J. Exp. Bol. (968), 49, 453-468 Wth 3 text-fgures Prnted n Great Brtan RESPIRATION IN THE AFRICAN LUNGFISH PROTOPTERUS AETHIOPICUS II. CONTROL OF BREATHING* BY KJELL JOHANSENf AND CLAUDE LENFANT Departments of Zoology, Medcne and Physology, Unversty of Washngton and Insttute of Respratory Physology, Frland Sanatorum, Seattle, Washngton, U.S.A. (Receved 6 March 968) INTRODUCTION The process of respratory gas exchange and gas transport n vertebrates shows adaptve adjustments correlated wth aquatc and aeral modes of breathng. Such adjustments are explct structurally n the desgn of the gas-exchangng organs and functonally n a number of features such as respratory propertes of blood and ventlaton characterstcs (Lenfant & Johansen, 966; Lenfant, Johansen & Grgg, 966; Steen & Kruysse, 964; Rahn, 966; Hughes, 966; Robn & Murdaugh, 966). Relatvely lttle work has appeared on the phylogenetc development of respratory control mechansms (Hughes & Shelton, 962). The lungfshes hold key postons n ths development n as much as they utlze both aquatc and aeral gas exchange wth dfferent emphass on these among speces from the three genera avalable. Respratory control has earler been evaluated for the Australan lungfsh, Neoceratodus forster, a predomnantly aquatc breather (Johansen, Lenfant & Grgg, 967). Ths present paper ams at a smlar evaluaton for Protopterus aethopcus whch depends prmarly on aeral breathng for gas exchange. In a prevous paper we have descrbed the respratory propertes of blood and normal pattern of gas exchange n P. aethopcus (Lenfant & Johansen, 968). MATERIAL, METHODS AND EXPERIMENTAL PROCEDURES Eghteen specmens of P. aethopcus were used n the present nvestgaton. The fsh ranged n weght from 500 g. to 6 kg. Part of the materal was transported by ar from Lake Vctora, Uganda, to Seattle, Washngton, U.S.A., whle some parts of the nvestgaton were made on freshly caught materal from Lake Vctora, at the Makerere Unversty College n the Department of Physology n Kampala, Uganda. J All fshes were fastng durng the perods of observaton and expermentaton. Ths work was supported by grant GB 4038 from the Natonal Scence Foundaton and grant HE-08405 from the Natonal Insttute of Health. t Establshed Investgator of the Amercan Heart Assocaton. Supported by the Northeastern Chapter, Washngton State Heart Assocaton. % We are ndebted to Professor P. G. Wrght n the Department of Physology at Makerere Unversty College for provdng laboratory facltes and for hs nterest and help throughout the course of ths work.
454 KjELL JOHANSEN AND CLAUDE LENFANT Blood gases and ph were measured by means of a Beckman 60 gas analyser usng an oxygen macro-electrode and a Severnghaus-type CO 2 electrode mounted n specal mcro-cuvettes. Blood ph was measured wth a Beckman mcro-assembly. Heart rate and blood pressures were measured usng Statham pressure transducers connected drectly to chroncally ndwellng catheters. Blood velocty was measured usng an ultrasonc Doppler-shft blood-flow meter after Frankln, Watson, Person & van Ctters (966). Branchal breathng rate was measured by recordng of pressure changes behnd an operculum va a chroncally mplanted catheter. The same catheter allowed samplng of exhaled water and njecton of drugs to the mmedate vcnty of the glls. The frequency of ar breathng was apparent as dstortons from the normal pattern n the blood-pressure record, but could also be counted by vsual observaton. All recordngs were made on an Offner Dynograph recorder. Dorsal aorta Coelac artery Pulmonary artery Vena cava Pulmonary Branchal arteres Blood velocty Blood samplng Fg.. Schematc drawng of the central crculaton n ProtopUrus. Stes of catheter cannulaton and placement of blood-velocty transducers are marked n. Blood samples were obtaned from a pulmonary artery, a pulmonary ven, a systemc artery and the vena cava. In some experments addtonal blood samples from an afferent branchal artery and pulmonary ar samples were acqured. All samples were obtaned va chronc cannulatons whch proved patent for perods extendng up to 2 days. Detals of anaesthesa, surgcal technques and methods of cannulaton and placement of blood-velocty transducers are found n earler papers (Lenfant & Johanson, 968; Johansen, Lenfant & Hanson, 968). Fgure shows a schematcal arrangement of the central crculaton n Protopterus wth stes of cannulaton and bloodvelocty tranducer placements. Followng recovery from anaesthesa several hours were allowed to pass before any samplng or expermental procedures were carred out. Measurements related to breathng and crculaton were recorded and correlated wth samplng of blood and lung gases. Studes were made on restng fsh n aerated water and durng ar exposure, as well as durng hypoxc, hyperoxc and hypercarbc condtons n the ambent water or gas phase. Addtonal experments ncluded pharmacologcal stmulaton of breathng.
