The ventilatory response of rodents to changes in arterial oxygen content

RESPIRATION PHYSIOLOGY ELSEVIER

Respiration Physiology 96 (1994) 199-211

The ventilatory response o f rodents to changes in arterial oxygen content Rhonda J. Garland, Richard Kinkead*, William K. Milsom Department of Zoology, University of British Columbia, 6270 University Blvd., Vancouver, British Columbia, Canada

Accepted 24 January 1994

Abstract A strong correlation between the arterial oxygen partial pressure (Pa%) values for the threshold of the hypoxic ventilatory response (HVR) and the shoulder of the oxyhemoglobin equilibrium curve (OEC) is retained in heterothermic rodents as body temperature changes despite changes in hemoglobin-oxygen affinity. It has been suggested that this may reflect either temperature-induced changes in the response characteristics of arterial chemoreceptors or an ability to sense changes in arterial 0 2 content (Ca%). This study examined the extent to which changing Cao2 independent of Pa% with carbon monoxide could contribute to the HVR in heterothermic (golden-mantled ground squirrels) and non-heterothermic rodents (rats). The HVR of isocapnic, anaesthetized rodents was assessed during both hypoxic hypoxia, which alters Pa% and Cao,- simultaneously, and carbon monoxide hypoxia, which alters Cao2 independent of Pa%. While both species exhibited ventilatory responses to hypoxic hypoxia and carbon monoxide hypoxia, the HVR of the squirrel was consistently stronger than that of the rat. Reductions in Cao2 independent of Pao2 could still produce 60% of the full HVR seen with hypoxic hypoxia in both species. Simultaneous changes in Pao2, however, were necessary to produce the full response. While it seems likely that the results can be explained by the changes in tissue P% which would occur at receptor sites under the various conditions, such an explanation is not totally supported by other studies. Key words: Carbon monoxide; Control of breathing, hypoxic ventilatory response, arterial 02 content; Hypoxia, vs carbon monoxide, ventilatory response; Mammals, ground squirrel (Spermophilus lateralis, rat

1. Introduction

Exposure to reduced ambient 02 concentrations decreases both arterial oxygen content (Cao2) and partial pressure (Pao2). In m a n y species the P a % at which venti* Corresponding author. Tel.: (604) 822-5799; Fax: (604) 822-2416; E-mail: [email protected]. 0034-5687/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 3 4 - 5 6 8 7 ( 9 4 ) 0 0 0 1 8 - U

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lation begins to increase is correlated with the Pao2 of the inflection point on the oxygen equilibrium curve (OEC) where Cao2 begins to decrease significantly (for reviews, see Wood, 1984; Milsom, 1990; Webb and Milsom, 1990). It is generally assumed that the stimulus for arterial chemoreceptors arises from the change in Pao2. Although the correlation between the threshold of the hypoxic ventilatory response (HVR) and the inflection point on the OEC is suggestive of the presence of chemoreceptors responsive to changes in Cao2, most authors have concluded that this correlation is a consequence of natural selection acting on the Pao~ threshold of the HVR in a manner which optimally protects arterial oxygen saturation (Van Nice et aL, 1980). Birehard and Tenney (1986) directly investigated the correlation between Pao2, Ca% and the HVR by experimentally altering the intraspecific P% at which blood is 50~o saturated (Ps0) in rats by infusion of sodium cyanate. These authors found that, despite a leftshifted OEC accompanied by a 10 Torr decrease in the intraspecific Ps0, the Pa% threshold of the HVR was unchanged, supporting the idea that the HVR is mediated solely by changes in Pao2. In contrast to the findings of Birchard and Tenney (1986), however, the relationship between the threshold of the HVR and the inflection point of the OEC is retained in species that experience natural fluctuations in Ps0. As body temperature fails in ectotherms (Glass et al., 1983) and hibernating endotherms (McArthur and Milsom, 1991) the hypoxic threshold shifts to increasingly lower values of Pao:, the values at which arterial blood begins to desaturate. It is possible that temperature-induced changes in the response characteristics of arterial chemoreceptors could account for this variation in the hypoxic threshold. It is also possible that these species possess chemoreceptors capable of responding to changes in Cao~. It has been suggested that for animals that undergo wide fluctuations in body temperature and exhibit a left-shifted OEC accompanied by an intraspecific decrease in Ps0, Cao: would be a better indicator of the oxygen status of the blood than would Pao2 (Wood, 1984). Based on these observations, the present study was undertaken to test the hypothesis that heterothermic rodents do respond to changes in Cao~ alone. To this end, it examined the comparative ventilatory responses to concurrent decreases in Pao: and Cao: as well as to decreases in Cao: alone, of rodent species which can (golden-mantled ground squirrels) and cannot (rats) hibernate.

