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Stimulus interaction in the responses of carotid body chemoreceptor single afferent fibers
Respiration Physiology (1975) 24, 249-266;
STIMULUS
North-Holland Publishing Company, Amsterdam
INTERACTION IN THE RESPONSES OF CAROTID CHEMORECEPTOR SINGLE AFFERENT FIBERS’
BODY
S. LAHIRI and R. G. DeLANEY Cardiovascular-Pulmonary Division, Department of Medicine and Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, U.S.A.
Abstract. The characteristics
of steady-state
to independent
changes
was maintained
within the normal
to changes
both
in arterial
in arterial
responses
of single afferent
Po, and Pco, were investigated limits (115-130
Po, and
Pco,.
torr).
fibers of carotid
in cats. The arterial
Single chemoreceptor
The relationship
between
afferent
the activity
chemoreceptors blood
pressure
fibers responded
of chemoreceptors
and changes in arterial Pc,, was linear at a constant arterial Po,. The two stimuli showed multiplicative interaction. The activity approached zero (threshold) as arterial P,,, was decreased at a These response constant arterial PO,, a decrease in arterial Po, decreased the arterial Pco, threshold. characteristics mechanism formational reversibly
of a single fiber suggest by the two stimuli. change
that
The data
the sensory fit into
in the membrane-bound
receptor
an idea that
polymeric
may
be activated
the mechanism
chromophore
group
may which
through involve reacts
a single a conwith
0,
and shows a Bohr-shift. Arterial
Pco,
Interaction
Arterial
Po,
Mechanism
Carotid
chemoreceptors
Threshold
between
O2 and COZ stimuli
of excitation
Despite several studies on the response of carotid chemoreceptor to changes in hypoxic and hypercapnic stimuli, independently of each other or in combination, (Euler et al., 1939; Eyzaguirre and Lewin, 1961; Hornbein et al., 1961; Biscoe et al., 1970; Sampson, 1970; Fitzgerald and Parks, 1971) the quantitative relationship between the effect of one stimulus on that of the other is not clear. This, in part, is due to the fact that single fiber preparations were not used in the investigation of the stimulus-response relationship. The interplay between the two stimuli might occur in a single fiber and involve recruitment of new fibers which are possibly activated at a higher level of arterial stimulus than others. If the two processes occurred simultaneously in a multi-fiber preparation quantitation of each type of response would not be possible. Therefore, the investigations of Hornbein et al. (1961) and Fitzgerald and Parks (1971) on the relationship between changes in Accepted for publication 4 April 1975. 1 Supported in part by USPHS Grant
HL-08805. 249
250
S. LAHIRI
AND R. G. DeLANEY
arterial Po, and Pco, and the average activity of the entire carotid sinus nerves in the cats provided little information as to the sensitivity of individual receptors and did not distinguish between the response of a single receptor and recruitment of receptors. Eyzaguirre and Lewin (1961) studied activity of carotid chemoreceptors in multi-fiber preparations but the interpretation of their experiments was further complicated by relating the nervous response to inspired gas concentration rather than to arterial PO,, Pco, and [H+] which provide a better basis of quantitative description of stimulus level. Biscoe er al. (1970) mvestigated the response of single carotid chemoreceptor afferent fibers to either changes in arterial Po, or to PcoZ and [H+] but did not examine the question of interplay between the two stimuli. Joels and Neil (1961) and Eyzaguirre and Lewin (1961) did address themselves to the question but did not provide a quantitative description. They merely showed that a single afferent fiber was capable of responding to both stimuli. In the present study we investigated the characteristics of response of carotid body single afferent fibers to the two stimuli while holding arterial blood pressure within normal limits. We found that each single fiber responded to changes both in arterial PO, and Pco2, and that the response to one stimulus was enhanced by the other. Although there were differences in mean frequency of discharge, there was a basic similarity in the pattern of response among the chemoreceptors. The recent report of Goodman (1974) is in accord with this observation. Also, the receptors showed a threshold for stimulation by either arterial Pco2 or [H+]. A decrease in arterial PO, decreased arterial Pc,,/[H+] threshold. Materials and methods Experiments were done in 55 adult cats (2.5-3.0 kg) anesthetized with a-chloralose (60 mg . kg- r, intravenously). Supplemental doses of the anesthetic were administered as needed. Arterial vessels were prepared for monitoring blood pressure, sampling blood, and a brachial vein for the administration of fluids. The rectal temperature was maintained between 38-39 “C. The trachea was intubated and the larynx and esophagus were removed to expose both carotid bifurcations. PREPARATION
OF CHEMORECEPTOR
AFFERENTS
The left carotid sinus nerve was exposed ventromedially, a small strand of the sinus nerve was snipped off at the glossopharyngeal junction, and single afferent units were obtained by conventional methods. RECORDING
SYSTEM
Saline wick electrodes were employed to record carotid chemoreceptor activity. Action potentials were preamplitied in two steps, and the output was then split and fed to: (1) a four-trace amplifier (Tektronix 3A74) in the dual-beam oscilloscope set to trigger in the alternate mode on the rising phase of the chemoreceptor action potential so that the size, shape and duration of single spikes could be characterized
CAROTID
CHEMORECEPTOR
02-co2
INTERACTION
251
