375

J. Physiol. (1976), 261, pp. 375-386 With 4 text-figure8 Printed in Great Britain

RESPIRATORY AND THERMOREGULATORY RESPONSES OF RABBITS BREATHING CARBON DIOXIDE DURING HEAT EXPOSURE

BY M. MASKREY AND S. C. NICOL From the Department of Physiology, University of Tasmania, G.P.O. Box 252C, Hobart, Tasmania 7001, Awstralia

(Received 24 February 1976) SUMMARY

1. Rabbits were clipped and exposed in turn to three environmental conditions: control (C), cold exposure (CE) and water deprivation (WD). Following each type of treatment, the rabbits were exposed to an ambient temperature (T&) of 35 'C for 1 hr. Throughout this period they breathed either normal atmospheric air or 6 % C02 in air. 2. During heat exposure, measurements were made of the respiratory responses and of the 02 consumption (Iv) of the rabbits. Rectal temperature (Tle) was measured immediately before and again immediately after heat exposure. 3. When subjected to cold exposure or water deprivation the rabbits showed an initial decrease in respiratory frequency (RF) and an initial increase in VT when compared with controls. There was no difference in JE. Rabbits breathing 6 % C02 showed an increase in VT and PE and a decrease in RF when compared with rabbits breathing atmospheric air. In all cases a change in VT or RF was associated with a reciprocal change in the other parameter. 4. The respiratory responses to breathing 6 % C02 were essentially similar in treated and control rabbits, from which it is concluded that neither cold exposure nor water deprivation alter the sensitivity of the medullary respiratory centre to the respiratory drive from the central chemosensors. 5. The increase in Tre during heat exposure was significantly less in rabbits breathing 6 % C02 than in rabbits breathing atmospheric air. between rabHowever, there was no significant over-all difference in bits breathing C02 and those breathing air. From this it is concluded that increased ventilation induced by C02 causes a greater dissipation of heat than does thermally-induced panting. 6. It is concluded that VT is controlled by the level of blood Pco,

ro,

M. MASKREY AND S. C. NICOL 376 whereas RF is controlled by thermoregulatory requirements. It is further concluded that the reciprocal relationship between VT and RF is regulated in such a way as to maintain IE at the appropriate level for effecting gaseous exchange and evaporative heat loss. INTRODUCTION

In animals which pant the respiratory system subserves two separate functions: respiratory gas exchange and temperature regulation. Thus respiration is controlled by inputs from the chemosensors and from thermoregulatory inputs. In only a few studies have animals been exposed simultaneously to high levels of CO2 and to a high ambient temperature (T.) and the majority of such observations have been made on the anaesthetized dog (e.g. Anrep & Hammouda, 1933; Albers, 1961; Kappey, Albers & Schmidt, 1962). The respiratory centre is also influenced by long term effects. Thus Maskrey & Nicol (1975), showed that thermal tachypnoea in the rabbit could be blocked by previous cold exposure or by water deprivation. Questions concerning the site of this block are still to be resolved. The main purpose of the present study is to investigate the effects on respiration of the interaction between the demands for gaseous exchange and for heat loss in rabbits exposed to a hot environment to which CO2 has been added. In addition it was hoped to observe whether this interaction is modified by previous cold exposure or water deprivation. METHODS Six rabbits of both sexes comprising animals of the New Zealand White and Himalayan breeds were used in the study. Their body weights were 3 0-5-0 kg. At the beginning of the investigation, the fur was removed from the trunk and hind quarters of all rabbits. The animals were then kept in this condition by regular clipping throughout the experimental period. Between experiments the animals were individually housed in wire cages at a T. of 20 ± 2° C. Rabbits were fed on dry bran pellets throughout. Each of the six rabbits were subjected in turn to three different sets of conditions as follows: Controls (C). Under control conditions, rabbits received water ad libitum up until the time of the experiment. Until the time of testing the animals remained housed at 200 C. Cold-exposure (CE). Cold-exposed rabbits also received water ad libitum up until the time of the experiment. Immediately before testing they were kept for 24 hr in a climatic chamber at T. of 50 C. Water-deprivation (WD). Under conditions of water deprivation rabbits were denied drinking water for 72 hr before testing. This treatment causes significant dehydration (Turlejska-Stelmasiak, 1974; Maskrey & Nicol, 1975). Water-deprived rabbits were kept at 200 C. Each rabbit was then tested singly, the following procedure being adopted. The rectal temperature (T,.) of the rabbit was measured using a mini Z electro-

