the differential cardio-respiratory responses to ambient hypoxia and systemic hypoxaemia in the...

Ž .Comparative Biochemistry and Physiology Part A 130 2001 677�687

The differential cardio-respiratory responses to ambienthypoxia and systemic hypoxaemia in the South American

lungfish, Lepidosiren paradoxa

Adriana Sancheza, Roseli Soncinia, Tobias Wanga,c,�, Pia Koldkjaera,d,Edwin W. Taylora,b, Mogens L. Glassa

aDepartment of Physiology, Faculty of Medicine of Ribeirao Preto, Uni�ersity of Sao Paulo, Sao Paulo, Brazil˜ ˜ ˜bSchool of Biosciences, The Uni�ersity of Birmingham, Birmingham, UK

cDepartment of Zoophysiology, Aarhus Uni�ersity, DK 8000 Aarhus C, DenmarkdSchool of Biological Science, The Uni�ersity of Li�erpool, Li�erpool, UK

Received 19 September 2000; received in revised form 16 June 2001; accepted 26 June 2001

Abstract

Ž .Lungfishes Dipnoi occupy an evolutionary transition between water and air breathing and possess well-developedlungs and reduced gills. The South American species, Lepidosiren paradoxa, is an obligate air-breather and has thelowest aquatic respiration of the three extant genera. To study the relative importance, location and modality ofreflexogenic sites sensitive to oxygen in the generation of cardio-respiratory responses, we measured ventilatoryresponses to changes in ambient oxygen and to reductions in blood oxygen content. Animals were exposed to aquaticand aerial hypoxia, both separately and in combination. While aerial hypoxia elicited brisk ventilatory responses, aquatichypoxia had no effect, indicating a primary role for internal rather than branchial receptors. Reducing haematocrit andblood oxygen content by approximately 50% did not affect ventilation during normoxia, showing that the specificmodality of the internal oxygen sensitive chemoreceptors is blood PO per se and not oxygen concentration. In light of2previous studies, it appears that the heart rate responses and the changes in pulmonary ventilation during oxygenshortage are similar in lungfish and tetrapods. Furthermore, the modality of the oxygen receptors controlling theseresponses is similar to tetrapods. Because the cardio-respiratory responses and the modality of the oxygen receptorsdiffer from typical water-breathing teleosts, it appears that many of the changes in the mechanisms exerting reflexcontrol over cardio-respiratory functions occurred at an early stage in vertebrate evolution. � 2001 Elsevier Science Inc.All rights reserved.

Keywords: Lungfish; Dipnoi; Lepidosiren; Hypoxia; Hyperoxia; Ventilation; Breathing pattern; Heart rate; Ventilatory response;Oxygen modality; Chemoreceptors

� Corresponding author. Tel.: �45-8942-2694; fax: �45-8619-4186.Ž .E-mail address: [email protected] T. Wang .

1095-6433�01�$ - see front matter � 2001 Elsevier Science Inc. All rights reserved.Ž .PII: S 1 0 9 5 - 6 4 3 3 0 1 0 0 3 9 5 - 6

( )A. Sanchez et al. � Comparati�e Biochemistry and Physiology Part A 130 2001 677�687678

1. Introduction

Ž .The lungfishes Dipnoi belong to an ancientlineage that occupies the evolutionary transitionbetween water and air breathing. In addition totheir well-developed lungs, they have retainedreduced gills and exhibit bimodal respiration,combined with a significant contribution of cuta-

Žneous gas exchange see Abe and Steffensen,.1996, for data on Lepidosiren . Among the three

extant genera, the Australian lungfish, Neocerato-dus, is a facultative air-breather that ventilates its

Žlungs only when exposed to hypoxic water Johan-.sen et al., 1967; Fritsche et al., 1993 , while the

South American lungfish, Lepidosiren paradoxa, isan obligate air-breather with highly reduced gillsŽ .Johansen and Lenfant, 1967 .