2. Materials and methods

Adult Wistar rats (Rattus norvegieus; mean weight 247.8 g + 6.6 g) and goldenmantled ground squirrels (Spermophilus lateralis; mean weight 212.4 g _+ 8.8 g) of either sex were obtained from a commercial supplier. The animals were housed in a controlled-environment chamber at an ambient temperature of 20 ° C + 1 ° C under a 12L: 12D photoperiod and provided with food and water ad libitum. Surgicalprocedures Both species were initially induced into a light plane of anaesthesia with 3.5 ~o halothane in air. Intraperitoneal injections of sodium pentobarbital (rats: 6.5 mg/100 g; squirrels: 9.5 mg/100 g) and atropine sulfate (0.35 mg/100 g) were then

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administered to maintain anaesthesia and minimize mucus secretion, respectively. Following exposure of the trachea by blunt dissection, a small incision was made between two cartilaginous rings and a 3 cm length of polyethylene tubing (PE-240) was inserted approximately 1 cm into the trachea and secured in place. Both femoral arteries and one femoral vein were then exposed and cannulated with polyethylene tubing (PE-50) filled with heparinized saline. Body temperature was monitored with a rectal thermistor and servo-controlled at 37 °C with an infrared heat lamp. Following the surgery, the animals were placed supine in a 6 L plexiglass chamber with ports on either end to allow gas flow through the chamber. A small pneumotachograph was attached to the tracheotomy tube and the differential pressure changes arising from air flow during ventilation were monitored with a differential pressure transducer (Validyne model DP 103-18). This signal was amplified and electronically integrated (Gould Universal Amplifier and Integrating Amplifier) to give tidal volume. The system was calibrated by injecting known volumes of air into the pneumotachograph. One arterial cannula was connected to a pressure transducer to monitor arterial blood pressure. The other arterial cannula was connected in series to the venous cannula via O2, CO2 and pH microelectrodes. Continuous movement of arterial blood through the extracorporeal loop and back into the animal through the venous cannula was ensured by a peristaltic pump (Gilson) operating at a rate of 1 ml/min. A constant plane of anaesthesia, indicated by a weak limb-withdrawal reflex, was maintained throughout the experimental procedure using supplements of sodium pentobarbital (1.0-3.5 mg/30 min). Experimentalprocedures

Arterial Po2, Pco2 and pH were continuously measured with miniature 02, CO 2 and pH electrodes (Microelectrodes) contained in a thermostatted cuvette and displayed on a Radiometer PHM-73 pH/blood gas monitor. The Pc% electrode was calibrated using water equilibrated with pre-analyzed gas mixtures (Medigas). The calibration of the Po2 electrode utilized water equilibrated with pre-analyzed gas mixtures as well as 5 mM sodium bisulfate while precision buffers (Radiometer) were used to calibrate the pH electrode. All calibrations and measurements were done at 37 °C with all solutions passing through the electrodes at a rate of 1 ml/min. Measurements of total Ca% were made on 20 #1 samples of blood according to the method of Tucker (1967) using a Radiometer P% electrode in a sealed chamber at 37 ° C. The gas mixtures administered to the animals were obtained by mixing air with 02, N2, CO2 or CO with flow meters. Prior to reaching the experimental chamber, the gas mixture was hydrated by bubbling it through a flask of water. The experimental gas mixtures were also monitored continuously using a Beckman OM-11 O2 analyzer and LB-2 CO2 analyzer calibrated with pre-analyzed gas mixtures. The differential pressure, tidal volume, Pao2, Paco: and pHa and arterial blood pressure were all displayed continuously on a chart recorder (Gould). Analytical procedures

The animals were maintained under normoxic normocapnic conditions until blood gas and respiratory variables stabilized. For each species, the animals were divided into two groups. The first group was made progressively hypoxic