and recorded on moving film (Kymograph Camera, C4N, Grass Instrument Co., Quincy, Mass.); (2) a galvanometer (flat response curve to 1650 Hz) in a recording system (Honeywell 1508A Visicorder, Denver, Colo.); (3) a Grass AM8 audiometer; and (4) a pulse height discriminator and an integrator (Frederick Haer & Co., Ann Arbor, Mich.) which summed impulses per second. For analytical use, the impulses were also manually counted over a 60-second period and expressed as a mean activity per second. TESTS FOR CHEMORECEPTOR
ACTIVITY
Action potentials were considered to arise from a chemoreceptor if their activity changed promptly upon changing inspired P,,. The chemoreceptor fiber also responded promptly and briskly to a breath of CO,-enriched air. In some experiments, the cats were paralyzed with gallamine triethiodide (Flaxedil: American Cyanamid Co.), 10 mg.kg-‘, intravenously. As ventilation finally stopped, the discharge rate of the fiber increased. This activity decreased upon ventilating the animal by a respiratory pump. INSPIRED
AND END-TIDAL
GAS
The inspired gas mixtures were administered to the cat through a T-tube connected to the pneumotachograph when breathing spontaneously or through a respiratory pump(Harvard Apparatus, Model 607) when paralyzed. The tracheal concentrations of 0, and CO;? were continuously monitored by a Pulmonary Function Oxygen Monitor (Westinghouse Model 211, Pittsburgh, Pa.) and an infrared capnograph (Godart, Statham Instruments, Oxnard, Calif.) respectively. The O2 and CO, sensors were calibrated. SYSTEMIC
ARTERIAL
BLOOD
PRESSURE
The systemic blood pressure was measured by a strain gauge transducer-amplifier system which was calibrated using a mercury manometer. The mean blood pressure was maintained within f 10 torr of the initial value by the administration of 0.9% NaCl or blood from a donor cat by slow intravenous drip throughout the experiment. ARTERIAL
Blood
BLOOD
samples
GASES AND
pH
(1.5 ml) from the brachial
artery
were withdrawn
at appropriate
times, and their P,,, Pco2 and pH were immediately determined at 38 “C using a Radiometer system (Radiometer, Copenhagen). The 0, and CO* electrodes were calibrated with blood equilibrated with gas mixtures of two different compositions. The pH electrode was calibrated with buffers of two different pH. PROCEDURE
The relationship between steady-state arterial PO,, PcoZ and pH and single afferent fiber activity was determined in two types of animal preparations: either in a spontaneously breathing cat or in a paralyzed cat respired by a pump. In the
252
S.
LAHIRI
AND R. G. DeLANEY
spontaneously breathing cats arterial blood gas changes were made by changing the inspired gas mixture alone. In this series the lower range of arterial PcoZ was limited to a few torr below normal, e.g. about 24 torr. In the pump-assisted preparation the arterial blood gas changes were made both by changing the rate and stroke volume of the pump and by varying the inspired gas composition. In these animals arterial Pco2 was lowered to values of the order of 10 torr, and thus investigation of chemoreceptor response over a wide range of stimulus was possible. A change in end-tidal gas tensions to pre-determined levels was associated with a change in the chemoreceptor activity which stabilized in less than 5 min. At this time an arterial sample was taken in order to relate mean activity of the afferent fiber to arterial Po2, Pco2 and [H+]. The agreement between end-tidal and arterial Pco2 values was usually very close but the arterial Po2’s were lower than the end-tidal Po2’s. The end-tidal values were used as a guide to arterial values. For defining the stimulus-response relationship over a wide range, arterial Po, was changed while arterial Pcoz was held constant. Subsequently, the arterial Pco2 was changed to another level where it was held constant while arterial Po, was again varied. For analytical purpose, the mean activity was then related to arterial Pco, at constant arterial Po,. A complete study required about 2 hours of recording. STABILITY
OF THE PREPARATION
The stability of response of the chemoreceptor preparation to a given stimulus, and the stability of acid-base of arterial blood need mention. Some of the afferent fibers failed after an initial response to hypoxia, and the study on these fibers could not be completed. To assess any development of instability during the progression of the experiment, arterial Po, was changed from one level to another maintaining arterial Pco2 constant. These transient responses also formed a part of another experimental design. Those chemoreceptor responses which were not reproducible were not used. There were occassions when the response to the same stimulus changed with time. The acid-base status of arterial blood was checked frequently by using Pro* and pH data. Within the test period the arterial acid-base did not change appreciably. However, after severe hypoxia ( < 30 torr) metabolic acidosis developed. Since this acidosis distorted the relationship between arterial Pco2 and chemoreceptor activity, the particular experiment was terminated as soon as it appeared. Results IDENTIFICATION
OF SINGLE
FIBER
ACTIVITY
The single fiber spikes were determined by the identity of their size, shape and duration. Figure 1 illustrates activity of two single fibers. The spikes never superimposed in our single fiber preparation. Figure 2 illustrates the response of a single fiber (large spikes) to changes in arterial Po, and PcoI. The effect of a decrease in arterial Po, at two levels of arterial Pco2 (28 and 34.5 torr, respectively) under steady-state conditions are shown. The
CAROTID
Fig. 1. Identification different
CHEMORECEPTOR
of single afferent
preparations.