Respraton n lungfsh. II 455 RESULTS Undsturbed breathng n aerated water It was demonstrated n an earler paper (Lenfant & Johansen, 968) that P. aethopcus has a bmodal gas exchange when lvng n water. Oxygen absorpton s manly carred out by the lungs, whle CO2 s chefly elmnated through glls and skn. A marked rregularty was apparent n the rhythms of both branchal and aeral breathng when the fsh rested n aerated water. Most commonly the ntervals between ar breaths ranged from 2 to 6 mn., but ntervals as short as 30 sec. and longer than 2. 3 35- CA _o CO r I r Strrng water tm a: T r n Tme 0 sec. Fg. 2. Top tracng: couplng of branchal (BRR) and pulmonary(b) breathng n undsturbed Protopterw. Lower tracng: ncrease n rate of branchal breathng dependng on agtaton of the water. hr. also occurred. Branchal breathng was equally lable both n frequency and vgour. In general the ventlatory movements were so shallow that water ventlaton was too small to be measured wth accuracy. The frequency of branchal pumpng was normally hgher than the frequency of ar breathng. At tmes long perods prevaled when branchal pumpng ceased altogether. It became apparent that the two modes of breathng were normally nterrelated n ther pattern. It was partcularly noteworthy that an ncreased rate and depth of branchal water pumpng commonly preceded an ar breath. The ntensfed branchal breathng started 20-60 sec. pror to the ar breath (Fg. 2). It was known from prevous work on the South Amercan lungfsh, Lepdosren paradoxa (Johansen & Lenfant, 967), that an extremely marked lablty n the branchal breathng rate could depend on mechancal agtaton of the water. Fgure 2 (lower tracng) demonstrates a smlar dependence of branchal breathng rate on strrng of the water for P. aethopcus.
456 KjELL JOHANSEN AND CLAUDE LENFANT Breathng behavour durng ar exposure The Afrcan lungfsh s known to aestvate out of water for several months durng perods of drought. The ablty of the fsh to wthstand ar exposure as measured by the pattern of gas exchange has also been studed expermentally (Lenfant & Johansen, 968). Expermental ar exposure by dranng the tank water elcted a conspcuous ncrease n the frequency of ar breathng (Fg. 3). 40 60 Tme (mn.) 80 00 Fg. 3. Change n frequency of arbreathng as a result of ar exposure n Protopterus. The dramatc ncrease n the rate of ar breathng appeared to be elcted by the physcal act of the exposure to ar. The onset of the response was too prompt to allow tme for nternal changes to occur. The powerful stmulus of ar exposure was also revealed n fsh recoverng from anaesthesa n water. If the head and mouth of such fsh were ar-exposed by gentle lftng, an ar-breath was taken long before voluntary motor movements and before any spontaneous attempt to surface for an ar breath. Ar breathng could also be evoked by ar exposure durng recovery from anaesthesa before a wthdrawal response to panful stmul, lke prckng wth a needle, was apparent. Blood gas tensons durng undsturbed breathng and durng ar exposure It was consdered essental to correlate the undsturbed breathng pattern n restng non-anaesthetzed fsh n water and also the breathng pattern n ar, wth gas tensons n the blood and n the pulmonary ar. Fgure 4 A and B show two plots relatng pulmonary gas and blood gas tensons to tme durng undsturbed breathng n aerated water. The samplng was started rght after an ar breath and was repeated at close ntervals tll the next breath. The vertcal arrows ndcate the tme of other ar breaths. It s apparent that the P o drops at about the same rate n all cycles. Note, however,