Experimental protocol

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by decreasing the fraction of inspired 02 (FIo 2) in the air through the addition of N 2 (hypoxic hypoxia). In the second group hypoxia was induced through the addition of carbon monoxide to the air (CO-hypoxia). In both groups the animals were maintained isocapnic by the addition or removal of CO2 from the gas mixture. Animals exposed to CO-hypoxia were maintained isoxic through alterations of the N 2 concentration in the gas mixture. At each progressive level of graded bypoxia sufficient time (approximately 5 min) was allowed for all variables to reach new stable levels. At that time, approximately 30 #1 of blood was collected for the determination of hematocrit and oxygen content. The level of hypoxia was increased until the 02 saturation of arterial blood decreased to approximately 50~o. On average, each animal was subjected to 4 intermediate levels of hypoxia en route to this final level. In order to broaden the Pao2 range, some animals were initially exposed to hyperoxia. In those cases, all variables were allowed to return to normoxic values before the onset of the experiment. Approximately 30 sec of breathing trace was recorded at high speed (5 mm/sec) at each level of hypoxia. Breathing frequency (fR) was determined for each 10-s interval, tidal volume (VT) was measured for fifteen consecutive breaths and ventilation (VE) was calculated as the product of the mean fR and the mean VT. Inter- and intraspecific comparisons between treatments were performed using two way ANOVAs and Tukey post hoe tests. Intraspecific comparisons within each treatment utilized paired t-tests. The fiducial limit of significance was set at P < 0.05.

Data analysis

3. Results

The mean values of blood gas and respiratory variables for rats and squirrels breathing air before exposure to either hypoxic hypoxia or CO-hypoxia are summarized in Table 1. There were no significant intraspecific differences between groups in any of the variables. Interspecific comparisons revealed that fR and VE of rats were consistently higher than those of squirrels in both groups.

Normoxia

Reducing the FIo2 from normoxia in the experimental gas mixture resulted in a decrease in Pao2 from 63 to 23 Tort and from 63 to 29 Torr in the squirrel and rat, respectively. Concurrent reductions in Cao2 from 27.0 to 13.5 ml O2/100 ml in the squirrel and 24.7 to 15.8 ml O2/100 ml in the rat (Table 1) confirmed that comparable levels of hypoxemia were produced in both species. The effects of changing both Pao2 and Cao.2 on ventilation in both rodent species are shown in Fig. 1. The relationships between VE, fg and VT and Pao~ are all nonlinear. The mean ~ZErose from the normoxic value of 35.3 to 253.7 ml/(min" 100 g) at the most severe level of hypoxia in the squirrels and from 58.3 to 178.7 ml/(min. 100 g) in the rats, due to significant increases in fR and VT in both species (Table 1). The relative magnitude of the ventilatory response between species differed; VE increased by 10-fold in the squirrels but only 6-fold in the rats (Fig. 1). This difference was a consequence of the lower resting VE of the squirrel since the absolute changes in VE were not significantly different between species. When expressed as a function of Cao:, the ventilatory response curves Hypoxic hypoxia

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Table 1 The effects of carbon monoxide hypoxia and hypoxic hypoxia on selected blood and respiratory variables Rat

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N2-induced hypoxia (n = 8)

CO-induced fiypoxia (n = 6)

Na-induced hypoxia (n = 8)

CO-induced hypoxia (n = 6)

pHa

normoxia hypoxla

7.49 +_0.02 7.51 _+0.03

7.46 +_0.08 7.41 _+0.02

7.46 _+0,02 7.44 + 0.02

7.43 + 0.02 7.41 + 0.02 a

Paco 2 (Torr)

normoxi~ hypoxla

41 + 3.6 40+3.5

44 + 4.2 44+4.0

46 + 3.5 43_+4.5

53 _+4.2 52_+3.9 a

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normoxla hypoxla

63 -+4.5 29 _+2.4 a

59 + 4.6 58 + 4.9 ~

63 _+4.5 23 _+1.3 ~

60 _+8.2 63 + 8.7 c

Cao 2 (ml 02/100 ml)

normoxla hypoxm

25 +_1.3 16+ 1.0"

28 + 0.9 15+2.0 a

27 + 0.6 14+0.8 ~

24 _+2.0 12+ 1.0 a

Hematocrit (%)

normoxl hypoxla

48 + 1.6 45 _+1.2

46 _+1.3 41 + 1.5 a

45 + 0.9 41 + 2.2

41 + 2.9 38 _+2.3

Blood pressure (cm H20 )

normoxl hypoxm

154+7.6 134_+ 10.5

161 + 11.6 147_+9.5

131 + 10.2 134_+ 13.6

126+ 8.0 127_+ 10.8

fR (breaths/min)

normoxl hypoxla

89+6.5 b 154_+ 13.9 a

95+9.1 b 158+9.1 ~

42_+9.6 116_+35.8 a

49_+9.4 101 + 10.6 a

VT (ml/100 g)

normoxl hypoxla

0.7_+0.13 1.2+0.17 ~

0.6+0.04 0.9 + 0.04 a

1.0-+0.15 2.5_+0.41 ~

0.7-+0.13 1.6_+0.44

9E (ml/(min • 100 g))

normoxm hypoxm

58+4.9 b 179 -+ 30.9 a

54_+7.9 b 149 _+13.3 a

35-+4.6 254 _+57.2 a

28-+3.4 147 _+25.6 ~

The values are expressed as means _+SEM. Hypoxic values were obtained at the most severe level of hypoxia (5090 drop in Cao, ) in both groups, aindicates a value significantly different from normoxia in same species with same treatment; b significantly different from normoxia value of squirrels with same treatment; ° significantly different between normoxic and hypoxic values within a species between treatments (P< 0.05).