The large
spikes
because
0 &o
chemoreceptor
fiber discharge.
A and B depict
in each case were considered
of their identity
253
2 INTERACTION
to originate
spikes from two
from a single fiber
of shape, size and duration.
paC02
Pa
A
02 tarr 490
9
95
1 C
43
tarr
28
I
II
IfI
II
I
1I
I
‘I
I
D
495
E
97
F
42
35
I G
., _., . .__
._.
-.
.
..
.
I . . .._.. _ 25 loop
I SEC.
Fig. 2. Response changes
in activity
of a single fiber (large as arterial
spikes)
PO, decreased
to changes
in arterial
PO, and Pc,,.
from 490 to 43 torr at arterial and G from 495 to 24.5 torr at arterial Pro2 of 34.5 torr (see table
A, B and C show
P,, of 28 torr, and D, E, F 1 and text for details).
relevant blood gas data and the mean activities of the single fiber are given in table 1. It is clear that as arterial P,, decreased the rate of activity in the single fiber increased (at a constant arterial Pc,,): A, B and C at arterial Pc,,=28 torr; D, E, F and G at arterial P co2 = 34.5 torr; the rate of increase was greater at lower arterial P,,. Hypercapnia potentiated the effect of hypoxia. Of interest is the observation that at at arterial P,, of about 500 torr only one unit was active (compare A and D in fig. 2), its response increasing with the level of hypercapnic stimulus. A decrease in arterial P,, was not only associated with a similar increase in the activity of the unit (compare B and D in fig. 2) but was also accompanied by evidence of discharge from a new unit.
254
S. LAHIRI
AND R. G. DeLANEy
TABLE Stimulus
response
Serial
Impulses
no.
(set-‘)
of single afferent Arterial
fiber of carotid
1
body in the cat (These data correspond Arterial
Brachial
PH
pressure
Arterial
(mean 0.2
490
28.0
7.385
115
2.1
95
28.3
7.380
113
5.3
43
28.0
7.390
119
1.9 4.4
495 97 42
35.0 34.5
7.310
115
34.5
7.320 7.315
114 120
34.0
7.310
105
15.0 24.1
24.5
25r--------
to fig. 2).
arterial torr)
p"O, = 35 torr
/
20-
15IMPULSES S-SC-'
IO-
20
30 PaCO,,
Fig. 3. Effect on single fiber activity of changes in arterial level of arterial PO,, an increase in arterial Pc,, increased at lower arterial
torr P,,? at four levels of arterial P,,. At any the rate of activity, this effect being greater P,,.
CAROTID
QUANTITATIVE
EFFECT
CHEMORECEPTOR
OF CHANGE
0 2-co
IN ARTERIAL
Pcol
255
2 INTERACTION
AND
PO,
To examine the effect of hypocapnia and alkalosis on the activity in a single unit, the animals were paralyzed and hyperventilated by a respiratory pump. One example of this relationship between arterial Pco2 and the mean activity of a single unit at four levels of arterial Po, is shown in fig. 3. It is clear that at all levels of arterial P,, with a decrease in arterial Pco2 a decrease in the mean activity of the chemoreceptor was approximately linear. At an arterial P,, of 400 torr, the activity approached zero as arterial PcoZ was decreased to about 13-14 torr (threshold stimulus). At lower levels of arterial PoZ, activity of the chemoreceptor unit also decreased towards zero when arterial Pco2 was decreased to values lower than 13 torr. Hypoxia also modified the effect of arterial Pco2 in the following conspicuous ways: at any level of arterial Pco2, the activity was greater at a lower arterial P,? and the activity for a given change of arterial Pco2 was also greater during hypoxia. Thus, there was a clear multiplicative interaction between the two stimuli. It is interesting to note that at Pao2 of 35 torr during hypercapnia above 30 torr the increase in response was less than linear. Figure 4 shows the effect of change of arterial Pco2 and PO2 over a wide range. At arterial PCoZof the order of 10 torr the response to moderate hypoxia (48 torr) practically disappeared. The response characteristics appeared similar to those seen in fig. 3. The receptor showed a strong multiplicative stimulus interaction. Also, the
Pa = 02
CA T# x-x
25
6/
torr
IMPULSES set“
0
40
20
60
80
100
Fig. 4. Effects on single fiber activity of wide changes in arterial Pc,, and Po,. In addition to the effects described in fig. 3 these data show that at low levels of arterial Pco, the rate of activity approaches zero indicating
threshold
stimuli.
