in spite of differences ingross morphology.

INTRODUCTION

During the past 30 years evidence has accumulated supporting the view that thelungs of mammals are lined with a layer of lipids and proteins, probably as lipo-protein, whose main function is to reduce the surface tension of the curved alveolarsurfaces. The first indication of such a lining was probably that obtained by vonNeergaard in 1929 when he demonstrated that the pressure necessary to enlarge thelungs to a given volume was less when they were filled with liquid than when theywere inflated with air. Replacement of the air-alveolar interface by a liquid-alveolarinterface evidently produced a significant reduction in the retractive forces operatingon the lung, von Neergaard concluded that surface tension forces were importantin the lungs. From pressure/volume experiments approximate calculations weremade which suggested that a considerable surface tension (about 40-50 dyn/cmf) is

• Present address: Department of Pharmacology, The Medical School, University of Bristol,Bristol BS8 iTD.

t 1 dyn/cm = 1 mN/m.

G. M. HUGHES AND G. A. VERGARA

present at such air-alveolar interfaces (Radford, 1954; Brown, 1957). Brown (1957)who knew about the lining film, calculated a much lower surface tension in deflatedlungs. Since these initial experiments this hypothesis has been supported by severalother lines of evidence including electron-microscopic studies of the lung lininglayers, chemical analyses of lung tissue and of washings from the lungs and also fromdirect measurements of the surface tension using a Wilhelmy balance (Clements,1957), or a bubble technique (Pattle, 1958). Corresponding studies with the lungsof lower vertebrates are as yet fragmentary, but in general support the view thatsimilar lining layers are present in lungfish, amphibians, reptiles and birds (Brooks,1970; Clements, 1962; Clements, Nellengoben & Trahan, 1970; Hallman & Gluck,1976; Hughes, 1967, 1970, 1973; Pattle, Schock, Creasey & Hughes 1977). However,there have been few studies (Cragg, 1975) of pressure-volume relationships corre-sponding to the initial work of von Neergaard. The purpose of the present investigationswas to determine these relationships for lungs filled with air and saline and as a resultto make estimates of the surface tension of the lung lining. A preliminary report of thiswork has been given (Vergara & Hughes, 1977).

MATERIALS AND METHODS

Experiments were carried out on frogs (Rana pipiens) of 15-35 g body weight.The animals were maintained in the laboratory at 13-14 °C and before cannulationof the lung they were anaesthetized by an intra-peritoneal injection of a o*i mlsaline solution containing 10% MS 222. A cannula (PP120) was flared at one endand inserted into the glottis where it was tied firmly. In some experiments inflationand deflation of the lung was carried out using the intact frog. In most cases, however,the frog was dissected carefully from the ventral side and one of the lungs isolatedtogether with its connexion to the cannula. With most preparations, it was onlypossible effectively to cannulate a single lung in any given specimen. The isolatedlung was connected into the apparatus shown in Figs. 1 and 2 and usually was usedfor air inflation and deflation experiments before being filled with saline. The appara-tus and procedures were similar to those used by Young, Tierney & Clements (1970)and by Cragg (1975). Results obtained in the last of the air P/V curves were com-pared with those obtained for the second of the saline curves in order to calculatesurface tension. Many lungs contained parasites and in no case were such lungs usedfor the experiments.

In the air experiments the cannulated lung was at its normal volume when con-nected to the apparatus (Fig. 1) and all taps were open to atmospheric pressure.When the taps were closed, air was injected via a syringe (I/D). Then the pressurebetween the two water manometers was adjusted (syringe P) so that there was nodifference in the water levels of manometer II. The pressure necessary to maintainconstant volume (PTU) of the tubes connecting manometer II to the lung was read onmanometer I. Volumes of o*i or 0-2 ml were used for each step of a given inflation/deflation cycle and the pressure reading was made 15 s after the injection or with-drawal of air. A correction was made for the compression or rarefaction of the airby means of the following equation:

Frog lung pressure-volume curves

M II

Fig. 1. Diagram of apparatus used for the inflation and deflation experiments using air. SPis the syringe used for adjusting the levels of M II; S I /D is used to change the volume ofthe frog lung. Pressure is read on M I.