RESPONSES TO C02 DURING HEAT EXPOSURE

377

thermometer (model MZ-C) after which the animal was immediately placed in a water-jacketed metabolic chamber in which the T. was maintained at 35 +±1 C with a relative humidity ofbetween 60 and 70 %. The composition ofthe inlet gas to the chamber was controlled using two flowmeters, one supplying atmospheric air, and the other pure CO2. The settings of the flowmeters were adjusted so that the chamber received either normal atmospheric air or 6 ± 1 % CO2 in air. The percentage of CO2 in the outlet air was monitored by means of a Morgan infrared CO2 analyser. The rabbits remained in the chamber for exactly 1 hr during which period their respiratory responses and 02 consumption (To2) were measured as follows. Respiratory responses. Respiratory responses were measured by whole-body plethysmography. Changes in pressure within the chamber were measured with a Kyowa type PG-IOGC pressure transducer. The respiratory excusions were displayed on a Rikadenki type T02NI-H flat bed recorder. VT was calculated using the equation derived by Drorbaugh & Fenn (1955). 02 consumption. VO2 was recorded using an open circuit technique. Outlet air from the metabolic chamber was dried and passed through a Servomex OA. 184 02 analyser used in the ratio mode with an 02 range of 16-21 %. The difference between outlet 02 concentration and atmospheric 02 concentration was displayed on a Rikadenki B341 potentiometric recorder, as were chamber temperature and outlet CO2 concentration. When rabbits were breathing atmospheric air their Io2 was calculated using the tables of Hill (1972). When the inlet air contained added CO2, 702 was calculated as set out below. The symbols used are in accordance with the usage of Hill (1972) with some additions: VO2 the 02 consumption of the animal in volume of dry 02 at s.t.p. per unit time, T, °2 the volume of 02 entering the metabolic chamber, °2 the volume of 02 leaving the metabolic chamber, RE total the total volume of all gases leaving the metabolic chamber, T, air the volume of atmospheric air entering the chamber through the air flowmeter, RI Co2 the volume of CO2 entering the chamber through the CO2 flowmeter, tVMg CO2 the volume of CO2 produced by the animal, FI, 02 the fractional concentration of oxygen in atmospheric air (= 0.20953), FE 02 the fractional concentration of 02 in the outlet air, FE CO2 the fractional concentration of CO2 in the outlet air, R.Q. the respiratory quotient of the animal as estimated from the data of Lee (1939). (01) V-02 VI, and because 02 is entering via the air flowmeter only

V,

0°2

=

Now 1

total

And

Fl.02 ;,air FE,02

(2)

Etotal'

I,air+ IC02 V02 + 1,C02 -

(3)

V2

M.C2

(4)

=

02

R.Q.(4

Substituting for V02 in eqn. (3) 7E, total

VI,,air + 7IC2

R Q. + VXC02

(5)

378

M. MASKREY AND S. C. NICOL

Now

co. +

I'M, CO

(6)

=

Therefore,

Substituting for

rmco2

M7,C002 JI,total E C02 JCO in eqn. (5), and rearranging terms: TYtt

Substituting for

Et

(7)

aIr+ (co2,RQ.)

(8

I-FEco, + (FE,co,/R.Q.)

P:toi in eqn. (2) 1O = 0-20953 VTI-F 0E 2R.!(9) B. 2 (1c+(1sIR.Q.)\ I--F c2(E

Following heat exposure, the rabbits were removed from the metabolic chamber and their Tr. again measured. In the analysis of the results the significance of differences between means was computed using Student's t test.

TALE 1. A summary of the respiratory responses obtained from control (C), coldexposed (CE) and water-deprived (W]) rabbits breathing either atmospheric air or 6 % C02 in air during the first 30 min and the second 30 min of exposure to 35° C lst 30min 2nd 30 min RF

RF

(breaths.

VT

min-Il)

(ml.)