for h y p o x i c h y p o x i a w e r e l i n e a r for b o t h s p e c i e s (rat: VE = - 6.38 C a % + 642 ( r = 0.77); squirrel: V E = 11.9 C a o + 1273 ( r = 0 . 8 1 ) ) o v e r t h e e x p e r i m e n t a l r a n g e o f C a % (Fig. 2). I n b o t h s p e c i e s , p H , P a c o ~ , H c t a n d b l o o d p r e s s u r e w e r e m a i n t a i n e d relatively c o n s t a n t t h r o u g h o u t t h e e x p e r i m e n t a l series ( T a b l e 1).

Carbon monoxide-induced hypoxia D u r i n g e x p o s u r e t o C O , t h e Pao2 d i d n o t d e v i a t e significantly f r o m t h e n o r m o x i c level in e i t h e r s p e c i e s ( T a b l e 1). S i m i l a r d r o p s in C a % w e r e p r o d u c e d in b o t h s p e c i e s (squirrel: f r o m 24.2 t o 12.0 m l O 2 / 1 0 0 m l ; rat: f r o m 27.7 t o 14.6 m l O 2 / 1 0 0 ml). T h e effects o f l o w e r i n g Cao2, i n d e p e n d e n t o f Pao~, o n t h e v e n t i l a t i o n o f s q u i r r e l s a n d r a t s a r e p r e s e n t e d in Fig..3. T h e r e l a t i o n s h i p s b e t w e e n fg, VT a n d VI~ a n d C a Q w e r e l i n e a r i n b o t h s p e c i e s (rat: V z = - 3.55 Cao~ + 360 (r = 0.82); squirrel: VE = - 7 . 2 8 C a % + 7 8 4 ( r = 0.74)). T h e 12 m l O 2 / 1 0 0 m l d r o p in Cao2 in s q u i r r e l s w a s a c c o m p a n i e d b y a 5-fold i n c r e a s e in VE f r o m 2 7 . 9 t o 146.5 m l / ( m i n . 100 g) a c h i e v e d t h r o u g h a s i g n i f i c a n t i n c r e a s e in fg. I n t h e r a t , a 13 m l O 2 / 1 0 0 m l fall in

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Fig. 1. Relationship between Pao2 and ventilation, breathing frequency and tidal volume, expressed as % change from normoxic values, during graded hypoxic hypoxia (rats = 0 ; squirrels= [7). Cao2 was accompanied by significant increases in both fR and VT resulting in VE rising from 54.2 to 148.6 ml/(min. 100 g). Again, the absolute changes in VE were not significantly different between species but when expressed in relative terms, the squirrels appeared to undergo a proportionately greater increase in ~'E (Fig. 3). In both

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Rhonda J. Garland et al. / Respiration Physiology 96 (1994) 199-211

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Cao2 (% control) Fig. 4. R e l a t i o n s h i p b e t w e e n C a o 2 a n d ventilation, e x p r e s s e d a s % c h a n g e f r o m n o r m o x i a , d u r i n g g r a d e d h y p o x i c h y p o x i a ( H H ) a n d c a r b o n m o n o x i d e h y p o x i a ( C O H ) . T h e original d a t a p o i n t s f r o m Figs. 2 a n d 3 h a v e b e e n r e p l a c e d with least s q u a r e s linear r e g r e s s i o n c u r v e s for the r a t ( . . . . ) a n d the squirrel ( - - ) . T h e inset d e p i c t s the r a t i o o f the ventilatory r e s p o n s e to c a r b o n m o n o x i d e h y p o x i a ( C O H VE) to t h a t o f h y p o x i c h y p o x i a ( H H "¢E) f r o m 4 0 - 9 0 % o f the c o n t r o l Cao2 range.