M I

Fig. 2. Diagram of apparatus used for inflation and deflation of a frog lung with saline.Changes in volume during inflation and deflation are measured in S I / D ; SP is syringefor adjusting pressures.

where VLi = lung volume of any step, PB = barometric pressure, PH>O = watervapour pressure, Px = pressure for the minimum lung volume, Ps = pressure forany volume step, VL1 = minimum lung volume, P i = volume of syringe I/D forthe minimum lung volume, f£2 = volume of syringe I/D for any volume step,Vra = volume of tubing between lung and manometer II.

Inflation with saline (0-9 % NaCI) was carried out using the apparatus in Fig. 2.First of all the pressure in the isolated lung was reduced to — 20 cm H,0 using theair apparatus (Fig. 1). It was then attached to the saline apparatus at this reduced

152 G. M. HUGHES AND G. A. VERGARA

I

10 -

0-5 -

10

Pressure (cm H2O)

Fig. 3. Plots of pressures in a balloon when inflated and then deflatedto different volumes with air (O) or saline (A).

pressure with the lung at a minimal volume. After adjusting (syringe P) the pressurewithin the saline apparatus to the same level (— 20 cm H2O) connexion was made tothe lung. Inflation of the lung with saline (syringe I/D) was once more carried out atsteps of o-i-o-2 ml, 3 min being taken for each step. As saline was injected into thelung the pressure was read off on MI, as it approached atmospheric pressure andthe condition indicated in Fig. 2. Inflation above atmospheric pressure was continuedand followed by deflation. During the saline inflation/deflation experiments the lungremained submerged in saline.

Before the apparatus was used for experiments with frog lungs some control studieswere made to determine the pressure/volume curves of rubber balloons using airand saline (Fig. 3). The results indicated that there was no hysteresis in the pressure/volume relationship because during both inflation and deflation with either air orsaline, the sigmoid-shaped curves were perfectly matched. Similar curves could beobtained provided the balloons were used on no more than three or four occasions.

Lungs were fixed at different inflation volumes by immersion in a 2-5 % glutaralde-hyde solution in a cacodylate buffer (pH 7-2) before embedding in an Epon-Araldite

Frog lung pressure-volume curves 153

1 2 3 4 5 6 7 8 9Pressure (cm H2O)

Fig. 4. Typical pressure/volume curves during inflation and deflation of a lung isolated froma frog of 27 g. Separate curves are plotted during inflation/deflation with air and saline.

mixture. Large sections, covering the whole cross-section, were cut with a LKBultratome and stained with toludine blue. These sections were analysed morpho-metrically (Hughes & Weibel, 1976) by means of intersection and point countingusing a projection microscope.

RESULTS

1. Air pressure /volume relationships

During inflation of the lung from atmospheric pressure, the increment in pressureper unit volume change decreased rapidly until there was very little change in pressurealthough lung volume increased very significantly. Above this level the curve showedits sigmoid nature as the pressure rose more steeply during the final 1 ml of inflation.The deflation curve was similar in general shape but was shifted to the left relativeto the inflation curve (Fig. 4). Consequently the pressure at any given volume is lessduring deflation than during inflation. This marked hysteresis was found in all pre-parations. Differences in the precise nature of the curves were observed when re-peated inflation and deflation cycles were carried out.


Pressure (cm H2O)

Fig. 5. Curve showing the relationship between pressure and volume during inflation of afrog lung with air and saline. The dashed Line shows the pressure (PJ used for calculating theconstant K and the surface tension. Weight of frog, 27 g.

2. Pressure/volume relationships of saline-filled lungs

The general form of the curves is similar to that obtained with air but during bothinflation and deflation the pressure changes were very much less for any given volumechange. The sigmoid shape of these curves was less pronounced with saline-filledlungs as also was the hysteresis (Fig. 4). Although variations were found in the precisenature of these curves according to the size of the frog, the same qualitative differenceswere always observed. There was no significant change in the shape or the hysteresisof the P/V curve when the rate of change of volume was altered. In Fig. 5 the in-flation of the curves are plotted and a line drawn to indicate the difference in pressureat one particular volume.

3. Pressure/volume curves from, lungs in situ

Pressure/volume curves showing hysteresis for lungs in the whole animal areshown in Fig. 6 for comparison with those obtained for isolated preparations. Thecurves obtained from the intact animal (Fig. 6 a) did not change very markedly whenthe frog was opened up and the air pressure/volume relationship once more deter-mined (Fig. 6 b). But when the heart, pericardium and liver were removed the resting


50

4 0

30

2 0

10

-

-

-

1 |

/

itff1

• Intact animal± In situ, intact• In situ, without

liver and heart

i i i i i

- 3 - 2 - I 0 1 2 3 4

Pressure (cm H2O)

Fig. 6. Plots showing P/V loops for a frog during progressive isolationof the lung. For explanation see text.

volume of the lung at atmospheric pressure was reduced (Fig. 6 c). Thus in the wholeanimal a greater pressure was required for the same volume change - presumablybecause of the limitations imposed by the viscera and body wall.