Rabbits breathing air C 235±46 5-7±0-5 CE 116 20 13-1 1-8 WD 102±26 12-7+ 1-5 All 151+23 10-5+1-1 Rabbits breathing 6 % CO2 C 139+ 28 21-2± 4-1 CE 91+16 26-0+4-5 WD 66 ± 10 35-7 ± 4-9 All 99+ 13 28-6+2-8

JIB

(1. mi-')

(breaths.

min-')

VT (Mi)

1-23±0-18 297±48

4-3±0-4

0-13 273 40 1-04+0-08 261+44 1-19+0-09 277±37

5-9 +1-2 5-5+ 1-3

1-31

2-44± 0-25 2-27+0-25 2-25 ± 0-36 2-32+0-16

153 ± 29 114+19 125± 23 131+21

(1.

JI

min')

5-3+0-7

1-19±0-12 1-42+ 0-15 0-99+0-11 1-20±0-08

19-4± 3-2 25-1+4-1 22-3+ 3-3 22-3+2-1

2-46 ± 0-30 2-20±0-21 2-40 ± 0-38 2-35±0-23

The figures shown are means + s.E. of mean. RESULTS

The results are summarized in Tables 1 and 2. Respiratory responses When breathing atmospheric air, both cold-exposed and water-deprived rabbits showed a significant decrease in RF (P < 0-05) and a significant increase in VT (P < 0-01) when compared with controls. This was the case for the first 30 min of heat exposure only (Fig. 1A, B). During the second 30 min of exposure there was no significant difference between

RESPONSES TO C02 DURING HEAT EXPOSURE 379 cold-exposed, water-deprived and control rabbits with respect to RF or

VT.

rE following cold exposure or water deprivation did not differ significantly from controls. This held true throughout the whole period of heat exposure (Fig. 1C). A