Potential stimuli for air-breathing in fish in-clude hypoxia, and hypercapnia, both modulatedby increased temperature and exercise, which in-crease oxygen demand and CO production2Ž .Johansen, 1971; Smatresk, 1994; Graham, 1997 .Until recently it was not established whether theincreases in air-breathing observed under thesecircumstances are stimulated solely by changes inoxygen availability, delivery, or demand, orwhether lungfish also respond to changes in bloodpH or PCO . In the bowfin Amia cal�a, air2breathing is only stimulated by changes in water

Ž .or blood O status McKenzie et al., 1991 , and2they do not appear to possess central chemosensi-tivity controlling gill ventilation or air breathingŽ .Hedrick et al., 1991 . In contrast, both Pro-

Ž .topterus the African lungfish and Lepidosirenincrease air breathing frequency during aquatic

Žhypercapnia Johansen and Lenfant, 1968;.Sanchez and Glass, 2001 . Sanchez and Glass

Ž .2001 also showed that Lepidosiren displays amarked post-hypercapnic hyperpnea as described

Ž .for ectothermic tetrapods Milsom, 1995 . Collec-tively, these observations indicate resemblancebetween Dipnoi and the land vertebrates withrespect to the ventilatory responses to hypercap-nia.

The relative importance and location of re-flexogenic sites sensitive to oxygen in the genera-tion of cardiorespiratory responses to changes inambient or blood gas composition have not yetbeen clearly elucidated for Lepidosiren. Conse-quently, the present study set out to evaluate towhat extent the hypoxic drive to pulmonary venti-lation arises from stimulation of external or inter-

nal receptors. This was explored by exposing ani-mals to aquatic and aerial hypoxia, both sepa-rately and in combination. Furthermore, themodality of the oxygen sensitive chemoreceptorshas not been investigated in lungfish. Thus, it isnot known whether they exhibit ventilatory re-

Ž� �.sponses to reduced blood oxygen content O2Žlike some teleost fish Randall, 1982; Smith and

.Jones, 1982; Soncini and Glass, 2000 , or regulateventilation in relation to oxygen partial pressureŽ . Ž .PO like amphibians Wang et al., 1994 . To2investigate this question, we compared the venti-latory responses during aerial hypoxia with theresponses measured following similar reductionsin blood oxygen content induced by reductions inhaematocrit during normoxia.

2. Materials and methods

2.1. Animals

ŽLungfish Lepidosiren paradoxa, Fitz; 800�2000.g of undetermined sex were collected in the

Pantanal region near the city of Cuiaba in the´state of Mato Grosso do Sul, southwestern Brazil,and transported to The University of Sao Paulo,

Ž .at Riberao Preto state of Sao Paulo . Here theywere maintained for several weeks prior to exper-iments in 1000-l tanks, containing dechlorinated,aerated tapwater at a temperature of 25�3�C.The water was continuously renewed and a 12h:12 h light:dark cycle was maintained. The ani-mals were fed, mainly on chopped liver, severaltimes a week, but food was withheld for morethan 48 h prior to experimentation.

2.2. Surgical procedure

The fish used for blood sampling and manipula-tion of blood oxygen carrying capacity wereanaesthetised by immersion into a 1-g l�1 benzo-caine solution until they ceased to exhibit respon-ses to pinching of the skin. The fish were thentransferred to a surgical table where the gills onone side were irrigated by a continuous flow ofwater containing 0.25 g l�1 benzocaine. The gill

Ž .arches on the contralateral right side were ex-posed by a 2-cm incision above the operculumand the afferent vessel on the fifth and last gillarch was dissected free and occlusively cannu-

� Ž .lated see Romer 1970 for gill structure and� Žnumbering . The catheter Clay Adams PE50 con-

( )A. Sanchez et al. � Comparati�e Biochemistry and Physiology Part A 130 2001 677�687 679