4. Lung compliance

From the pressure/volume curve described above it is possible to obtain values forlung compliance. It is evident that the compliance during saline filling is greaterthan during filling with air, the pressure changes for a given volume of saline injectionbeing much less than those following a comparable volume change produced by airinjection. The compliance and area of the hysteresis loop during air filling were almostidentical both before and after the lung had been inflated with saline.

Estimates of compliance usually indicate the slope of some part of the pressure/volume curve. In the frog little data is available concerning the range of volumechanges during normal ventilation. Calculations were therefore made on all partsof the pressure/volume curves for the isolated and in situ lungs. Fig. 7 shows plotsof the compliance in relation to the relative volume; each point represents the com-pliance at intervals of 1 cm H2O pressure change. The maximum compliance wasfound when the volume of the lung was about half its maximum inflation, i.e. 50%

156

200

100


-Inflation ^ — h 6 Deflation-

100 100

Humana•a Human

• Cat "

Rabbit1

Frog

Lizard

B Mouse

200

100

10

01

0 10 20 30 40 50 60 70 80 90 100 100 90 80 70 60 50 40 30 20 10 0Relative volume (%)

Fig. 7. Compliance at different volumes (% maximum inflation) obtained from P/V curvesof isolated lungs from a variety of vertebrates (references 2,3, 5, 7, 8, 11 in Table 1). In orderto clarify the change in compliance which occurs between the end of inflation and the begin-ning of deflation, the region of maximum volume (i.e. 100% relative volume) ha» beenexpanded in this figure.

of the volume at which an increase in pressure gave scarcely any increase in volume.The minimum values occurred at the end of inflation and the beginning of deflation(i.e. 100% relative volume). During deflation the value rose to approximately thesame maximum as during inflation. An approximately tenfold range of compliancewas found during inflation for any frog lung. Analysis of the pressure/volume curvesfrom intact preparations (Fig. 8) gave essentially the same results, but the whole rangeof values was slightly lower. It is evident from the plots given in Fig. 7 that valuesfor the frog are about ten times greater than corresponding values for the mouse orlizard of approximately the same body mass.

DISCUSSION(a) Lung compliance

As a result of the investigations on the frog lung an interest developed in the rangeof compliance values obtained in different parts of the pressure/volume curve, and acomparison with results obtained for mammals. Values normally given for themammal are in relation to the functional residual capacity (shown as the first pointplotted in Fig. 7), but for comparative purposes a number of published pressure/volume curves have been analysed. Maximum and minimum values for compliancederived from such curves are summarized in Table 1. In making comparative studiesscaling factors must always be taken into account and for mammals (Stahl, 1967)the relationship has been shown to be:

lung compliance (at F.R.C.) = 0-0021 W1'08 (W in g).

Frog lung pressure-volume curves :57

200 - - 200

1 0 0 : . . - . "•<. . • • • ' " ' • • • • . . = 1 0 0

s iob <~atir \ - . .-• / dio

e

: Human • . " • '

Protopterus

. Cat*--^\ ' " •

Frog Lizard r -^^^

~ Frog*^^

. . . . I

sx . • ' • ' • € Human \

^ Cat

/ Protopterus ~

^ ^ y \ S _ / Frog _ I

^ / ^ !

I I I I I I I 1 1 I 1 I I I

r 1= . . ^ ^ — - ^ \f ^ 'Lizard d l2 Froi

.2 r ^ •*•"Z.o

01b A \ \ / / dOl

0 10 20 30 40 50 60 70 80 90 100 100 90 80 70 60 50 40 30 20 10 0

Relative volume (%)

Fig. 8. Compliance at different volumes (% maximum inflation) obtained from P/V curves ofin situ lungs of a variety of vertebrates (references a, 4, 8, 9, 10 and 11 in Table 1).

During the present survey a similar relationship has been shown for maximumcompliance (= 0-00244 W10*9) determined from the P/V curves of mammals anda relationship having a similar slope (= 0*036 W0*9*) was obtained for lower verte-brates (Fig. 9). The tenfold difference in the intercept values for these regressionlines indicates that at almost all body sizes a lower vertebrate lung would be expectedto have a compliance that is approximately ten times that for a mammal of comparablebody size. Thus the deduction based upon the relationships between frog and mouselungs (Fig. 7) is given further support.