At arterial
Po,=25
torr, the activity
P co1 increased.
appeared
to level off as arterial
256
S. LAHIRI
AND R. G. DeLANEY
15PCQ = 24 forr
I
IOIMPULSES
set-’ 5-
CAT # 67
0
30
20
IO
40
ARTERIAL
Fig. 5. Relationship Increases
between
in the mean activity arterial
rate of single fiber activity are associated
PCo2. This relationship
[H+],
50
nmoles,
and arterial
with the increases is enhanced
[H+]
in arterial
by decreases
3
I-’
at five levels of arterial [H+] induced
in arterial
by increases
Po,. in
P,,.
receptor showed Pace? threshold which was lower with lower Pao2. The experimental observations above arterial Pcoz of 50 torr were only a few, and therefore the description of the linear response over this range of arterial Pco2 may not be justifiable. Clearly, there was a plateau in the response to PaLco2at a Paoz of 25 torr. Although we did not explore the extent to which the effect of arterial Pco2 was due to the CO, molecule per se or its effect on arterial [H +I, we can examine the response of the chemoreceptor unit to arterial [H+] that occurred concomitantly with the change of arterial Pco,. The relationship between chemoreceptor response to arterial [H+] at five levels of arterial P,, is shown in fig. 5. At each level of arterial Po, the relationship, beginning from near zero activity, appeared linear. It is also apparent that at the level of [H ‘1 of 15-30 nmoles.ll’ the chemoreceptor activity decreased nearly to zero. This arterial [H ‘1 threshold appeared to depend on arterial Po2, the threshold [H+] being lower at lower arterial Po, (see later). VARIATION
OF COMBINATION
EFFECTS OF ARTERIAL
P,,
AND PcoJ
An apparent variation of the interactive effects of arterial Po, and arterial Pco2 was found among several of the units investigated. There was one unit which responded feebly to a rise in arterial PC-.,. Increases of arterial Pco2 from 35 to 50 torr increased the activity by less than 1 impulse. set- ’ at arterial Po, of 89 and 515 torr. At an arterial P,, of 40 torr, the impulse frequency went up by 3 set-’ for a similar increase in arterial Pcol from 35 to 50 torr. This unit, however, responded vigorously to a change in arterial Po, from 515 to 26 torr at Paco2 of 35 torr by increasing the activity from 0.6 to 18 impulses. set ’ . Thus, there appeared to be little multiplicative
CAROTID
CHEMORFKEPTOR
0 2&o
257
2 INTERACTION
stimulus response in this fiber. However, it is possible, though unlikely, that a plateau was already reached in its response to Pacoz. Unfortunately, this fiber was not studied at Paco2 lower than 35 torr. Other types of responses were encountered in fibers which showed inherently a low frequency of discharge. In one multi-fiber strand the submaximal activity of a single fiber in steady-state seldom exceeded 3 set-‘. In the same strand, there was another fiber which responded with a frequency of 18 see-’ at the same stimulus level. However, both of them showed the same pattern of multiplicative stimulus interaction. In summary, all 38 single afferent fibers showed an increase in the rate of discharge as arterial PO, was decreased from 500 to 30 torr at the normocapnic level. All except three of these fibers showed considerable multiplicative stimulus interaction, and their sensitivities to any change in arterial stimulus were very similar, although there were differences in mean frequency of impulses. THRESHOLD
STIMULI
Most of the single fibers examined showed an arterial Pco2 or [H+] threshold, i.e. a value of arterial Pco2 and [H+] above which chemoreceptor activity began to increase and below which the activity approached zero in the steady state. In the artificially ventilated animals these values were actually reached at several levels of arterial Po,. The dependence of arterial PcoZ and [H+] threshold on arterial P,, was obvious. The results from one experiment are shown in a threshold Paco2-Pao2 diagram in fig. 6. Each point in the diagram represents a pair of Paco2 and Pao2 at which the chemoreceptor was practically silent. Clearly, Paco2 threshold decreased with a decrease of Paoz. The decrease in the threshold Paco2 was greater for a given decrease in Pao, as the intensity of hypoxia increased. In the same preparation
20
1
Pace, XA
torr IO -
_-x-+-
x/ x CdTX6,
0 0 Fig. 6. Threshold sents a combination
I 20
stimulus
I 40 for carotid
of Pa,,,
I 60
I 80
chemoreceptors
and Pa,,
I 100
1 120
Pao2,
torr
shown
, 140
in Pa co,-Pa,,
I , 180 j,,
I 160
diagram.
at which the receptor was just active. was lower at a lower Pa,,.