Respraton n lungfsh. II 457 the great varablty n the duraton of the breathng cycles and the consequent varablty n the arteral P o, at whch another breath s taken. These data are suggestve that the absolute level of arteral O 2 tenson does not represent a strong trggerng stmulus to aeral breathng. 60 50 50 00 50 Tme from breath (sec.) A o PV AB a DAO 200 30 20 0 Tme from breath (mn.) B Fg. 4. Relatonshp of O tenson n pulmonary gas or blood durng the nterval between consecutve ar breaths n Protopterus. The vertcal arrows ndcate tme of another breath. A and B represent analyses from two dfferent ndvduals. AB, afferent bronchal; DAO, dorsal aorta; PV, pulmonary ven. Fgure 5 A and B correlate ph n arteral blood wth arteral oxygen tenson for a seres of breath cycles of varable duraton. The tme course of changes n the blood ph between breaths presents a varable
458 KjELL JOHANSEN AND CLAUDE LENFANT pcture but suggests no tendency for the occurrence of a new breath to be correlated wth a reducton n arteral ph. These results ndcate that ar exposure was assocated wth a general ncrease n the level of O 2 tenson and CO 2 tenson n all vessels sampled (Fg. 6). 7-8 I 77 O I Q 7-6 7-5 50 0 20 30 Tme from breath (mn.) A 50 CD ] 30 20 0 _L 0 20 30 40 50 Tme from breath (mn.) Fg. 5. Tme course of arteral ph (A) and systemc arteral O, tenson (B) durng consecutve ntervals between ar breaths n Protopterut. The vertcal arrows mark the tme of another breath. DAO, dorsal aorta. B The tme requred for blood samplng lmts the possblty of correlatng ar breathng closely wth changes n blood gas composton. A fsh may typcally take an ar breath wthn a few seconds after ar exposure, wth successve breaths followng at close ntervals commonly up to 20 tmes more frequently than pror to ar exposure. Return to water after prolonged ar exposure was assocated wth an ncreased vgour and frequently of branchal breathng whle the rate of ar breathng rapdly returned to ts value pror to ar exposure. Breathng response to hypoxc water Lungfshes were exposed to hypoxc water by bubblng ntrogen n the aquara whle montorng the declne n water P Os, or by transferrng fsh to tanks wth O 2 defcent water of known concentraton. Durng these procedures the branchal respratory rate and the frequency of ar breathng were recorded and correlated wth
Respraton n lungfsh. II 459 changes n blood gas tensons and ph. Exposure to hypoxc water never evoked any compensatory breathng response, suggestve that the P o, of ambent water plays a normal role n the control of breathng. 90 80 70 B B I I 60 exposure 50 20 0 0 20 30 40 50 Tme (mn.) Fg. 6. Tme course of pulmonary O! tenson and blood O, tenson before and durng a perod of ar exposure n Protopterus. The arrows ndcate ar breaths. A, ar n lung; DAO, dorsal aorta; PA, pulmonary arrery; PV, pulmonary ven. 6- - 0 S 3 I 2 I 5 20 B 30 o 60 I 20 40 60 Tme (mm.) 80 00 Fg. 7. Change n frequency of ar breathng when ntrogen was njected nto the lung of Protopterus. Breathng response to hypoxc gas n the lung Fshes were made to surface nto an hypoxc atmosphere, or hypoxc gas mxtures were carefully njected drectly nto the lungs va chroncally ndwellng catheters.