.taining heparinised Ringer was forwarded intothe ventral aorta and secured to the gill arch bysutures, after which the catheter was exteriorisedand secured to the body wall. The incision wasclosed by sutures and tissue glue, and the animalwas transferred to aerated water where it wasallowed to recover for approximately 24 h beforeexperimentation. There was no detectable effectof operation on the behaviour of the animals. Theexperimental chamber was covered to avoid visualdisturbances to the animal

2.3. Analytical techniques

Blood samples of 0.7 ml were withdrawn anaer-obically from the ventral aortic cannula and anal-

Žysed immediately following analysis, 0.5 ml of theblood and 0.2 ml of Saline was re-injected into

.the animal . Haematocrit was determined fol-lowing 3-min centrifugation at 12 000 rpm in cap-illary tubes. Blood PO and pH were determined2

Žusing a FAC 204A O analyser FAC Instru-2.ments, Sao Carlos, Sao Paulo State, Brazil and a˜ ˜

Ž .Micronal B374 pH meter Sao Paulo, Brazil . The˜oxygen electrodes were calibrated with pure ni-trogen and humidified atmospheric air, taking theblood�gas sensitivity ratio into accountŽ .Siggaard-Andersen, 1976 . The pH electrode was

Žadjusted using high precision buffers S1500 and.S1510; Siggaard-Andersen, 1976 . The electrode

chamber was maintained at the temperature ofŽ .the fish water 25�C . Total content of O was2

Ž .measured as described by Tucker 1967 .

2.4. Measurement of �entilation and cardiac�ariables

Ventilation at 25�C was measured directly, us-Žing a plethysmographic method Lomholt and

.Johansen, 1974 . In short, the animal was con-fined within a 10-l chamber, shaped like an in-verted funnel and filled with water to slightlyabove the cylindrical neck. Due to the expansionof the lungs, inspiration increased the water levelwithin the neck of the funnel, while the animalfloated upwards. Oppositely, expiration decreasedthe water level. These vertical movements of thewater column were recorded as pressure changes,

Ž .using a Niho Kohoen Polygraph System Japan ,consisting of a venous pressure transducer, anAP6216 carrier amplifier and the recorder. Cali-bration was obtained by injection and withdrawal

of known volumes of water, which provided adirect relationship between pressure change atthe bottom of the water column and the amountof water added or withdrawn from the system.

Between periods of blood sampling, the catheterwas connected to an arterial pressure transducerthat formed part of the Niho Kohoen system.Cardiac frequency and blood pressure wererecorded, using the polygraph system. Calibrationof the pressure transducer was performed dailyusing a static water column of known elevation.The experimental chamber was continuouslyflushed either with air or with gas mixtures ob-tained by feeding pure gases to a GF-3�MP gasmixer. The total flow was set at 1 l min�1 andgases were supplied to the air space in the funneland�or to the water space containing the fish.Using this system, water PO could be changed2within 10�15 min.

2.5. Experimental protocols

Experiments were performed as three separateseries, as described below. Series 1 addressed theeffects of repeated exposure to hypoxia; series 2described the effects of aerial and�or aquatichypoxia; and series 3 determined the effects ofreduced blood oxygen content during normoxiaand hypoxia.

2.5.1. Series 1The effects of repeated exposure to aerial hy-

poxia was determined in nine lungfish. Each un-restrained and un-operated lungfish was placedinto the experimental chamber overnight andventilation during normoxia was recorded the fol-lowing day. When a stable ventilatory pattern hadbeen recorded for a minimum of 30 min, hypoxiaŽ .aerial PO �69, then 35 mmHg was applied as2described below, after which the animals werereturned to normoxia. Following 2�3 h in nor-moxia, the hypoxic exposures were repeated.