Clearly
Each point Pa,,,
repre-
threshold
258
S.
another
fiber showed
pattern
of the threshold
LAHIRI
a higher Pa,,,-Pa,,
AND R. G. DeLANEY
Paco2 threshold
at the same
Pao,,
although
the
curve was similar.
Discussion MULTIPLICATIVE
STIMULUS
INTERACTION
This study showed response characteristics of single afferent fibers from the carotid chemoreceptors in the cat. Apparently, there was a spectrum of stimulus-response relations for the carotid chemoreceptors. There were variations in multiplicative stimulus interaction, arterial P co2-PoZ threshold and in mean frequency of discharge. However, if differences in mean frequency of discharge were taken into account, the similarities between the response curves of most of the receptors were quite clear. This finding is in conformity with the claim of homogeneity of response of chemoreceptor fibers (Goodman, 1974). Occasional deviation from a homogeneity indicated that the basic phenomenon might be influenced by many factors, such as variations of innervation, size and types (medullated us. nonmedullated) of fibers, number of steps involved in the excitation process, etc. In general, the observed stimulus-response curves resembled those reported by Hornbein et al. (1961) Eyzaguirre and Lewin (1961) and Fitzgerald and Parks (1971) in multi-fiber or whole nerve preparations. None of the fibers showed any inhibition of activity . with increase in arterial Pco2, unlike the observation of Paintal and Riley (1966) on aortic chemoreceptor fiber. However, Sampson and Hainsworth (1972) and Fitzgerald( R. S. Fitzgerald, personal communication) found that increases in arterial activity. P car increased aortic chemoreceptor An explanation of the phenomenon of multiplicative stimulus interaction at the level of carotid body chemoreceptors requires an examination of the effect of the stimuli on carotid body blood flow and oxygen uptake. Purves (1970a) reported small changes in blood flow with changes in the stimulus level but it has been reported that a considerable change in blood flow did not change carotid body tissue P,, (Acker ef al., 1973) nor the activity of carotid chemoreceptors (see Biscoe, 1971). Thus it is unlikely that any change in blood flow associated with the experimental procedure materially contributed to the chemoreceptor response reported in this paper. According to Purves (1970a) both hypoxia and hypercapnia decreased carotid body oxygen uptake, and he supported the previously postulated contention (see Howe and Neil, 1972) that this decreased oxygen uptake might be the mechanism of chemoreceptor stimulation. But it is not clear if his data on 0, uptake corresponded to the hypoxic-hypercapnic multiplicative interaction we observed in the activity of chemoreceptors. Also, oxygen uptake of the carotid body after sympathectomy was found to be independent of Pao, (Purves, 1970b), although it is known that the pattern of response of chemoreceptors is not changed by sympathectomy (see Biscoe, 1971; Torrance, 1974). Thus, oxygen uptake need not be crucial to receptor excitation.
CAROTID
THRESHOLD
CHEMORECEPTOR
02&oz
INTERACTION
259
STIMULUS
Various changes
authors who investigated the response of peripheral chemoreceptors to in arterial blood gas tension addressed themselves to the question of arterial Po, threshold. Defining threshold as the Po, at which the rate of discharge begins to change with the changes of Po, Biscoe et al. (1970) assigned a range of arterial PO, of 140-440 torr (presumably at normal arterial Pco, and pH) for carotid chemoreceptors, and Paintal and Riley (1966) at inspired P,, of 8&160 torr for aortic chemoreceptors. Our results are generally in conformity with those of Biscoe et values of Paintal and Riley could al. (1970). The unusually low P,, threshold be due to low arterial Pco2 and pH in their preparation. However, according to those authors the effect of arterial CO,-H+ on aortic chemoreceptors was not stimulatory, and therefore any low arterial CO*-H f could not have influenced arterial P,, threshold. On the other hand, in the absence of a potentiating effect of C02-H+, arterial P,, threshold for the aortic chemoreceptors could be lower than that of the carotid chemoreceptors. The variation of arterial Po, threshold for different receptors at a constant arterial P co2 and pH observed in this work could be due to quantitative differences in the mechanistic component(s) ultimately responsible for the initiation of impulse. However, an explanation might be found in the relation between arterial PO, and receptor Paz. The POr at different receptors may not be the same at the same arterial Po2, although it may bear a constant relation with arterial PO,. Unfortunately, we do not know the site of the receptor, nor its PO,. There are, however, two reports of measurements of carotid body tissue PO, using 0, electrodes (Whalen and Nair, 1973; Acker et al., 1973). But the meaning of their findings is not clear. Whalen and Nair (1973) reported that, in a cat breathing air, tissue PO, of carotid body decreased from 80 to 40 torr as the electrode was driven from the outer to inner zone. But Acker et al. (1973) found opposite results; that is, O-10 torr at the periphery and 60-70 torr at the center. However, they seem to agree that tissue PoL, although it may bear a constant relation with arterial PO,. Unfortunately, we do not know the site of the receptor, nor its PO,. There are however, two reports of could vary from one location to another. This variation of receptor PO2 might account for the observed differences in the apparent Pa,,-Pa,,, threshold. We do not know if carotid body tissue PO, is altered by changes in arterial Pco, and pH but we found that arterial PO, threshold was dependent on arterial Pco2 and [H+]. Low enough arterial Pco2 (about 10 torr) and arterial [H+] (about 12-22 nmoles.l-‘) either abolished or nearly abolished the response of most of the chemoreceptors to moderate hypoxia. Joels and Neil (1960) briefly reported that perfusate pH of 7.75 or [H+] of 17.8 nmoles.ll’ nearly inactivated carotid chemoreceptors. We also observed that chemoreceptor stimulation by hemorrhagic hypotension was considerably diminished by alkalosis (unpublished). At a constant normal blood pressure a multiplicative stimulus interaction determined receptor threshold (see fig. 6). It is unlikely that this effect of CO,-H+ on Pao2 threshold is