460 KjELL JOHANSEN AND CLAUDE LENFANT Both procedures elcted an ncreased frequency of ar breathng. Fgure 7 llustrates a typcal response. It s noteworthy that the accelerated breathng followng njecton of hypoxc gas nto the lung was elcted at arteral Po, levels hgher than most of those occurrng n the late phase of normal spontaneous breathng cycles. Breathng response to ncreased ambent CO 2 When 5 % COj, n ar was bubbled carefully through the aquarum a consstent reducton n the rate of branchal water pumpng occurred. Sometmes the response was evoked promptly, at other tmes more slowly. Correlated wth the reduced o, CO, P 25 0 0 AO 28 0 23-3 PV 3 0 2-7 PA 28 0 24 o, 250 42 0 420 30 0 co, 200 23 7 23 9 25-7 250 CO, 0 6r 3 JS 2 a A- 0 I 5 2 20 a 2 30 60 & 20 40 60 80 00 Tme (mn.) Fg. 8. Change n frequency of ar breathng n Protopteru durng bubblng of 5 % CO, n ar nto the aquarum. At the top of the fgure s lsted gas tensons n ambent water (P), dorsal aortc blood (AO), pulmonary venous blood (PV) and pulmonary arteral blood (PA). branchal breathng was an ncrease n the frequency of ar breathng. The data dsclosed that no sgnfcant changes occurred n the CO 2 tenson of arteral blood, whle the O 2 tenson commonly showed an ncrease n all vessels sampled (Fg. 8). Fgure 9 provdes addtonal nformaton on blood ph changes and branchal breathng rate whle CO a was bubbled through the water. Agan a stmulaton of ar breathng s apparent whle branchal breathng s depressed. The accelerated ar breathng does not correlate wth a reduced ph, but such a reducton occurs n the recovery perod. Fgure 0 correlates branchal breathng rate and frequency of ar breathng wth blood velocty n the pulmonary artery. The top tracng was recorded when the fsh rested n aerated water wth slow bubblng of CO 2 through the water. The water ph had been reduced by one unt durng the CO a bubblng. The branchal breathng rate was two per mnute before bubblng, but ceased altogether n response to the ncrease of CO 2 n the water. The rate of ar breathng ncreased concurrently, reducng the nterval between breaths from more than 4 mn. to about mn. The pulmonary blood
Respraton n lungfsh. II 46 flow was reduced, but t s not known whether the reduced blood flow resulted from a general reducton n cardac output or from a redstrbuton of the regonal blood flow. 7-9 r- X 78 76 7-5 Water ph 6-45 BW, 2/mln.? 34 5 7 5 75 5 85 600-)"6-43 n 30 mn. 6/mn 2/mn. 60 20-0 - 0 20 30 40 50 60 70 80 90 00 0 Tme (nun.) Fg. 9. Tme course of arteral ph and P o, n Protopterus before and after bubblng of 5 % CO, n ar nto the water. Branchal breathng rate (BRR) and rate of ar breathng (arrows) are marked n. DAO, dorsal aorta. Water ph 6-45 5-,! * Branchal respratons 7 <H I I I I I I I I I I I I I I I I I I I I I I I Water ph 5-50 No branchal respratons " r Tme 0 sec. Fg. 0. Correlaton of branchal breathng rate (small arrows), ar breathng rate (B) and pulmonary blood velocty n Protopterus before and durng bubblng of 5 % CO, n ar through the water.