2.5.2. Series 2The ventilatory responses to aerial and�or

aquatic hypoxia were measured in eight unre-strained and un-operated lungfish. Each animalwas placed in the experimental chamber the daybefore experimentation. The animal never strug-gled and a regular breathing pattern soon devel-oped. After a normoxic control period, es-tablished by 2 h of vigorous aeration of the cham-


ber, the water PO was, in sequence, changed to269, 35 and then, 208 mmHg, corresponding to 10,5 and 30% O , respectively, delivered from the2gas flow meter. Meanwhile, the aerial phase wasmaintained normoxic. Secondly, after a normoxicrecovery period, the same sequence of gas mix-tures was delivered to the aerial phase, while thewater was kept normoxic. Thirdly, the hypoxicsequences were applied simultaneously to thewater and to the aerial phase. Each experimentalcondition was maintained for 1 h. Meanwhile,tidal volume and frequency were continuouslyrecorded.

2.5.3. Series 3Cannulated fish were placed in the experimen-

tal chamber in order to record heart rate andventilation. After obtaining a blood sample dur-ing normoxia, hypoxia was applied as described

Ž .above Series 1 and blood samples were obtainedat the end of each exposure. The animals were

Žreturned to normoxia, and a volume of blood 2%.of body weight was removed to render the ani-

mal anaemic, down to a haematocrit of approxi-mately 50% of normal. Blood volume was main-tained by replacing the removed blood with anequal volume of lungfish Ringer. Ventilation andheart rate were measured under normoxic condi-tions, 2�3 h after the reduction in blood oxygencarrying capacity.

2.6. Statistics and data analysis

The effects of repeated exposure to hypoxiaŽ .Series 1 , and the effects of reducing blood oxy-gen carrying capacity on blood gases and the

Ž .cardiorespiratory responses to hypoxia Series 3were evaluated by two-way ANOVAs for re-peated measures. The effect of aquatic and aerialhypoxia on ventilation in the uninstrumented ani-

Ž .mals Series 2 was evaluated by a one-wayANOVA for repeated measures. In all cases,means that were significantly different from thenormoxic level were subsequently identified by aStudent�Newman�Keuls post-hoc test. We ap-plied a fiducial limit for significance of P�0.05and all data are presented as mean�1 S.E.M.

3. Results

3.1. The effects of ambient hypoxia on un-operatedand unrestrained animals

The responses of un-operated and unrestrainedanimals to repeated exposure to aerial hypoxiaŽ .Series 1 are illustrated in Table 1. The fre-

Ž .quency of air breathes f increased significantlyRduring both sets of exposures. Coupled with aninsignificant trend towards an increased tidal

Ž .volume V , this resulted in a highly significant,TŽ .three-fold increase in ventilation volume V at aI

P O of 38 mmHg, compared to the normoxicI 2control value. There was no significant differencebetween the responses measured during the firstand the second exposures to hypoxia.

ŽIn a separate series of single exposures Series.2 , illustrated in Fig. 1, aerial hypoxia, with or

without aquatic hypoxia, caused a progressive in-crease in f reaching a four-fold increase at aRPO of 35 mmHg, compared to the control, nor-2moxic rate. As the amplitude of air breaths was

Table 1Effects of repeated exposure to aerial hypoxia in unrestrained and un-operated lungfish

First hypoxic exposure Second hypoxic exposure runŽ . Ž .PO in mmHg PO in mmHg2 2

150 72 38 150 72 38Ž . Ž . Ž . Ž . Ž . Ž .9 9 9 9 9 7

�1Ž .V ml kg 26.5�1.8 28.3�2.8 34.7�4.1 31.2�2.3 29.3�2.5 33.5�4.6T�1Ž .f h 10.4�1 17.2�3.5 23.3�5.6 9.1�1.8 18.3�4.3 26.5�5.7R

�1 �1Ž .V ml kg h 272�32.5 483�88.3 791�194 259�61 566�133 834�174I

Values are presented as mean�1 S.E.M., and the number of animals is listed in brackets; there was no significant differenceŽ .between the first and second set of exposures two-way ANOVA for repeated measures .