S. LAHIRI
260
AND R. G. DeLANEY
due to its direct effect on the receptor Po:. Fitzgerald and Parks ( 197 1) found an arterial Pco2 and [H+] threshold in their whole nerve preparation in the cat at about 21 tot-r and 27.5 nmoles.ll’, respectively, but they apparently did not consider the effect of arterial PoZ on these threshold values. In considering the chemoreceptor threshold, we have not made a strict distinction between near silence and absolute silence of receptors. This distinction is difficult to make because of the random nature of chemoreceptor impulse. If it can be made, one may find that the threshold response is a continuous function of the receptor p,, as defined by l/( PoZ -C) (Lloyd and Cunningham, 1963). MECHANISM
OF RECEPTOR
EXCITATION
Most of the proposed mechanisms of carotid body chemoreceptor activation by hypoxia have been linked, in one way or another, to changes in oxidative metabolism by hypoxia. Many metabolic inhibitors have been found to stimulate the carotid body (see Howe and Neil, 1972). It has also been reported that oxygen uptake is decreased with a decrease of Po, of arterial blood, or of perfusing or bathing fluids (Fay, 1970; Purves, 1970a; Leitner and Liaubet, 1971). The observations on the relationship between arterial P,, and oxygen uptake by other tissues have shown that arterial Po, has to be decreased to a considerably lower value (less than 40 torr) before oxygen uptake is compromised (e.g. Lubbers, 1968; Tuteur and Lahiri, 197 1; Whalen et al., 1973). Also, oxygen uptake of man is not diminished as a result of tolerable hypoxia (e.g. Pugh et ul., 1964). The critical Po, of isolated mitochondria from tissues like kidney has been found to be of the order of 1 torr (Stolp et al., 1971). The oxygen affinity for the usual terminal oxidase cytochrome a, has been reported to be less than 0.1 torr (e.g. Chance, 1957). Thus, if the foregoing relationship is applicable to the carotid body its 0, uptake would not be expected to be impaired with the high blood by a small decrease of arterial Po, from 100 torr particularly flow that the carotid body is known to have (Daly et al., 1954). To satisfy the hypothesis of metabolic change as a trigger one could postulate that PO2 at the receptor site is very low in the face of high arterial and venous Paz, or the mitochondrial oxidative system is modified in such a way that metabolism is reduced even at a high PO2 or an appropriate combination of both. It is possible that the high oxygen uptake is not directly linked with the excitation process. The component of 0, uptake which is sensitive to small changes in arterial Po, might be concerned with the metabolism, for example, of catecholamines. This process in turn may lower receptor Po, leading to a decreased oxygen uptake and excitation of the receptor. On the other hand, it could be that diminished 0, uptake has little to do with the initiation of impulses, because the sympathectomized carotid body which retains a normal pattern of stimulus response shows an oxygen uptake independent of a considerable change in Pa,, (Purves, 1970b). Recently, Mills and Jijbsis (1972) produced spectrophotometric evidence that there are two components of cytochrome a3 in the carotid body in the cat - one with high 0, affinity, the usual component, and another, with low O2 affinity. According
CAROTID
CHEMORECEPTOR
02-co,
INTERACTION
261
to Mills and Jobsis, the cytochrome a3 with low 0, affinity in the carotid body functions as the O2 sensor. They reported that with a decrease of perfusate PO2 from the normal value this oxidase was reduced and this was associated with the increase in the activity of carotid chemoreceptors. But there is no certainty that the receptor Po, was close to the perfusate Po2, as the authors presumed. On the other hand, the finding that oxygen uptake of the saline perfused carotid body was lower (Fay, 1970) than that in vivo suggests that there might have been a large extravascular Po, gradient in some areas of the perfused carotid body of Mills and Jobsis ( 1972). The Po, at the receptor whose activity was recorded could, in effect, have been very low. Mills and Jobsis (1972) did not take the effect of CO,-H+ on chemoreceptor activity into account. If CO,-H + were to act through the sensor cytochrome a3 it would be expected that O2 equilibria of the cytochrome would be influenced by CO,-H+. No such data on this component of cytochrome a3 are available but the usual cytochrome a3 does not show any measurable effect of [H+] in the physiological range (Wald and Allen, 1957; Chance and Conrad, 1959). It is possible, however, that CO,-H+ stimulates the same chemoreceptor by a completely different mechanism (see Fitzgerald and Parks, 1971). But there is