It is also possible to compare the result obtained from these static pressure/volumerelationships with the P/V curves obtained by West & Jones (1975) for the dynamicrelationships recorded during pulmonary ventilation. Calculations of compliance fordifferent parts of their curves suggest a similar range of values (Fig. 8). Thus it canbe concluded that the values of compliance obtained in this study give a good indica-tion of the type of compliance relationship which operates in the normal pulmonaryventilation of Rana pipiens. Presumably the greater compliance of the frog lung re-duces the work of breathing.

(b) Surface tension

The general form of the pressure/volume curves obtained using both air and salinewas very similar and showed a hysteresis in all cases. The pressure required forinflation to a given volume using saline was always less than with air. These obser-vations support the view that the lung lining contains a substance which reducessurface tension forces within the lung. The finding that no such differences in

6 EXB 76


Table 2. Summary of measurements made on the surface tensionof material obtained from vertebrate lungs

Species

Lamb

Dog

Dog

DogCatRat

Cat

RatCatDog

Rat

Guinea pig

Pigeon

Chicken

Turtle

Frog

Frog (R. pipieni)

Clawed toad(Xenopus laevit)

Chicken (Gailus)

Mammal

Toad

Material

Lung extract

Lung washings

Lung extract

'Lung'

'Lung'

Lung extract

Compressionfresh lung

Lung extractand washings

Lung extract




'Lung'

Squeezedfresh lung

Squeezedfresh lung

Squeezedfresh lung

Lung extract

Method

W.B.*

W.B.

W J .

P/V curves

P/V curves

W.B.

W.B.

W.B.

W.B.

W.B.

W J .

W.B.

P/V curves

Bubble

Bubble

Bubble

W.B.

Surfacetension

(mN/m)

Max: 36Min: 6(Area iS%)

Max: 30Min: 0-3(Areai7%)

Max: 35Min: 13(Area 20%)

Max: 50Min: 5

Reference

deLemos et al. (1969)

Finley et al. (1968)

Modes & Vergara (1973)

Brown, Johnson & Clements (1959)

(Area 10-20%)

Max: 50Min: o-a(Area ao %)

Max: 40-30Min: 10-15(Area 30%)



Min: 18(Area 20%)Max: 52Min: 29(Area 20%)Max: 52Min: 28(Area 20%)

Max: 55Min: 30(Areaao%)

Max: 50Min: 0-3(Area 25 %)

Stabilityratio: o-8i

Stabilityratio: 0-76

Stability

Fisher, Wilson & Weber (1970)

Brown et al. (1959)

Miller & Bondurant (1961)

Motles et al. (1971)

Klaus et al. (1962)




This study

Pattle & Hopkinson (1063)

Pattle & Hopkinson (1963)

Pattle & Hopkinson (1963)ratio: o-6-o-87

Min: 18(Area 20%)

• Wilhelmy Balance.

Klaus et al. (1962)


lOOOp

100oX

u

8 10

D.

Ou

i 1000

Human

K=0036WOW4

r=0-957

10

K=10ml\ F r o g .

Fr°g"/V=10mlLizard• X

i (t Mouse i

= 00024 W104»

r=0-969

i i i 11 in i i i i 11n

103

Body weight (g)10* 103

Fig. 9. Log-log plot of maximum compliance of the lungs of vertebrates against body weight.Regression line* are shown for mammals and lower vertebrates. A dashed line shows therelationship obtained by Stahl (1967) for mammalian lung compliance at resting volume(i.e. F.R-C).

pressure/volume curves were observed in balloons inflated with air or saline is alsoin agreement with this interpretation.

Assuming that the forces due to tissue elasticity remain the same when the lungis filled with air or saline and that there are no changes in the basic geometry of thealveoli and other air spaces, then it is probable that the only mechanisms responsiblefor the difference in pressure/volume relationships are due to the surface forces atthe air-liquid interface.