C

B 151-5

300 E

C200

100

0

CC

E

~~~~~E

C

0

E 10

0-5

5

C CE WD

0

C CE WD

0

C CE WD

Fig. 1. Respiratory responses of rabbits breathing atmospheric air during the first 30 min of heat exposure (T. 350 C). In this and following Figures: C stands for control rabbits; CE, cold-exposed; and WD, water-deprived. The histograms represent mean values and the bars indicate s.E. of mean. A, respiratory frequency. B, tidal volume. C, minute volume.

When breathing 6 % CO2 rabbits showed a significant decrease in RF (P < 0.05) and a highly significant increase in VT (P < 0-001) compared with when breathing air (Fig. 2A, B). This led to a significant increase in JE (P < 0.05) in rabbits breathing 6 % C02 (Fig. 20C). Rectal temperatures The mean initial Tre was significantly reduced in cold-exposed rabbits when compared with controls (P < 0-05), whereas there was no significant difference in initial T between rabbits under control conditions and under water-deprived conditions. All rabbits showed a moderate increase in 1Te during the I hr at 35° C. This increase was significantly less when rabbits breathed 6 % C02 than when they breathed atmospheric air (P < 0-05). This is illustrated in Fig. 3.

380

M. MASKREY AND S. C. NICOL

02 consumption Previously cold-exposed rabbits breathing atmospheric air showed a significant increase in J2 compared with controls (P < 0.05). However, in cold-exposed rabbits breathing 6 % CO2 this difference was not signifi-

a

A

I

300 IC

301-

0

D 200 U C

_

3-01-

I

E 20 0 E

I

tr

T.L I^ E

-II

2-01-

I

El

lI

I

0

"

C

B

I

C

100

lo

I l

1-01

-

._

0

Air

Co2

-

0

Air

CO2

-

0I

Air

CO2

Fig. 2. Comparison of the respiratory responses of rabbits when breathing atmospheric air (open columns) with those when breathing 6% CO2 in air (cross-hatched columns). In each case the first column shows results from the first 30 min of heat exposure (T. 350 C) while the second column shows results from the second 30 min of heat exposure. A, respiratory frequency. B, tidal volume. C, minute volume. TABLE 2. A summary of the rectal temperature and 02 consumption measurements obtained from control (C), cold-exposed (CE) and water-deprived (WD) rabbits exposed to 350 C and breathing either atmospheric air of 6 % CO2 Initial Tr. (O C) C 39-01 ± 0-07 Rabbits breathing air CE 38-69±0-09 WD 38-90±0-06 Rabbits breathing 6 %

ATr.

CO2

C CE WD C CE WD

r0

(O C) (ml g-' hr-1) 0-72 ± 0-08 0-483 ± 0-029 0-80+0-10 0-615 ± 0-016

0-85±0-05 0-448 + 0-027 0-58 + 0-08 0-473 + 0-031 0-70 + 0-10 0-537 + 0-032 0-57 ± 0-07 0-403 ± 0-026

The figures shown are means + s.E. of mean.

RESPONSES TO C02 DURING HEAT EXPOSURE 381 cant. Water deprivation did not significantly alter 1,, neither did breathing 6 % C02 significantly alter J0 under control or water-deprived conditions. These results are illustrated in Fig. 4.

1*0 I-

1 I I

G 1-

I

I I 05F-

I 0

C

CE WDI C CE WD Air CO2

Fig. 3. Change in rectal temperature of rabbits breathing atmospheric air (open columns) and breathing 6% CO in air (cross-hatched columns) while exposed to an ambient temperature of 350 C. DISCUSSION

The results show that in rabbits hypercapnia during exposure to 350 C causes an increase in VT and EX, and a decrease in RF, while reducing the rise in Tr which normally occurs during heat exposure. It appears that RF and VT are controlled by separate inputs, which interact to control 1E. Thus the inhibition of thermal tachypnoea during the first 30 min of heat exposure in rabbits previously subjected to cold exposure or to water deprivation, which was reported previously (Maskrey & Nicol, 1975), is shown in the present study to cause no change inl r. This is because the reduction in RF is compensated by a ris3 in VT. Similarly, while exposure to C02 caused an increase in 1E under all experimental conditions by increasing VT, this was accompanied by a fall in RF. During the second 13

PHY 261

382 M. MASKREY AND S. C. NICOL 30 mi of heat exposure all rabbits breathing atmospheric air were panting, while the rise in VT seen in hypercapnic animals was accompanied by an inhibition of thermal tachypnoea. The findings on RF agree with those for the anaesthetized dog (Anrep & Hammouda, 1933; Albers, 1961) and for the conscious dog (Albers, Usinger & Scholand, 1975). Jennings & Macklin (1972) reported that unanaesthetized panting dogs always showed

06

K

T 011

T A5

II

I

II II

I-

I

III

LE 0-41-

II

I I

C

II

0

9 I

a. E

I

C

8

0

I I

0-2

_I-IF

III-1

0

|I

h

I

I

C

CE WD C CE WD Air CO2 of rabbits Fig. 4. °2 consumption breathing atmospheric air (unshaded columns) and breathing 6 % CO2 in air (cross-hatched columns) while exposed to an ambient temperature of 350 C.