Abbre�iations: V � tidal volume; f �respiratory frequency; V � inspired volume.T R I


Ž . Ž .Fig. 1. Changes in ventilation rate f , tidal volume V andR TŽ . Ž .ventilation volume V of lungfish Lepidosiren exposed toI

two levels of hypoxia and to hyperoxia. The changes in PO2Ž .were imposed in the water alone open circles , in the air

Ž .space alone grey circles , or in both media simultaneouslyŽ .solid circles . While aquatic hypoxia alone was without effecton ventilation, hypoxia imposed in the air space from whichthe fish breathed caused significant increases in f and V ,R Twith or without aquatic hypoxia. Mean values that are signifi-cantly different from the normoxic value are marked with anasterisk.

unaffected, this resulted in a proportional in-crease in V . Aquatic hypoxia, with access toInormoxic air, had no significant effect on themean rate or amplitude of the air-breaths, so thatventilation volume was unaffected. Neitheraquatic nor aerial hyperoxia alone, had any affecton the frequency or amplitude of air breaths.However, there was a 30% reduction in mean fR

in fish exposed to aerial hyperoxia in hyperoxicwater that, when combined with a small decreasein V , resulted in a significant reduction in meanT

Ž .V to 60% of the control, normoxic value Fig. 1 .IIn this second series of experiments, the

breathing frequencies were generally lower thanthose obtained in series 1 and 3. Because theanimals studied in series 1 were un-operated, asin series 2, we are not able to identify a simplecause for this difference. Since series 2 was per-formed on a separate group of fish during thewinter season, it is possible that seasonal effectsmay account for the lower breathing frequency.

3.2. Changes in blood gas �alues during hypoxia andfollowing induced anaemia

The changes in blood gas values during a two-step reduction in ambient PO are shown in Fig.22. The oxygen partial pressure changed in directproportion to ambient hypoxia and the other dataare related to these values. Hypoxic exposurecaused a progressive reduction in blood oxygen

Ž .content CaO , while haematocrit and pH were2unaffected. The substitution of approximately halfof the blood volume by Ringer resulted in a 50%reduction in normoxic haematocrit, a proportio-nal decrease in blood oxygen content, but did notaffect pH.

3.3. Ventilation and heart rate in relation to �entralaortic PO during hypoxia and following induced2anaemia

The changes in ventilation and heart rate inrelation to changes in ventral aortic PO during2progressive hypoxia and in response to anaemia,induced by a 50% reduction in haematocrit, areillustrated in Fig. 3. Hypoxia had no effect onheart rate or V , but caused a significant increaseTin f , with the result that V was markedly in-R Icreased to more than double its normoxic value.Anaemia induced a 10% increase in normoxicheart rate, but had no effect on the respiratoryvariables.

4. Discussion

4.1. Critique of blood sampling

The blood samples were obtained through acannula inserted into the fifth gill arch, from


Fig. 2. Blood gas values in lungfish exposed to hypoxic gasmixtures in the air space of the experimental chamber andsubsequently, to induced anaemia. Blood oxygen content wasmarkedly reduced both by hypoxia and by anaemia. Hypoxiawas without effect on haematocrit, but it was reduced byapproximately 50% by anaemia. Neither imposed variableaffected pH. Mean values that are significantly different fromthe normoxic value are marked with an asterisk.

where it was advanced into the bulbus. This can-nulation is relatively non-invasive and the animalsrecovered quickly from the surgery. The bloodperfusing the functional gill arches predominantlystems from the systemic venous circulation, and ispreferentially distributed towards the pulmonary

Žcirculation e.g. Johansen et al., 1968a; Lenfant.and Johansen, 1968; Fishman et al., 1985, 1989 .