462 KjELL JOHANSEN AND CLAUDE LENFANT Pharmacologcal stmulaton of breathng Studes of mammalan chemoreceptors have shown that chemcals known to stmulate sympathetc ganglon cells by a ncotne-lke acton also exert a stmulatory effect on perpheral chemoreceptor cells nvolved n respratory control. There s no prevous data to ndcate whether chemoreceptors of lower vertebrates possess smlar sensory qualtes, but t was decded to test the effect of ncotne on the breathng pattern n Protopterus. These experments revealed that ncotne, n the form of Ncetamde, elcted an ncrease n both aeral and branchal breathng. The response occurred whether the drug was admnstered ntravenously or ntroduced beneath the Ncetamde Breath 3 * 3 5 ^ 5 I I r~rn! I Tme 0 sec. Fg. I I. Change n branchal breathng rate (BRR) when Ncetamde was ntroduced beneath an operculum n Protopterus. The lower tracng represents systemc arteral blood pressure. Pror to applcaton of the drug, branchal breathng rate was shallow and nfrequent (less than per mm.). opercula n the water bathng the glls. In addton, the drug had a hypotensve effect. When admnstered ntravenously the fall n arteral blood pressure more or less concded wth the appearance of the branchal breathng response. However, f the drug was ntroduced beneath the opercula n the water close to the glls, the accelerated branchal breathng appeared long before the blood-pressure drop (Fg.n). If chemoreceptors n respratory control have homologous response mechansms n all vertebrates the results ndcate that such receptors do exst n Protopterus. The locaton of these receptors n Protopterus s not known, but t merts attenton that the response s evoked sooner from admnstraton of the drug n the water close to the glls than from gvng t ntravenously. Hstamne s not known to nterfere drectly wth chemoreceptor stmulaton. The drug, however, exerted a marked stmulatory effect on ar breathng n Protopterus, when admnstered ether ntravascularly or externally n the water. Hstamne had the addtonal effect of producng restlessness and coughng. The response may well be related to a general constrcton of vascular smooth muscle n the glls, or to a constrctve effect on the profusely dstrbuted smooth musculature n the lung parenchyma of Protopterus (Johansen & Rete, 967). Couplng of respratory and crculatory events It was consstently observed that spontaneous breathng caused marked changes n heart rate, blood pressure and blood flow. Fgure 2 shows blood velocty tracngs from
Respraton n lungfsh. II 4^3 the vena cava (top) and the pulmonary artery (bottom) n conjuncton wth a spontaneous ar breath. Note the marked ncrease n blood flow assocated wth the breath. The bottom tracng from the pulmonary artery shows that the flow ncrease could occur n antcpaton of the actual breathng act. The blood flow ncrease n the pulmonary artery occurred largely n the form of an ncreased dastolc flow component whle the peak systolc velocty remaned essentally unaltered. If long ntervals prevaled between ar breaths there was often a marked slowng of the heart rate wth reduced flow towards the end of a breathng cycle. 5 CH J' r r Tme 0 sec. Fg z Blood-velocty tracngs from the vena cava and pulmonary artery n two dfferent Protopterus. Both records demonstrate an ncrease n blood velocty assocated wth an ar breath. B. DISCUSSION In an earler paper t was shown that the Afrcan lungfsh employs a bmodal gas exchange. The fsh depends almost exclusvely on ts lungs for O2 absorpton, whle the coarse, atrophed glls and skn reman responsble for the major part of CO2 elmnaton (Lenfant & Johansen, 968). It s to be expected that such bmodal gas exchange s reflected n the respratory control system of the fsh. On ths assumpton t becomes mperatve to dstngush between external stmul n the water and m the atmosphere (gas phase) surroundng the fsh, when attemptng to evaluate ts respratory control.. The remarkable varablty n both branchal and aeral breathng rates was strkng. Wthout apparent changes n external condtons the tme nterval between spontaneous ar breaths could vary from 30 sec. to more than hr. Fgures 4 and 5 demonstrate that the varablty of ntervals between ar breaths shows no correlaton wth the arteral O2 tenson at whch another breath s taken. The varablty was large enough to suggest that the actual level of arteral blood gas tensons or ntrapulmonary Exp. Bol 49, 2