From each pair of air and saline pressure/volume curves calculations were made ofthe surface tension using a method based upon that of Fisher, Wilson & Weber(1970) and Bachofen, Hildebrandt & Bachofen (1970). In this method it is necessaryto assume a maximum value for the surface tension, and for this purpose thevalue used (50 mN/m) was based upon measurements of the surface tension ofwashings from mammalian lung using a Wilhelmy surface tension balance (Table 2).It has often been assumed that the internal surface area of a vertebrate lung (mammal)is proportional to the two-thirds power of the lung volume. In a lung containinglarge numbers of alveoli approximately spherical in shape, such an assumption isreasonable, but for a lung so different in basic organization as that of the frog it wasdecided to investigate this relationship in more detail. Morphometric estimates of theair volume were obtained from point counts of sectioned material and an estimate ofthe internal surface area was obtained from intersection counting. Results obtained

6-2

i6o G. M. HUGHES AND G. A. VERGARA

100-

0 10 20 30 40 50

Surface tension (dyn/cm)

Fig. io. Plot of surface tension against relative surface area for a frog lung.For detailed explanation see text

for lungs of different inflation volumes showed that the surface area is more closelyrelated to the one-half power of the volume (SA oc V°~a).

(c) Calculation

PaU and Pganne are the pressures of the air and saline-filled lungs; Pair/water *8 thepressure due to surface forces = PB (Fig. 5), i.e. pressure differences between airand saline curves at any volume.

The work done on the surface lining can be expressed as

W=Ps.dV or W=y.dA,

where dV = increment in volume; dA = increment in area, and y = surface tension.From morphometric analysis:

A = K.Vi, (1)which on differentiating:

. . K.dVdA = -

Frog king pressure^volume curves 161

tiTable i . Maximum and minimum values for lung compliance from pressure/volume

curves of vertebrates: volumes and pressures at maximum inflation are also given

Species

HumanTot. reap. syst.

HumanLungs aloneTot. resp. syst.

DogBoth lungs

Monkey*Dog*Cat

Tot. resp. syst.Cat*Rabbit*Rat*Mouse*Lizard

Both lungs(Lacerta ticula)

Tot. pulm. syst.{Lacerta viridit)

Frog (R. pipiens)Tot. pulm. syst.

Frog (JR. pipiens)Both lungsTot. pulm. syBt

Fish (Protopterw)Tot. pulm. syst.

Maximumcompliance

(ml/cm H,O)*

Inn.

250

170

" 3

2013331

193aa

6-5o-35O'll

0-21

O-75

Deft.

3 1 0

180142

2034435

48940980-47O-IO

0-38

i-oo

Minimumcompliance

(ml/cm H,O)

Inn.

40

n o58

4«12

7

5743-3O-I3OO2

o-io

O-O5

Den.

1 0

80

4a

1621

i-71I-I

o-ioo-oi

o-oa

o-oa

Maximuminflation

Volume Pressure(ml) (cm H,O)

5300

27003000

2000400390

33o300

757-31-9

0 5 a

7 1

25

136136

24317-5as-a

34-0ai-53119-83O-O

2 5

a3S

Bodyweightf

(g)

(76400)

(44650)(44650)

(30460)(6670)(6570)

(5S6s)(5090)(1375)

28528

5

3 9

Ref.

1

3a

1

33

43567

8

8

x-78

a-651-37

33-8

140

300

240

0-16 0-06 a-8 60

028038

0-050-03

5-556

5-884

50-0 9-0

80

2636

500

11

11

• Probably both lungs.t Weights in parentheses have been estimated from lung volumes given by original authors, using

the relationship: total lung capacity = 53-5 W1"0* (Stahl, 1067).t References: 1, Mead, Whittenberger & Radford (1957); 2, Butler (1957); 3, Bachofen et al. (1970);

4, Radford (1964); 5, Lempert& Macklem (1971); 6, Weiss & Jumia(i97i); 7, Morstatter et al. (1976);8, Cragg (1975); 9, West & Jones (197s); 10, G. M. Hughes (unpublished); n , this study.

Then

2.

Hence

and

r = K '

KP

(3)

To calculate K, the maximum pressure difference (P,,m»x) between the air and

saline inflation curves was taken together with the volume at which this occurred

(Fig. 5). The mean value of K for five preparations was 261; S.E. ± 18. The corre-

sponding value for cat lung is 1225; S.E. ± 100 (Fisher et al. 1970).


It is evident from the calculations summarized above that insertion of values feuK and appropriate volumes and Ps in equation (3) gives the surface tension at differerlvolumes. Some of the values obtained are given in Table 2, together with the resultsof surface tension measurements on a variety of tetrapod lungs using differentmethods.