In addition, it is uncertain whether the insertionof the cannulae interfered with the blood flow

separation in the bulbus. Therefore, the bloodgases measured in our study are likely to reflect amixture of systemic venous blood and blood re-turning from the lungs. Thus, the ventral aorticblood gases reported here are unlikely to repre-sent arterial systemic blood. Arterial blood isnormally sampled for descriptions of cardiorespi-ratory response curves. However, the location ofoxygen-sensitive chemoreceptors within the car-diovascular system are unknown in lungfish andthe primary purposes of cannulation was to moni-tor the manipulation of blood oxygen carryingcapacity and to establish whether vascular PO2levels were significantly affected. In this context,the collected blood samples answered the ques-tions addressed.

4.2. Ventilatory pattern and responses to hypoxia inwater and air

During normoxia, and when undisturbed, Lepi-dosiren remained quietly submerged and surfacedat regular intervals to breathe. Each breath con-sisted of a single prolonged and continuous expi-ration that was followed immediately by a bout of

Žconsecutive small inspirations between 5 and 14,.with a mean of approx. 9 inspirations . The same

breathing pattern was recently observed in caecil-Ž .ian amphibians Gardner et al., 2000 and resem-

bles the inflation bouts of anuran amphibiansŽ .Kruhøffer et al., 1987 . From a functional pointof view the repeated inspirations reduce the func-tional dead space. In the African lungfish, Pro-topterus, the inspirations are caused by a buccal

Žforce pump mechanism McMahon, 1969; De-.laney and Fishman, 1977 and visual inspection

during the present study suggests that this is alsothe case for Lepidosiren. During hypoxia, the ven-tilatory pattern continued to consist of singlebreaths and the increased overall lung ventilationwas accomplished by a reduction of the breath-hold periods, whereas tidal volume only increasedvery slightly. This points to an important role ofpulmonary stretch receptor feedback exerting theHering�Breuer reflex, where inflation reflexlyterminates inspiration. Protopterus possesses pul-

Ž .monary stretch receptors Delaney et al., 1983 ,and manipulation of lung volume alters the dura-

Žtion of the non-ventilatory periods Pack et al.,.1992 . Stretch receptors in the air-breathing or-

gans are also important in determining tidalvolume and breathing pattern in other air breath-


Fig. 3. The effect of hypoxia and anaemia on ventilation and heart rate in lungfish, plotted against mean levels of oxygen partialŽ . Ž . Ž .pressure in the ventral aorta P O . While tidal volume V was unaffected, ventilation rate f increased during hypoxia, causingVA 2 T R

Ž .a proportional increase in ventilation volume V . Heart rate was unaffected by hypoxia, but increased by 10% in anaemic fish.I

Žing fish such as Amia and Lepisosteus Johansenet al., 1970; Smatresk and Cameron, 1982; Hedrick

.and Jones, 1999 . As pointed out by Hedrick andŽ .Jones 1993, 1999 , the control of tidal volume is

particularly important for aquatic animals wherethe degree of lung inflation significantly affectsbuoyancy.

In most species of teleost fish, gill ventilationincreases markedly during aquatic hypoxia, due tostimulation of receptors that are located princi-

Žpally on the first gill arch Daxboeck and Hole-.ton, 1978; Milsom and Brill, 1986 . Some of these

receptors are located externally and screen theinspired water, while others are internal and mon-

Žitor blood O levels Milsom and Brill, 1986;2Smatresk et al., 1986; Burleson and Smatresk,

.1990; Burleson et al., 1992 . As a consequence,ventilatory responses to hypoxic water may occur

Žwithout any changes in blood O levels Glass et2.al., 1990; Soncini and Glass, 2000 . Pulmonary

ventilation in Lepidosiren did not increase in re-sponse to aquatic hypoxia and it does not appear,

therefore, that branchial O sensitive receptors2are involved in the control of pulmonary ventila-tion in this animal. The existence of externalreceptors cannot, however, be excluded, since ad-dition of nicotine or cyanide into the opercularcavity stimulates both branchial and pulmonary