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Rhonda J. Garland et al. / Respiration Physiology 96 (1994) 199-211

species, pH, Pacts, Hct and blood pressure were maintained relatively constant throughout the experimental series (Table 1). Comparisons A summary of the ventilatory responses for both species to hypoxic hypoxia and CO-hypoxia, expressed as a function of Cat2, is shown in Fig. 4. For clarity, this figure presents the least squares linear regression curves for the individual data points seen in Fig. 2 and Fig. 3 for each treatment and species. While the slopes of the regression lines describing the ventilatory responses to hypoxic hypoxia are significantly different between species, the slopes of the ventilatory responses to COhypoxia are not (Fig. 4). Comparisons between the slopes describing the two forms of hypoxia revealed no intraspecific differences. The effect of altering Pat2 and Cat2 simultaneously consistently exceeded the effect of altering Cat2 alone regardless of the species. The inset in Fig. 4 expresses the VE during CO exposure as a ratio of the VE during N 2 exposure. In both species, changing Cao~ alone could still produce 60% of the ventilatory response that occurred when both Pao~ and Cat2 were altered together.

4. Discussion Critique o f method The Pat2 values obtained for rats and squirrel under normoxic conditions in the present study were much lower than those reported in the literature for animals under similar conditions. The values of Cat2 were not. Assuming that these animals have normal hemoglobin concentrations (for both, rat and squirrel: [Hb] = 15 g/100 ml of blood at a hematocrit of 46~o (Altman, 1961; Maginnis et al., 1994)), the Cat2 values appear correct and the Pao~ values appear incorrect. This is supported by the fact that the resting levels of ventilation recorded in both species were consistent with the animals being normoxic. It would appear that despite precautions, the flowthrough method used to continuously measure Pat2 led to an underestimation of the real values. The reasons for this are not clear. Although this suggests that the curves shown in Fig. 1 are somewhat left-shifted, it does not affect any of the other figures or the overall conclusions. Carbon monoxide has recently been shown to be a naturally occurring gas (along with nitric oxide) that may be released from neurons and act as a neuromodulator (Snyder, 1992). Little is yet known, however, about location, specificity or magnitude of effect making it impossible to predict how exogenous CO may affect endogenous mechanisms. Indeed, Lahiri et al. (1993) report that low levels of CO attenuate carotid chemoreceptor responses to hypoxia in vitro, while high levels of CO (Pco > 500 Torr) have an excitatory effect. Thus, at this time, we can simply note that interpretation of experiments utilizing exogenous CO may turn out to be more complicated than previously believed.

The normoxic respiratory variables in Table 1 are similar to those found previously in rats (Cragg and Drysdale, 1983) and squirrels (Davies and Schadt, 1989) under pentobarbital anaesthesia. Slight differences in values for fR and/or VT due to the depressant effects of barbiturate anaesthetic are Hypoxic ventilatory responses in m a m m a l s

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apparent in both species when the values of the present report are compared to those recorded for conscious animals (rats: Walker et al., 1985; Frappell et al., 1992; squirrels: Davies and Schadt, 1989; McArthur and Milsom, 1991). The significantly higher resting ventilation of the rats compared to the squirrels is consistent with the relative hypoventilation of semifossorial species (Boggs et al., 1984). The ventilatory responses to hypoxic hypoxia presented in Fig. 1 are similar to those reported in the rat (Cragg and Drysdale, 1983) and squirrel (McArthur and Milsom, 1991). Since the animals in this study were maintained isocapnic, the ventilatory responses shown here represent true hypoxic ventilatory responses. The slight left-shift in the hypoxic ventilatory response curve of the squirrel compared to the rat and the higher HbO2 affinity of squirrel blood (Ps0 = 18.1 Torr; Magginnis et al., 1994) compared to that of rat blood (Ps0 = 35.5 Torr; Birchard and Tenney, 1986) would conform to reports that lower Pao2 thresholds exist for the HVR of animals with lower values of Ps0 (Van Nice et al., 1980). During CO-hypoxia, both species showed a strong ventilatory response. Although we are not aware of any previous accounts of the ventilatory responses of rodents to CO inhalation, similar observations have been reported for cats and goats (Santiago and Edelman, 1976; Chapman et al., 1982; Gautier and Bonora, 1983; Gautier et al., 1990). The average relative increase in ventilation for both squirrels (521~o) and rats (275 ~o) during CO inhalation exceeded the values reported previously ( 216 ~o increase in conscious goats: Santiago and Edelman, 1976; 130~ increase in anaesthetized cats: Gautier and Bonora, 1983; 130 To increase in conscious cats: Gautier et al., 1990). Since the average change in Cao2 reported for these studies is comparable to that produced in the present study, the difference cannot be explained by the level of CO administered and it seems most likely that the difference is an effect of species. The ventilatory responses of the squirrel were consistently greater than those of the rat regardless of the form of hypoxic exposure. When the ventilatory responses to both forms of hypoxia were expressed as a function of Cao2 (Fig. 4), it was clear that the ventilatory response to hypoxic hypoxia exceeded that to CO-hypoxia in both species; while 60~o of the full ventilatory response could be reached by changing Cao~ alone in both species (Fig. 4), concurrent changes in Pa% were required to elicit the full response. This does not necessarily imply that changes in Pa% alone account for only 40~o of the HVR. Since only Ca% was examined independently, speculation remains as to whether the interaction between the two potential stimuli is additive, multiplicative or redundant. Whatever the case, the data do suggest that both species can respond to CO-induced changes in Cao~ alone. Despite the obvious distinction between hypoxic hypoxia and CO-hypoxia at the arterial level, it has been argued that the ventilatory responses to both are actually mediated through changes in tissue Po2 (Mills and Edwards, 1968). Carbon monoxide alters the shape and position of the OEC. Besides reducing the amount of functional Hb available for binding 02, CO increases the Hb affinity for 0 2 (Haldane, 1912; Haab, 1990). The extent to which tissue Po2 is altered will depend not only on the level of carboxyhemoglobin (HbCO) in the blood but also on the degree of 02 extraction at the receptor level. In Fig. 5A and 5B, OECs Pao2, Cao2 and tissue Po2 as modulators o f ventilation