Changes in surface tension associated with changing surface area plotted out asa change in relative area, calculated on the basis of the J-power relationship to lungvolume are given in Fig. 10. This plot shows a hysteresis loop which encloses asmaller area than that obtained for a mammalian lung (Fisher et al. 1970).

When interpreting the curves relating surface tension to surface area for both thefrog and mammalian lungs it is apparent that the fall in surface tension is very rapidfollowing a decrease of only 15-20 % in surface area. This contrasts with the resultsobtained with the surface-tension balance, using washings or extracts of mammalianlungs, where a much greater reduction in area, by about 40-50 % of its original area,is necessary to obtain a comparable reduction in surface tension, i.e. to about 5-10 mN/m.When the lung volume, and consequently surface area, is reduced to about 50%of the total lung capacity, the surface tension falls to about 5 % of its maximum value.At small volumes, however, the differences in pressure between the air and salinedeflation curves approach zero and consequently the surface tension must alsoapproach zero. During re-expansion of the lung the surface tension rises once morebut at any given surface area is always less during deflation than during inflation.Miller & Bondurant (1961) studied the surface tension/area loop of extracts of froglung with a Wilhelmy balance. They found a minimum value of 30 dyn/cm whenthe surface was reduced to 20 % of its original area. The area of the hysteresis loopwas half that found for rat lungs under comparable conditions. No mention wasmade of the temperature at which these experiments were carried out. This para-meter has a very important effect on surface activity and has been shown to beespecially relevant in relation to the properties of frog lung surfactant (Pattle et al.1977). The experiments described in the present paper were carried out at 13 °C.However, it is difficult to compare satisfactorily the loops obtained with a surfacebalance by Miller and Bondurant and those described here using P/V curves, asmany authors have found a discrepancy between the results of the two methods formammalian lungs.

One advantage of the present study is that it is based on a morphometric deter-mination of the relationship between surface area and volume of the lung without anyassumption concerned with the shape of the basic units.

In conclusion it may be stated that evidence for the existence of a surfactant liningin the frog lung has now been increased by the results of these pressure/volumestudies. Added to the known evidence derived from electron microscopy, and measure-ments on bubbles squeezed from frog lungs, it seems safe to conclude that such alining is present. The possible function of such a lining is not very clear at first sight.The function usually applicable to the mammalian lung does not seem ideal becauseof the much larger dimensions of the alveolar units. Nevertheless, it is very probablethat the presence of a surface tension reducing film would play an important role inthe early stage of inflation of these lungs, especially in small frogs, for they are almostcompletely emptied during expiration.


It was noticeable during the inflation experiments that the lung tended to fill firstIn its more anterior regions, i.e. those regions closest to the glottis, and perhapsduring such a stage of the ventilatory cycle the lining film would be important.

The range of the pressure and volume changes employed in the present study areclearly in excess of those of the normal ventilatory cycle. However, it is well knownthat the lungs of frogs are inflated to volumes which greatly exceed the normal dimen-sions at full inspiration in certain stages of the life-cycle, e.g. vocalization duringthe breeding season. The lungs are also inflated supramaximally to serve a buoyancyfunction. Thus it can be concluded that the total range of pressure/volume relation-ships shown here approximates to that which normally occurs in such structures.

From comparison of washings obtained from frog lungs with washings frommammalian lungs (Hughes & Vergara, 1978), it appears that the lining layers aremore similar in their composition than was at first supposed and this becomes espe-cially apparent when the concentrations of the different phospholipid componentsare compared in relation to the area of their internal surfaces. Thus, in spite of thedifferences in alveolar dimensions, these similarities suggest that perhaps the lininglayers may perform similar functions. Consequently the supposed role of such layersin reducing the surface tension forces may have been over-emphasized. An 'anti-glue' function of a surfactant lining is clearly a possibility, as has been suggested forthe mammalian lung (Sanderson et al. 1976). In the case of the mammalian lung,some morphometric and physiological investigations (Weibel et al. 1973; Hills, 1971)have suggested that many alveoli collapse at different stages during the ventilatorycycle and the alveolar linings slide over one another as they open and close. Similargeometric modifications might also take place in lungs of the frog type (Hughes,1978); in fact material fixed at different levels of inflation has confirmed this possi-bility. Until further evidence is forthcoming such changes in overall lung morphologymust be speculative, but at least such an hypothesis has the advantage of enablingus to understand similarities between lungs which may have wide differences in theirsurface properties because of their dimensions alone.