Žventilation in Protopterus Johansen et al., 1968a;.Lahiri et al., 1970 . The inability of aquatic hy-

poxia to stimulate lung ventilation is consistentwith responses of Neoceratodus and Protopterus,the air-breathing teleosts Channa argus and Elec-trophorus electricus, and the aquatic caecilian Ty-

Žphlonectus natans Johansen et al., 1967, 1968b;Johansen and Lenfant, 1968; Glass et al., 1986;

.Gardner et al., 2000 . However, in Amia, An-cistris, Hypostomus and Lepisosteus, stimulation ofexternal oxygen sensitive receptors stimulate

Žbreathing Johansen et al., 1970; Graham andBaird, 1982; Smatresk et al., 1986; Hedrick and

.Jones, 1993 .The ventilatory response to aerial hypoxia con-

cords with the effects observed in the other species


Žof lungfish e.g. Jesse et al., 1967; Johansen et al.,1967; Johansen and Lenfant, 1968; Fritsche et al.,

.1993 . These responses must be attributed to in-ternal oxygen sensitive receptors, most likely sens-ing blood PO , but the specific location of these2

Ž .remain unknown Fishman et al., 1989 . It is notsurprising that Lepidosiren resembles the othergenera of lungfishes in this context, as the mem-bers of the Lepidosirenidae are obligate airbreathers, while the other genera are more or lessfacultative air-breathers. According to Johansen

Ž .et al. 1976 , adult specimens of Protopterus ob-tain 70% of total O uptake by pulmonary venti-2lation. Because the gills of Lepidosiren are con-siderably reduced so that the filaments are nearly

Žabsent on the first two gill arches Johansen and.Lenfant, 1967; Lenfant et al., 1970 , it would be

expected that branchial gas exchange is lowerthan in any other species of lungfish. However,

Ž .Abe and Steffensen 1996 showed that almost40% of the oxygen uptake of quietly resting Lepi-dosiren occur over the skin and gills.

Pulmonary ventilation decreased during expo-Ž .sure to hyperoxia in Lepidosiren Fig. 1 , showing

that oxygen provides a tonic drive to ventilationin normoxia. Similar effects have been observed

Ž .in Protopterus Jesse et al., 1967 , other air-Žbreathing fish, amphibians and reptiles e.g.

Lomholt and Johansen, 1974; Heisler, 1982;Hughes and Singh, 1971; Smatresk et al., 1986;

.McKenzie et al., 1992 . Gill ventilation in teleostsis also reduced during hyperoxia.

4.3. Heart rate during hypoxia

In Lepidosiren and Protopterus, earlier studieshave shown an increased heart rate during the

Žintermittent breathing episodes Axelsson et al.,.1989; Burggren and Johansen, 1986 and Fritsche

Ž .et al. 1993 also recorded increased pulmonaryblood flow associated with lung ventilation inNeoceratodus. In our study there were no appar-ent changes in heart rate during ventilation. Theheart rates recorded in the present study are ingeneral agreement with previous studies on Lepi-

Ž .dosiren Axelsson et al., 1989 , but as pointed outby these authors, these heart rates appear rela-

Ž .tively high. In the study by Axelsson et al. 1989 ,the respiratory-related tachycardia did occur inanimals with low heart rate, possibly due to a

larger vagal tone on the heart that can be re-leased during ventilation.