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Po2 (mm Hg) Fig. 5. Oxygen equilibrium curves (OEC) for rat blood. Panels A and B show OEC in the presence of different amounts of carboxyhemoglobin.The curves 1, 2 and 3 correspond to HbCO concentrations of 0, 20 and 50~o, respectively. In A, the effects of low (O) and high (O) extraction at the receptor level on tissue Po2 are shown for points with the same Pao2 but different levels of Cao2. In B, the effects of low and high extraction at the receptor level on tissue Po2 are shown for points with the same Cao2 but different levels of Pao2. In C, OEC for control rats (1), sodium cyanate treated rats (4, redrawn from Birchard and Tenney, 1986) and rats with an anemia-induced 509/0 reduction in Cao2 (5, calculated) are shown for blood with the same Pao2 but different levels of Cao2.

for rat blood containing 0-50~o H b C O have been calculated using the methods of Roughton and Darling (1944) and the standard curve o f Birchard and Tenney (1986); Fig. 5C contains the O E C for sodium cyanate treated rats (Birchard and Tenney, 1986) and a calculated O E C for the rat during anemia (50~o reduction in hematocrit), two conditions which also alter Cao2 at constant P a o . Assuming that metabolism and tissue p H remain constant, tissue Po2 at both high and low levels of 0 2 extraction can be estimated for blood having the same Pao2 but different Cao~ (Fig. 5A) as well as for blood having the same Cao2 but different Pao~ (Fig. 5B). The value for high extraction (25 ~o) is based on whole body arterio-venous Cao~ differences recorded in rats (Burlington and Milsom, unpublished data) while the low extraction value (5 ~o) is based on reports of carotid b o d y 02 consumption (Purves, 1969). Based on the ventilatory responses recorded in the present study, the ventilation at any given combination of Pao~ and Cao2 can be estimated. F r o m these estimates, inferences as to whether CO-induced changes in Ca% can be acting solely through changes in P% at the tissue level can be made.