We wish to thank the Medical Research Council for providing a research grantwhich made it possible to carry out this work.

We are grateful to Professor Robert Bils for skilfully cutting the large sections offrog lungs.

REFERENCES

BACHOFEN, H., HLLDEBHANDT, J. & BACHOFKN, M. (1970). Pressure-volume curves of air- and liquid-filled excised lungs - surface tension in situ. J. appl. Phytiol. 39, 422-431.

BIENKOWSXI, R. & SKOLNICK, M. (1974). On the calculation of surface tension in lungs from pressure -volume data. J. Coll. Int. Sci. 48, 350—351.

BROOKS, R. E. (1970). Lung alveolar cell cytosomes: a consideration of their significance. Z. Zelffortch.mikroth. Anat. 106, 484-497.

BROWN, E. S. (1957). Lung area from surface tension effects. Proc. Soc. exp. Biol. Med. 95, 168-170.BROWN, E. S., JOHNSON, R. P. & CLEMENTS, J. A. (1959). Pulmonary surface tension. J. appl. Pkytiot.

14, 717-730.BUTLBR, J. (1957). The adaptation of the relaxed lungs and chest wall to changes in volume. C/tn. Set.

16, 4*1-433-CLEMENTS, J. A. (1957). Surface tension of lung extracts. Proc. Soc. exp. Biol. Med. 95, 170-172.


CLEMENTS, J. A. (196a). Surface tension in the lungs. Sci. Am. 307, 120-130.CLEMENTS, J. A., NELLENBOOHN, J. & TRAHAN, H. J. (1970). Pulmonary surfactant and evolution of thd

lungs. Science, N. Y. 169, 603-604.CRAGC, P. A. (1975). Respiration and body weight in the reptilian genus. Lacerta: a physiological, ana-

tomical and morphometric study. Ph.D. thesis, Bristol University.DELKMOS, R., WOLFSDORF, J., NACHMAN, R., BLOCK, A. J., LBIBY, G., WILKINDSON, H. A., ALLEN, T.,

HALLER, J. A., MORGAN, W. & AVBRY, M. E. (1969). Lung injury from oxygen in lambs; the role ofartificial ventilation. Anathaiology 30 (6), 609-618.

FINLEY, T. N., PRATT, S. A., LADMAN, A. J., BREWER, L. & MCKAY, M. B. (1968). Morphological andlipid analysis of the alveolar lining material in dog lung. J, Lipid Res. 9, 357-365.

FISHER, M. J., WILSON, M. F. & WEBER, K. C. (1970). Determination of alveolar surface area and tensionfrom in situ pressure-volume data. Resp. Phytiol. 10, 159-171.

HALLMAN, M. & GLUCK, L. (1976). Phosphatidylglycerol in lung surfactant. III. Possible modifier ofsurfactant function. J. Lip. Res. 17, 357-263.

HILLS, B. A. (1971). Geometric irreversibility and compliance hysteresis in the lung. Resp. Physiol. 13,50-61.

HUGHES, G. M. (1967). Evolution between air and water. In Development of the Lung (ed. A. V. S. deReuck and R. Porter), pp. 64-80. Ciba Fndn Symp. London: Churchill.

HUGHES, G. M. (1970). Ultrastructure of the air-breathing organs of some lower vertebrates, yth Congr.int. Microsc. ilectronique, Grenoble 3, 599.

HUGHES, G. M. (1973). Ultrastructure of the lung of Neoceratodus and Lepidosiren in relation to the lungof other vertebrates. Folia Morph. Praha 31, 155-161.

HUGHES, G. M. (1978). A morphological and ultrastructural comparison of some vertebrate lungs. FoliaMorph. Praha (in the Press).

HUGHES, G. M. & VERGARA, G. A. (1978). Phospholipids in lung surfactant of Rana pipiens.J. Physiol.VIS, 48-49P.

HUGHES, G. M. &WEIBEL, E. R. (1976). Morphometry of fish lungs. In Respiration of Amphibious Verte-brates (ed. G. M. Hughes), pp. 213-232. London, New York: Academic Press.

KLAUS, M., REISS, O. K., TOOLBY, M. H., PIBL, C. & CLEMENTS, J. A. (1962). Alveolar epithelial cellmitochondria as source of the surface-active lung lining. Science, N. Y. 137, 750-751.

LEMPERT, J. & MACKLEM, P. T. (1971). Effect of temperature on rabbit lung surfactant and pressure-volume hysteresis. J. appl. Physiol. 31, 380-385.