There was no significant change in heart rateŽ .during hypoxia in Lepidosiren Fig. 3 . This in

agreement with earlier observations on Neocera-todus, although this species does increase pulmo-nary blood flow, possibly due to redistribution ofblood, during progressive aquatic hypoxiaŽ .Fritsche et al., 1993 . A reflex tachycardia inresponse to a reduced oxygen supply is character-istic of committed lung breathers such as mam-mals that show the so-called ‘secondary response’to hypoxia following stimulation of lung stretch

Žreceptors during hypoxic hyperventilation Daly,.1997 . In this respect, Lepidosiren shows physio-

logical responses that are typical for lungbreathers possessing pulmonary mechanorecep-

Ž .tors Delaney et al., 1983 . However, becauseheart rate remained elevated during the breathhold periods in the anaemic animals, stimulationof pulmonary stretch receptors is not obligatoryfor the hypoxaemic tachycardia. Lepidosiren didshow a significant tachycardia in normoxic hypox-aemia. The cardiac responses of lungfish are dia-metrically opposed to that of typical water-breath-ing fish, where environmental hypoxia normally

Ž .induces a reflex bradycardia Taylor, 1992 . Thisresponse is predominantly in response to stimula-tion of externally located branchial oxygen recep-

Ž .tors sensitive to water PO Burleson et al., 1992 .2Thus, the absence of a hypoxic bradycardia is inagreement with the lack of evidence for externalreceptors on the gills of Lepidosiren.

4.4. Modality of the oxygen response and the effectsof reducing haematocrit

The specific oxygen stimulus that elicits ventila-tory responses in vertebrates is a matter of con-

Ž .tention e.g. Boggs, 1995 , but most studies onair-breathing vertebrates point to PO being the2

Ždriving stimulus e.g. Lahiri et al., 1981; Wang etal., 1994, 1997; Boggs, 1995; McKenzie et al.,

.1991 . The responses of Lepidosiren are consis-tent with this view, as the large reduction inhaematocrit and ventral aortic oxygen contentduring normoxia did not augment ventilationabove the normoxic values previously attainedŽ .Figs. 2 and 3 . The fact that the lungfish studiedin series 1 maintained a brisk hypoxic ventilatoryresponse during the second hypoxic exposure


shows that they are still able to respond to reduc-tion in partial pressure. Thus, from our data itappears that the chemoreceptors responsible forthe hypoxic ventilatory response are sensitive toPO and that the lungfish resembles other air-2breathing vertebrates, in this respect. Again, thehypoxic responses of Lepidosiren are opposed totypical, water-breathing fish, where systemic hy-poxaemia, even in the absence of an associated

Ž .hypoxia e.g. anaemia appears to stimulate venti-Ž .lation Randall, 1982; Smith and Jones, 1982 .

Heart rate increased following the reduction inŽ .haematocrit in Lepidosiren Fig. 3 . A tachycardia

in response to lowered blood oxygen content, inthe absence or presence of ventilatory changes,appears to be a general response of water and

Žair-breathing vertebrates e.g. Wood and Shelton,.1980; Boggs, 1995; Wang et al., 1994, 1997 . In

mammals, this response may arise from stimula-Žtion of chemoreceptors in the aortic arch Daly,

.1997 and it seems that other vertebrates alsopossess oxygen sensitive chemoreceptors that pri-marily exert a cardiovascular control. However, instudies that involve withdrawal of blood, it isdifficult to dismiss the possibility that the heartrate changes are due to regulation of blood pres-sure rather than stimulation of oxygen-sensitivechemoreceptors per se.

4.5. Perspecti�es and conclusions

These and previous studies demonstrate thatthe location with respect to the oxygen cascadeand the functional characteristics of oxygen sensi-tive receptors controlling the heart and ventila-tion, plus the stimulus modality of these recep-tors, are similar in lungfish and tetrapods. Thus,many of the changes in the mechanisms exertingreflex control over cardio-respiratory functions,accompanying the transition between water andair breathing, may have occurred at an early stagein vertebrate evolution.

Acknowledgements

ŽThis study was supported by FAPESP Proc.. �no. 1998�06731-5 and CNPq Proc. no.

Ž .�300603�91-6 RN and The Danish ResearchŽ .Council SNF . We gratefully acknowledge tech-

nical help from Humberto Giusti.

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the differential cardio-respiratory responses to ambient hypoxia and systemic hypoxaemia in the...

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