464 KjELL JOHANSEN AND CLAUDE LENFANT gas tensons played no promnent role n settng the breathng rhythm. It appears that the fsh rele9 more on tolerance to large nternal varatons n gas tensons than on regulatons aganst them. The most powerful stmulus to. ncreased ar breathng n Protopterus was ar exposure tself. The persstence of ths response n fsh recoverng from anaesthesa whle stll unresponsve to tactle or panful stmul served to emphasze that ths apparent reflex has a marked degree of structural and functonal autonomy from hgher ntegratve actvtes of the nervous system. All ndcatons are that the sensory nput resultng n ths response s related to the removal of the head from the water, and possbly related to the sudden nfluence of net gravtatonal forces actng on the fsh n ar. A remote parallel to ths response n the lungfsh s present n the mammalan foetus when emergng from aquatc foetal lfe to a terrestral exstence based on ar breathng. Controlled experments have ndcated that the crucal ntaton of ar breathng n the newborn mammal cannot be drectly trggered by nternal chemoreceptor stmulaton but may be related to the stmulus of exposure to ar. The large lterature on breathng responses to changes n external gas composton n teleost fshes s practcally wthout excepton n attrbutng a marked stmulatory effect on ventlaton from both hypoxc water and CO 2 -rch water (Hughes & Shelton, 962). Protopterus dffers from all other fshes studed by beng unresponsve to deoxygenated water. Snce the glls n the adult fsh are of very lttle consequence n oxygen absorpton (Lenfant & Johansen, 968), a change n O a tenson of the ambent water wll only slowly become manfest nternally. A reducton n gll surface area as well as ncreased dffuson resstance across the gas-exchangng surfaces of the glls are common n ar-breathng fshes wth accessory or alternatve ar-breathng organs. In essence there exsts no effectve, functonal lnkage between nternally located chemoreceptors responsve to hypoxc stmul and the external water. After ths paper was ready for submsson an nvestgaton on lung and gll ventlaton n the Afrcan lungfsh by Jesse, Shub & Fshman (968) appeared. Thenresults dffer n mportant respects from ours. The fshes used by Jesse et al. responded to decreased oxygen tensons n the water by augmentaton of both branchal and pulmonary breathng. The juvenle status of ther fsh weghng on the average 65 g. aganst an average of 3 kg. for our fsh may explan some of the dfferences n the results. Juvenle specmens of lungfsh are known to depend more on gll breathng than adults. The survval of ther fshes for several days wthout access to ar substantates ths contenton, as numerous reports from studes of adult fsh clam that they succumb rapdly f prevented access to the atmosphere. Further comparson wth the work by Jesse et al. s also lmted by ther expermental procedures whch do not allow a dstncton between external stmul n the water and n the ambent gas phase. Neoceratodus, the Australan lungfsh, lves n water havng a generally hgh but somewhat varable O 2 level n water. Ths lungfsh has well-developed glls and utlzes them effectvely n O a absorpton (Lenfant et al. 966). Neoceratodus promptly responded to deoxygenated water by ntensfed breathng (Johansen et al. 967). For Protopterus condtons are markedly dfferent. Its habtat s much less conducve to oxygen absorpton and the fsh s structurally better adapted for oxygen absorpton
Respraton n lungfsh. II 465 from the atmosphere than from the water. When two sources of oxygen can be used smple consderaton of energetcs suggests that the most accessble source and the method of breathng requrng the least amount of energy be utlzed. The large spontaneous varablty n the normal undsturbed breathng rhythm of Protopterus, apparently uncorrelated wth nternal gas tensons and ph, seems to preclude any great mportance n the level of these parameters for the regulaton of breathng. Yet t was qute evdent that nspraton or njecton of hypoxc gas mxtures nto the lung prompted an ncreased frequency of ar breathng. It appears possble that the receptors trggerng ar breathng are exposed to stmul other than the P o level n the arteral blood (Fgs. 4, 5 and 9). ' ' ' ' ' ' ' l 0 20 30 40 50 60 70 80 90 00 0 20 Po t n pulm. ar (mm, Hg) Fg. 3. Composte plot of the relatonshp between O, tenson n pulmonary gas and n the pulmonary venous blood (PV). It seems approprate to comment brefly on the low senstvty of chemoreceptors respondng to arteral condtons n Protopterus n relaton to