no convincing evidence showing that the effect of CO,-Hf could be dissociated from the effect of hypoxia (see .Howe and Neil, 1972). Goodman (1974) who studied single carotid chemoreceptors simultaneously in pairs, concluded that the mechanism of impulse generation was common to both hypoxia and hypercapnia. In this context it is interesting to note that Paintal (1967) speculated that the Po, sensor of chemoreceptors functioned as a mechanoreceptor but his hypothesis does not take the effect of CO,-H+ and the multiplicative stimulus interaction into account. In a long series of papers Eyzaguirre and his colleagues (cf. Eyzaguirre et al., 1972) produced evidence that acetylcholine may be involved in generating the spike potential - a view not accepted generally (see Torrance, 1974). However, the question how hypoxia initiates the release of acetylcholine is not clear. Several authors have reviewed the subject recently (Biscoe, 1971; Howe and Neil, 1972; Torrance, 1974). None of the existing theories seems to explain adequately the results of the present study. We, therefore, propose a hypothesis in order to take into account the phenomenon of multiplicative 02C02 interaction as seen in the threshold and sensitivity of the chemoreceptors. An attempt is also made to explain the stimulatory effect of CO,-H+ on the activity of chemoreceptors at a nonhypoxic arterial Po2, and the plateau in the response to CO,-H+ during hypoxia. In proposing the hypothesis an assumption is made that a series of events occurs between chemoreception and final triggering of a spike potential. The hypothesis concerns the crucial question of how the stimulus is sensed by the receptor. The observations showing a multiplicative effect of one stimulus upon another suggest a mechanism common to both hypoxia and CO,-H+. This common mechanism may involve a single step or more than one step in the series of events leading to a discharge.
262
S. LAHIRI
AND R. G. DeLANEY
Because of the common nature, it could be argued that the sensor is primarily an oxygen receptor and the effect of CO,-H+ is secondary to it, or the sensor is a COzpHf receptor and oxygen regulates the stimulus strength at the receptor site. We postulate that the receptor unit is presumably located in the membrane of glomus cells. The unit is a chromophore group which reversibly binds with 0, and which may play a pivotal role in chemoreception. The receptor is sensitive to arterial Po, because arterial Po, determines oxygenation of the receptor unit, and thus its conformation. This structural change may then, for example, facilitate release of a transmitter which eventually activates sensory fibers. It is presumed that the receptor is situated close to arterial blood, and the receptor Po, bears a close relation with arterial Po, rather than Po, of the rest of the carotid body tissue. The observation that the steady-state tissue Po, of the carotid body was often autoregulated when arterial Po, was varied but the activity of chemoreceptors varied with arterial Po, (Acker et al., 1973; H. Acker, personal communication) lends support to such a notion. An increase in acidity at the receptor site decreases its affinity for 0, (analogous to Bohr effect), thereby decreasing the concentration of O,-bound receptor. This decrease in O,-bound receptor causes a conformational change initiating or altering activity of sensory fibers even though the receptor P,, may remain unchanged. The relationship betwgcn activity of a single unit and arterial Po, at different levels of arterial Pco, or pH could be analyzed in terms of 0, equilibrium curves. It is assumed that the maximum activity in steady states represents maximum steady-state O2 desaturation of the receptors during severe arterial hypoxia. The observed activity of the receptor at various intermediate levels of arterial Po, expressed as percent of the maximum activity is equivalent to desaturated active receptors. From these data O2 equilibrium curves could be constructed. The raw data from fig. 4 were used to construct these curves at three levels of arterial pH. The curves are shown in fig. 7. Application of the Hill empirical equation to these curves provides values of n which lie between 2.7 and 3.8. This result indicates that the number of binding sites interacting with ligand is greater than 3. These preliminary data suggest that the receptor unit could be a hemoglobin like polymeric chromophore molecule. It is interesting to note that the arterial Po, at 1000,;; 0, desaturation of the receptor at pH 7.25 was about 25 tort-. The corresponding receptor Po, might have been very low which excited the receptor maximally. However, the position of the curves would vary depending on the Pao, at which the maximum activity is observed. This example also illustrates that 100’yO0, saturation which silenced the receptor (threshold) was dependent on arterial pH as well. The foregoing hypothesis thus appears to account for the 02C02 multiplicative interaction which determines threshold and activity of the receptors. A similar hypothesis invoking a possible role of an extravascular pigment has been considered previously (Lloyd et al., 1968). The maximum plateau in COZ response during hypoxia suggests a maximal steady-state response at a maximal desaturated state of the receptors. Also, an
CAROTID
CHEMORECEPTOR
0 2-co
263
2 INTERACTION
Arterial
p-2
PH
60 RECEPTOR sop Ye 40
20
0 0
20
40
Fig. 7. Calculated arterial
O2 equilibrium
60
60 PO02
curves corresponding
to activity
pH. The data from fig. 4 were used for the calculation.