a decrease in RF on breathing CO2 whereas non-panting dogs exposed to comparable environmental conditions either showed no change or a transient rise in RF. The effect of hypercapnia in the absence of panting has also been studied in decerebrate cats (Rosenstein & Borison, 1963) anaesthetized cats (Widdicombe & Winning, 1974) and conscious kangaroo rats (Soholt, Yousef & Dill, 1973). In all these cases hypercapnia produced a rise in RF, but the first two are not physiologically comparable with the present study, while the kangaroo rat is a species which does not pant. The reduction in RF in the rabbit during hypercapnia is not dependent on prior cold exposure or water deprivation as control animals at

RESPONSES TO C02 DURING HEAT EXPOSURE 383 lower ambient temperatures showed a fall rather than a rise in RF when breathing CO2 (M. Maskrey & S. C. Nicol, unpublished observations). While there was some increase in RF from the first to the second 30 mm in rabbits breathing C02, this was accompanied by a fall in VT, and rE remained unchanged. Thus there appears to be a reciprocal relation between RF and VT, a rise or fall in one being normally accompanied by an opposite change in the other, hence tending to reduce the change in VE. This could be a mechanical effect in which RF is regulated through the Hering-Breuer reflex (Tang, 1967), or due to high C02 concentrations causing a direct inhibition of RF as well as stimulating VT. However, these results can be explained if the principal controller of VT is Pco2, while RF is mainly determined by temperature. This arrangement has previously been suggested by Bligh (1973) following the observations of Bligh & Allen (1970) on shorn sheep. Removal of excess C02 is accomplished by increasing alveolar ventilation. Thus hypercapnia causes an increase in rE by increasing VT through the direct action of C02 on the medullary respiratory centre. An increase in body temperature acts through the hypothalamic temperature regulating centre to increase respiratory evaporative heat loss by increasing E. An increase in alveolar ventilation would produce hypocapnia, resulting in a reduction of tidal volume. Thus the effect of heat stress is to increase RF and decrease VT, producing an increased opportunity for evaporative heat loss while minimizing the effects on blood Pco0. During C02 exposure a reduction of VT due to hypocapnia cannot occur, and moreover, the hypercapnic drive produces a rise in 1E by increasing VT. Rabbits breathing 6 % C02 showed a significantly smaller increase in Tre during heat exposure than did rabbits breathing atmospheric air. This difference could theoretically have been brought about either by a decrease in heat production by the animal or by an increase in heat loss. C02 has been shown to reduce body temperature in the rat (Szegvari & Varnai, 1962) the kangaroo rat (Soholt et al. 1973) and the guinea-pig (Schaefer, Messier, Morgan & Baker, 1975). In each of these reports the fall in body temperature was accompanied by a fall in t°2The increase in previously cold-exposed rabbits over that measured for controls has already been reported (Maskrey & Nicol, 1975). In the present study, C02 inhalation has been shown to reduce this difference. However, the io, in animals breathing 6 % C02 was over-all not significantly different from the io, in animals breathing atmospheric air. From this it would appear unlikely that C02 inhalation for 1 hr causes a significant change in heat production in the rabbit exposed to 35° C.

VO2in

13-2

384 M. MASKREY AND S. C. NICOL In unxanaesthetized dogs exposed to 400 C, the increase in body temperature was less when the animals breathed 4 % C02 than when they breathed atmospheric air, despite the fact that RF was reduced (Albers et al. 1975). The authors suggested that ventilatory heat dissipation in the dogs had become more effective when respiration was stimulated by CO2. It would appear from the results of the present study that rabbits react in the same way. The increased effectiveness of C02-induced hyperventilation in the dissipation of heat is probably due to the increased involvement of alveolar air in addition to dead space ventilation. This is made possible by the removal of the hypocapnic inhibition of VT. By increasing heat dissipation and thus decreasing the rate of rise of body temperature, the C02-induced hyperventilation will reduce the thermal stimulus to increase RE. Thus RF in animals breathing C02 will be lower than in animals breathing atmospheric air. An increase in alveolar ventilation also occurs in the second stage panting seen in some severely heat stressed animals: the rapid shallow breathing pattern of first stage panting is superseded by slower deeper breathing (Hales, 1967; Hales & Webster, 1967). In this case the increased alveolar ventilation causes disturbances of blood gas concentrations (Hales & Webster, 1967; Hales & Findlay, 1968). Presumably the heat stress is the dominant influence on the respiratory centre during second stage