the structural development of the lung as a gas exchanger. In hgher ar breathng vertebrates there are normally small gradents n O 2 and CO 2 tensons between the alveolar gas and the arteral blood. In Protopterus large gradents exst between pulmonary gas and blood leavng the lung and even steeper gradents between the pulmonary gas and the systemc arteral blood. The long ntervals between breaths result n a wde range n composton of pulmonary gas between breaths. The large gradents between gas and blood wll tend to mnmze the changes n the blood aganst the changes n pulmonary gas. Fgure 3 demonstrates an average plot of the gradents n O 2 tenson between pulmonary gas and pulmonary venous blood n Protopterus. Whle the pulmonary gas ranges from 20 to 30 mm. Hg n oxygen tenson the pulmonary venous blood has a correspondng range between 55 and 7 mm. Hg. A smaller gradent between gas and blood would have resulted n larger cyclc varatons n the latter. The large gradents may result from a crude structure of the lung wth large dffuson resstance and long dffuson paths or they can result from an uneven matchng of blood and gas n the lung. 30-2
466 KJELL JOHANSEN AND CLAUDE LENFANT Fsh lvng n temperate, well aerated waters have very low values of nternal CO tensons because of the relatve ease by whch CO 2 s elmnated n aquatc gas exchange. It has been argued (Hughes & Shelton, 962) that the only logcal advantage a fsh can expect from a senstvty to CO 2 tensons s as a measure of oxygen defcency n arteral blood. These authors saw, however, a conflct n ther statement, snce ncreased ventlaton of fsh n CO 2 -rch waters seems napproprate n as much as CO 2 then wll accumulate rather than beng elmnated from the fsh. In Protopterus the glls are much more effectve than the lungs for CO 2 elmnaton whle the fsh stays n water (Lenfant & Johansen, 968). In spte of ther crude and degenerated form the glls hence consttute a functonal lnkage for CO 2 between the nternal envronment and the ambent water. On that bass t seems a sgnfcant fndng that ncreased CO 2 tenson n the ambent water evokes a breathng response, whereas hypoxc water has no effect on the breathng pattern. It s noteworthy that the depressve effect on gll breathng elcted by bubblng 5 % CO 2 n the water was evoked before the arteral CO 2 tensons had changed apprecably (Fg. 7). Symbranchus marmoratus, a facultatve ar-breathng fsh nhabtng tropcal freshwater swamps n South Amerca, and the Australan lungfsh, show a smlar nhbton of branchal breathng n CO 2 -rch water (Johansen, 966; Johansen et al. 967). The nhbton was not removed n spte of ncreasng nternal CO 2 tensons untl the ambent CO 2 tenson was agan lowered. When ths was done, however, a hgh frequency of forceful branchal movements was establshed. Hypothetcally the sensory mechansm for CO 2 wll dstngush between the ambent and nternal CO 2 tensons and drve the effectors to keep the nternal CO 2 tensons as low as possble. However, t s obvous that when ncreased external CO 8 exerts a depresson on branchal breathng, nternal hypoxa wll ensue unless other correctve measures are moblzed. On that account t s of nterest that the response has only been descrbed for fsh capable of aeral breathng. Typcally n the Australan lungfsh, Neoceratodus (Johansen et al. 967), as well as shown presently for Protopterus, depresson of branchal breathng wll stmulate aeral breathng. It should be noted that our experments do not exclude that a dfferent response mght occur to lower CO 2 concentratons (Wllmer, 934; Jesse et al. 968). Fshes n general show a close correlaton between respratory and crculatory events (Satchell, 960). A functonal couplng between ventlaton and perfuson also appears to exst n lungfshes (Johansen et al. 968). The evdence suggests that such couplng may be mportant for the effcency of the matchng process between blood and the respratory meda. It has been shown for both Protopterus and Neoceratodus that lung nflaton wll elct a tachycarda and conversely lung deflaton wll produce a bradycarda. These responses were clearly dependent upon the prevalng heart rate and dd not occur f the rate was hgh pror to lung nflaton. In addton to the heart-rate change there was typcally an ncrease n total cardac output and sometmes n the rato of pulmonary flow to total flow. In all spontaneously occurrng events showng a couplng of respraton and crculaton, t was a change n respraton that nstgated a crculatory change.