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Regardless of 0 2 extraction at the receptor level, for any given Pao~, tissue Po~ would always be lower during CO-hypoxia than during normoxia (HbCO = 0 ~ ) (Fig. 5A). The higher ventilation recorded during CO-hypoxia compared to normoxia (Fig. 2), therefore, is consistent with changes in tissue Po2 being the stimulus for the HVR. In contrast, differences in tissue Po~ could only explain the reduced HVR with CO-hypoxia compared to hypoxic hypoxia (Fig. 4; Table 1) if extraction was low. Curves 1 and 3 in Fig. 5B show that for the same Cao2, high extraction results in a lower tissue Po~ during CO-hypoxia than during hypoxic hypoxia. As a consequence, ventilation should increase more with CO-hypoxia, which was not the case. Studies that have examined the effects of experimentally-induced changes in the intraspecific Ps0 on the HVR or the effects of anemia on the HVR, however, are not totally consistent with tissue PQ acting as the sole stimulus for the HVR with either high or low levels of tissue 02 extraction. The Pao~ threshold of the HVR remains unchanged in sodium cyanate treated rats exposed to hypoxic hypoxia (Birchard and Tenney, 1986) despite the fact that for any given Pao~, tissue Po~ would always have been lower in sodium cyanate treated rats compared to untreated animals (Fig. 5C). Furthermore, if tissue Po~ were acting as the stimulus for the HVR, the ventilation of anemic animals with reduced Caoz would always be expected to exceed that of nonanemic animals since Po~ at the receptor level will always be lower in anemic than non-anemic animals (Fig. 5C). The ventilatory responses of animals to anemia, however, have been somewhat equivocal. While Cropp (1970) reported that the ventilatory response to 10~o 02 was greater in anemic dogs than it was in non-anemic dogs and a similar observation was made in goats during transient hypoxia (inhalation of several breaths of N2), no difference was found between anemic and non-anemic goats during steady state hypoxia (12~o 02 for 7 rain) (Santiago et al., 1975). Thus, although much of the data is consistent with the hypothesis that the HVR is mediated solely through changes in tissue Po~ if extraction at the receptor site is low, not all data fits this picture. It remains possible, therefore, that some species can sense changes in Cao2 per se. Sites o f Oz-chemodetection The exact mechanism underlying the ventilatory response to CO-hypoxia remains obscure. It is generally accepted that the HVR is mediated by the carotid body chemoreceptors sensing changes in Pao2. The question then arises, if the HVR in rodents is not due solely to changes in tissue Po2 can carotid bodies in ground squirrels and rats sense changes in Cao~ or is the HVR to changing Cao~ mediated by some other receptor group? Both are possible. Many carotid body denervated animals retain or regain an HVR (Bisgard et al., 1980; Smith and Mills, 1980; Maskrey et al., 1981; Webb and Milsom, 1990), suggesting that a non-carotid body chemoreceptor is capable of eliciting an HVR in these species. Furthermore, the HVR of carotid body denervated animals resembles the hypoxic tachypnea characteristic of CO inhalation at constant Pao2 (Santiago and Edelman, 1976; Chapman e t a l . , 1982; Gautier and Bonora, 1983; Gautier et al., 1990). That is, the HVR is reduced in magnitude and due primarily to changes in breathing frequency. This is certainly true of the golden-mantled ground squirrel (Webb and Milsom, 1990). It follows, therefore, that some aspect of 02 delivery other than Pao~ may be responsible for the HVR, acting

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at n o n - c a r o t i d b o d y c h e m o r e c e p t o r s , d u r i n g C O - h y p o x i a in the g r o u n d squirrels a n d rats. This d o e s n o t rule out the possibility t h a t the c a r o t i d b o d i e s in t h e s e species c a n sense c h a n g e s in Cao2.

5. Conclusion It is clear t h a t C O - i n d u c e d c h a n g e s in Cao2 i n d e p e n d e n t o f Pao2 are c a p a b l e o f eliciting a v e n t i l a t o r y r e s p o n s e in b o t h h e t e r o t h e r m i c a n d n o n - h e t e r o t h e r m i c r o d e n t species. A l t h o u g h this study did n o t a d d r e s s m e c h a n i s m p e r se, it a p p e a r s that either tissue Po2 c o u l d be acting as the stimulus for the H V R d u r i n g C O - h y p o x i a or t h a t t h e s e a n i m a l s c a n s e n s e c h a n g e s in 0 2 c o n t e n t per se. A l t h o u g h it is easier to ascribe a d a p t i v e significance to this feature in h e t e r o t h e r m i c r o d e n t s , the d a t a c o l l e c t e d in the p r e s e n t s t u d y suggests it m a y be a c o m m o n feature o f all r o d e n t species.