panting, and the hypocapnia resulting from the increase in alveolar ventilation is then unable to exert a compensatory influence through its effect on the chemoreceptors. These aspects have been discussed in some detail by Hales (1974). The fact that neither cold exposure nor water deprivation significantly affect the response to C02 shows that these blocks do not alter the sensitivity of the central chemoreceptors but act on the component of the respiratory drive originating in the hypothalamus. The effect of this drive is principally on RF which normally increases during heat exposure, and is decreased by prior cold exposure or dehydration. However, animals which were water-deprived or previously cold-exposed, while showing a reduction in RF during heat exposure maintained a high rE by increasing VT. Because of this, heat loss was increased even in the blocked animals and was sufficient to ensure that the rise in Tre was not significantly greater than that seen in the controls. Thus the over-all regulation of respiration can be considered as the result of the interaction of two control systems. Normally one controls blood gas concentrations by altering VT in response to changes in blood PC0 , while the other responds to thermoregulatory requirements by altering RE. However, in the rabbit at least, if the normal response of an

385 RESPONSES TO CO2 DURING HEAT EXPOSURE increase in RF at high Ta is blocked, P]E will be raised by increasing VT. Thus total ventilation is maintained at the appropriate level by the reciprocal relation between VT and RF arising from these interacting functions.

REFERENCES

ALBERS, C. (1961). Der Mechanismus des WArmehechelns beim Hund. III. Die C02Empfindlichkeit des Atemzentrums wAhrend des WArmehechelns. Pfliigers Arch. gee. Physiol. 274, 166-183. ALBERS, C., USINGER, W. & SCHOLAND, C. (1975). Intracellular pH in unanaesthetized dogs during panting. Rewp. Physiol. 23, 58-70. ANREP, G. V. &HA OUDA, M. (1933). Observations on panting. J. Physsol. 77,16-34. BLIGH, J. (1973). Temperature Regukstion in MammaeM and other Vertebrates, pp. 6768. Amsterdam: North-Holland. BLIGH, J. & ALLEN, T. E. (1970). A comparative consideration of the modes of evaporative heat loss from mammals. In Physiological and Behavioural Temperature Regulation, ed. HARDY, J. D., GAGGE, A. P. & STOLWIJK, J. A. J., pp. 97107. Springfield: Thomas. DRORBAUGH, J. E. & FENN, W. 0. (1955). A barometric method for measuring ventilation in new-born infants. Pediatrics 16, 81-87. HiAT s, J. R. S. (1967). The partition of respiratory ventilation of the panting ox. J. Physiol. 188, 45-46P. HATEs, J. R. S. (1974). Physiological Responses to Heat. In MTP International Review of Science, Physiology Series 1, vol. 7, ed. ROBERTSHAw, D.,;pp. 107-162. London: Butterworths. EHATs, J. R. S. & FuiDL&Y, J. D. (1968). Respiration of the ox: normal values and the effects of exposure to hot environments. Resp. Phys-ol. 4, 333-352. HALES, J. R. S. & WEBSTER, M. E. D. (1967). Respiratory function during thermal tachypnoea in sheep. J. Physiol. 190, 241-260. HILL, R. W. (1972). Determination of oxygen consumption by use of the paramagnetic oxygen analyser. J. appl. Physiol. 33, 261-263. JENNINGS, D. B. & MACKLIN, R. D. (1972). The effects of 02 and C02 and of ambient temperature on ventilatory patterns of dogs. Reep. Physiol. 16, 79-91. KAPPEY, P. F., ALBERS, C. & SCHMIDT, R. (1962). Die ventilatorishe CO2-Reaktion des Hundes wahrend der Wdrmetachypnoe. Pflugers Arch. gee. Physiol. 275, 312-326. LEE, R. D. (1939). Basal metabolism of the adult rabbit and prerequisites for its measurement. J. Nuir. 18, 473-488. MAsKREY, M. & NIcOL, S. C. (1975). Inhibition of thermal tachypnoea in rabbits following exposure to cold and water deprivation. J. Physiol. 252, 481-490. ROSENSTEIN, R. & BORsON, H. L. (1963). Actions of carbon dioxide and sodium salicylate on central control of respiration in cats. J. Pharmac. exp. Ther. 139, 361-367.

SCHAPEER, K. E., MESSIER, A. A., MORGAN, C. & BAR R, G. T. (1975). Effect of chronic hypercapnia on body temperature regulation. J. appl. Physiol. 38, 900906. SOHOLT, L. F., YOUSEF, M. K. & DoL, D. B. (1973). Responses of Merriam's kangaroo rats, Dipodomys merriami, to various levels of carbon dioxide concentration. Comp. Biochem. Physiol. 45A, 455-462.

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SZEGVARI, G. & VANE~i, I. (1962). The effect of hypercapnia on heat production and colonic, muscle and subcutaneous temperature in the rat. The site of thermoregulatory heat production. Acta phy8iol. hung. 22, 65-72. TuRLEJs-STELMAs, E. (1974). The influence of dehydration on heat dissipation mechanisms in the rabbit. J. Physiol., Paris 68, 5-15. WIDDICOMBE:, J. G. & WINNING, A. (1974). Effects of hypoxia, hypercapnia and changes in body temperature on the pattern of breathing in cats. Resp. Physiol. 21, 203-221.