Respraton n lungfsh. II 467 SUMMARY. Factors controllng aeral and aquatc breathng have been studed n the ntact, free-swmmng Afrcan lungfsh, Protopterus aethopcus. Frequences of aeral and branchal breathng were correlated wth gas tensons n the lung and n blood from pulmonary and systemc arteres and vens. 2. Studes were made_ on fsh restng n aerated water, durng exposure to ar and durng hypoxc and hypercarbc condtons n the envronment. 3. Both branchal and pulmonary breathng were rregular durng rest n aerated water. The rate of branchal breathng normally exceeded that of ar breathng. An ncreased rate and vgour of branchal breathng commonly preceded an ar breath. There was no ndcaton that a new ar breath was related to the values of arteral P o and ph. 4. Ar exposure elcted a marked and mmedate ncrease n the rate of ar breathng. Hypoxc water never evoked any compensatory breathng responses whle breathng from a hypoxc gas mxture quckly ncreased the rate of ar breathng. Bubblng 5 % CO 2 n ar nto the aquarum caused a reducton n branchal breathng whle the rate of ar breathng ncreased. 5. Ncotne njected ntravenously or n the water close to the glls elcted an ncrease n both aeral and branchal breathng. 6. Respratory and crculatory events were correlated durng undsturbed breathng and durng artfcal lung nflaton. Increased cardac output and a shft n regonal blood flow to a hgher pulmonary flow occurred wth ar breaths. REFERENCES FRANKLIN, D. L., WARTON, N. W., PIERSON, K. E. & VAN CITTERS, R. L. (966). A technque for radotelemetry of blood flow velocty from unrestraned anmals. Am. J. med. Electron. 5, 24-8. HUGHES, G. M. (966). Evoluton between ar and water. In Development of the Lung, pp. 64-80. Cba Foundaton Symposum. HUGHES, G. M. & SHELTON, G. (962). Respratory mechansms and ther nervous control n fsh. In Advance! n Comparatve Physology and Bochemstry, vol., pp. 275 364. New York: Academc Press. JESSE, J., SHUB, C. & FISHMAN, A. P. (968). Lung and gll ventlaton of the Afrcan lungfsh. Respraton Physology 3, 267-87. JOHANSEN, K. (966). Arbreathng n the teleost, Symbranchus marmoratus Comp. Bochem. Physol. 8, JOHANSEN, K. & LENFANT, C. (967). Respratory functon n the South Amercan lungfsh, Lepdosren paradoxa (Ftz.) J. exp. Bol. 46, 205-8. JOHANSEN, K., LENFANT, C. & GRIGG, G. C. (967). Respratory control n the lungfsh, Neoceratodus forster (Krefft). Comp. Bochem. Physol. 20, 835-854. JOHANSEN, K., LENFANT, C. & HANSON, D. (968). Cardovascular dynamcs n lung-fshes. Z. vergl. Physol. June, 968. JOHANSEN, K. & REITE, O. B. (967). Effects of acetylchohne and bogenc amnes on pulmonary smooth muscle n the Afrcan lungfsh, Protopterus aethopcus. Acta physol scand. 7, 248-52. LENFANT, C. & JOHANSEN, K. (966). Respratory functon n the elasmobranch Squahs suckley. Respraton Physology I, 3 29. LENFANT, C. & JOHANSEN, K. (968). Respraton n the Afrcan lungfsh, Proptopterus aethopcus. I. Respratory propertes of blood and normal patterns of breathng and gas exchange. J. exp. Bwl. 49, 437-52. LENFANT, C., JOHANSEN, K. & GRIGG, G. C. (966). Respratory propertes of blood and pattern of gas exchange n the lungfsh, Neoceratodus forster (Krefft). Respraton Physology 3, -2. RAHN, H. (966). Aquatc gas exchange theory. Respraton Physology, 2. ROBIN, E. D. & MURDAMGH, H. V. (966). Qualtatve aspects of vertebrate gas exchange. In Development of the Lung, pp. 85-98. Cba Foundaton Symposum.
468 KjELL JOHANSEN AND CLAUDE LENFANT SATCHELL, G. H. (960). The reflex coordnaton of the heart beat wth respraton n the dogfsh. J. exp. Bol. 37, 79-3- STEEN, J. B. & KRUYSSE, A. (964). The respratory functon of teleost glls. Comp. Bochem. Pkytol. 3, 27-42. WILLMER, E. N. (934). Some observatons on the respraton of certan tropcal fresh water fshes. J. exp. Bol., 28-306.