6. References Altman, P.L. (1961). Blood and other body fluids, edited by D.S. Dittmer. Washington, D.C.: Federation of American Societies for Experimental Biology, 540 pp. Birchard, G.F. and S.M. Tenney (1986). The hypoxic ventilatory response of rats with increased blood oxygen affinity. Respir. PhysioL 66: 225-233. Bisgard, G.E., H.V. Forster and J.P. Klein (1980). Recovery of peripheral chemoreceptor function after denervation in ponies. J. AppL Physiol. 49: 964-970. Boggs, D.F., D.L. Kilgore and G.F. Birchard (1984). Respiratory physiology of burrowing mammals and birds. Comp. Biochem. PhysioL 77: 1-7. Chapman, R.W., T.V. Santiago and N.H. Edelman (1982). Brain hypoxia and control of breathing: role of the vagi. J. AppL Physiol. 53: 212-217. Cragg, P.A. and D.B. Drysdale (1983). Interaction of hypoxia and hypercapnia on ventilation, tidal volume and respiratory frequency in the anaesthetized rat. J. Physiol. (London) 341: 477-493. Cropp, G.J.A. (1970). Ventilation in anemia and polycythemia. Can. J. Physiol. PharmacoL 48: 382-393. Davies, D.G. and J.C. Schadt (1989). Ventilatory responses of the ground squirrel, Spermophilus tridecemlineatus, to various levels of hypoxia. Comp. Biochem. Physiol. 92A: 255-257. Frappell, P., C. Lanthier, R.V. Baudinette and J.P. Mortola (1992). Metabolism and ventilation in acute hypoxia: a comparative analysis in small mammalian species. Am. J. Physiol. 262: R1040-R1046. Gautier, H. and M. Bonora (1983). Ventilatory response of intact cats to carbon monoxide hypoxia. J. Appl. Physiol. 55: 1064-1071. Gautier, H., M. Bonora and D. Zaoui (1990). Effects of carotid denervation and decerebration on ventilatory response to CO. J. Appl. PhysioL 69: 1423-1428. Glass, M.L., R.G. Boutilier and N. Heisler (1983). Ventilatory control of arterial P O 2 in the turtle Chrysemys picta bellii: effects of temperature and hypoxia. J. Comp. PhysioL 151: 145-153. Haab, P. (1990). The effect of carbon monoxide on respiration. Experientia 46: 1202-1205. Haldane, J.B.S. (1912). The dissociation of oxyhemoglobin in human blood during partial CO-poisoning. J. Physiol. (London) 45: XXII-XXIV. Lahiri, S. R. Itturriaga, A. Mokashi, D.K. Ray and D. Chugh (1993). CO reveals dual mechanisms of 02 chemoreception in the cat carotid body. Respir. Physiol. 94:227-240 Magginnis, L.A., E.S. Lo and W.K. Milsom (1994). Effects of hibernation on blood oxygen transport in golden mantled ground squirrels. Respir. PhysioL 95: 195-208. Maskrey, M., D. Megirian and S.C. Nicol (1981). Effects of decortication and carotid sinus nerve section on ventilation of the rat. Respir. PhysioL 43: 263-273.

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McArthur, M.D. and W.K. Milsom (1991). Ventilation and respiratory sensitivity of euthermic Columbian and golden-mantled ground squirrels (Spermophilus columbianus and Spermophilus lateralis) during the summer and winter. PhysioL Zool. 64: 921-939. Milsom, W.K. (1990). Control and co-ordination of gas exchange in air breathers. In: Advances in Comparative and Environmental Physiology, Vol. 6, edited by R.G. Boutilier. Berlin, Heidelberg: SpringerVerlag, pp. 347-400. Mills, E. and M.W. Edwards (1968). Stimulation of aortic and carotid chemoreceptors during carbon monoxide inhalation. J. Appl. Physiol. 25: 494-502. Purves, M.J. (1969). Changes in oxygen consumption of the carotid body of the cat. J. Physiol. (London). 200:132P-133P. Roughton, F.J.W. and R.C. Darling (1944). The effect of carbon monoxide on the oxyhemoglobin dissociation curve. Am. J. Physiol. 141: 17-31. Santiago, T.V., N.H. Edelman and A.P. Fishman (1975). The effect of anemia on the ventilatory response to transient and steady-state hypoxia. J. Clin. Invest. 55: 410-418. Santiago, T.V. and N.H~ Edelman (1976). Mechanism of the ventilatory response to carbon monoxide. J. Clin. Invest. 57: 977-986. Smith, P.G. and E. Mills (1980). Restoration of the reflex ventilatory response to hypoxia after removal of carotid bodies in the cat. Neuroscience 5: 573-580. Snyder, S.H. (1992). Nitric oxide: first in a new class of neurotransmitters? Science 257:494. Tucker, V.A. (1967). Method for oxygen content and dissociation curves on microliter blood samples. J. Appl. Physiol. 23: 410-414. Van Nice, P., C.P. Black and S.M. Tenney (1980). A comparative study of ventilatory responses to hypoxia with reference to hemoglobin O2-affinity in llama, cat, rat, duck and goose. Comp. Biochem. PhysioL 66A: 347-350. Walker, B.R., E.M. Adams and N.F. Voelkel (1985). Ventilatory responses of hamsters and rats to hypoxia and hypercapnia. J. Appl. Physiol. 59: 1955-1960. Webb, C.L. and W.K. Milsom (1990). Carotid body contribution to hypoxic ventilatory responses in euthermic and hibernating ground squirrels. In: Arterial Chemoreception, edited by C. Eyzaguirre, S.J. Fidone, R.S. Fitzgerald, S. Lahiri and D.M. McDonald. New York: Springer-Verlag, pp. 337-343. Wood, S.C (1984). Cardiovascular shunts and oxygen transport in lower vertebrates. Am. J. Physiol. 247: R3-R14.