MEAD, J., WHTTTENBERGER, J. L. & RADFORD, E. P. JR (1957). Surface tension as a factor in pulmonaryvolume-pressure hysteresis. J. appl. Pkysiol. 10, 191-196.

MILLER, D. A. & BONDURANT, S. (1961). Surface characteristics of vertebrate lung extract*. J. appl.Physiol. 16 (6), 1075-1077.

M0R8TATTER, C. E., GLASER, R. M., JURRUS, E. R. & WEISS, H. S. (1976). Compliance and stability ofexcised mouse lungs. Comp. Biochem. Physiol. 54 A, 197-202.

MOTLES, E. & VEROARA, G. (1973). Influencia de la acidosis metabolica y respiratoria agudas sobre elsistema surfactante pulmonar del perro. Rev. mid. Chile 101, 3-11.

MOTLES, E., VEROARA, G., OYARZUN, M., PUIG, F. & VAINROJ, M. (1971). Efecto de la acetazolamidasobre el sistema surfactante del pulmon de cobayo. Arch. Biol. med. exp., Chile 8, 19-35.

PATTLB, R. E. (1958). Properties, function and origin of the alveolar lining layer. Proc. R. Soc. Land. B148, 217-240.

PATTLB, R. E. (1969). The development of the foetal lung. In Foetal Autonomy (ed. G. E. W. Wolsten-holme and M. O'Connor), pp. 132-146. Ciba Fndn. Symp. London: Churchill.

PATTLB, R. E. (1976). The lung surfactant in the evolutionary tree. In Respiration of Amphibious Verte-brates (ed. G. M. Hughes), pp. 233-155. London, New York: Academic Press.

PATTLB, R. E. & HOPKINSON, D. A. W. (1963). Lung lining in bird, reptile and amphibian. Nature, Lend.300, 894.

PATTLB, R. E., SCHOCK, C , CREASEY, J. M. & HUGHES, G. M. (1977). Surpellic films, lung surfactant andtheir cellular origin in newt, caecilian and frog.J. Zoo!., Lond. 183, 135-136.

RADFORD, E. P. JR (1954). Method for estimating respiratory surface area of mammalian lungs from theirphysical characteristics. Proc. Soc. exp. Biol. Med. 87, 58-61.

RADFORD, E. P. JR (1964). Static mechanical properties of mammalian lungs. In Handbook of Physiology:Respiration, Sect. 3, vol. 1, pp. 429-450. Washington, D.C., Am. Physiol. Soc

SANDERSON, R. J., PAUL, C. W., VATTER, A. E. & FILLEY, G. F. (1976). Morphological and physicalbasis for lung surfactant action. Rap. Physiol. 37, 379-392.

STAHL, W. R. (1967). Scaling of respiratory variables in mammals. J. appl. Physiol. 33, 453-460.VERGARA, G. A. & HUGHES, G. M. (1977). Pressure volume curves and a surfactant system in frog lungs.

Proc. 13th Int. Cong. Pkysiol. Sci., Paris, p. 785.VON NEEROAARD, K. (1929). Neue Auffassungen ttber einen Grundbegriff der atemmechanik: Die

Retraktionskraft der Lunge, abhfingig von der Oberflachenspannung in den Alveolen. Z. Gesamte


exp. Med. 66, 373-394. Translated in Pulmonary and Respiratory Phytiology (1976), part 1 (ed.Julius H. Comroe Jr), pp. 214-234. New York: Dowden, Hutchinson and Ross.

WEIBEL, E. R., UNTEBSEE, P., GIL, J. & ZULAUF, M. (1973). Morphometric estimation of pulmonarydiffusion capacity. VI. Effect of varying positive pressure inflation of air spaces. Rap. Phytiol. 18,285-308.

WEISS, H. S. & JURRUS, E. (1971). Starvation on compliance and surfactant of the rat lung. Rap. Phytiol.ra, 123-129.

WEST, N. H. & JONES, D. R. (1975). Breathing movements in the frog Rana pipiens. II. The power out-put and efficiency of breathing. Can.J. Zool. 53, 345-353.

YOUNG, S. L., TIERNEY, D. F. & CLEMENTS, J. A. (1970). Mechanism of compliance change in excisedrat lungs at low transpulmonary pressure. J. appl. Phytiol. 29, 780-785.

static pressure-volume curve for th lunes g of the frog...

Documents