7.25 and Po, of 25 torr was taken as 100% 0s which is presumably
located
pH 7.25 were expressed
in a cellular
as percent
desaturation
membrane.
of the maximum
loo
(mmlig)
equivalent
The observed activity.
of chemoreceptors
The maximal
From
activity
at three levels of at arterial
of a hypothetical
activities
at higher
pH of
chromophore levels of Paol at
these data at an arterial
pH of 7.25
and similar data at other pH values the curves were constructed. These curves correspond receptor activity but closely resemble 0, equilibrium curves of a polymeric chromophore
to chemogroup with
a Bohr shift (see text for details).
apparent plateau at other levels of PO, may correspond to 0, equilibrium curves of a polymeric chromophore if plotted in terms of O2 desaturation against Paco2 at a constant Pao,. A change from one CO2 plateau to another can be achieved by changing the level of Pa,,. The plateau observed, for example, by Fitzgerald and Parks (1971) can thus be explained. The effect of CO,-H+ on the activity of chemoreceptors in the absence of arterial hypoxia is not easily understood in terms of this hypothesis unless it is assumed that CO,-H+ lowers O2 content of the receptors. It is possible that the phenomenon is analogous to Root shift (Root, 1931) found in 0, equilibration of hemoglobin in some marine fish. According to the Bohr shift the receptor is expected to show a reciprocal Haldane effect (see Kilmartin and Rossi-Bernardi, 1973). That is, oxygenation of the receptor would increase the local [H+] which in turn would tend to desaturate and excite it. The net result is a balance of these two opposite effects which illustrates an example of biological feedback mechanism at a molecular level.
S. LAHIRI
264
AND R. G. DeLANEY
As mentioned, the applicability of these data to the proposed hypothesis is not necessarily unique. Torrance (1975) has advanced a hypothesis which explains multiplicative C02-O2 interaction in the response of carotid chemoreceptors. He has proposed that the afferent nerve endings in the space between Type I and Type II cells are sensitive only to H ‘, and [H+] in the space is maintained stable by a pump which is PO2 sensitive. The pump raises (HCO;) in the space more than in arterial blood during hypercapnia, and hypoxia decreases the effectiveness of the pump. This system would give rise to [H+] in the space with changes in Pa,-.oz at different levels of Pao2 resembling the fan of CO2 response curves of chemoreceptors. According to the author this hypothesis also predicts a multiplicative O,-CO, interaction at the threshold because of the variation in the local [H+] with arterial PO2 for any arterial [H+]. The crucial part of this attractive scheme is a P,, sensitive bicarbonate pump. Our hypothesis focuses on the Po2-sensitive mechanism for the initiation of events, release of a transmitter, secretion of bicarbonate or whatever, leading to an impulse. Our working hypothesis is that a polymeric chromophore at a membrane site may function as an internal probe for 0, and initiate the process of excitation. Its reaction with 0, is reversible, unlike other proposed mechanisms in which 0, is irreversibly consumed. This distinction of reversibility is a unique feature of the hypothesis and is amenable to experimental verification. Acknowledgments
We are grateful to Dr. A. P. Fishman, Dr. T. F. Hornbein and Dr. R. W. Torrance for their critical review of the manuscript. Our thanks are due to Mr. A. Mokashi for his technical assistance. References Acker, H., H. P. Keller, ship between
D. W. Lubbers,
neuronal
activity
D. Bingmann,
of chemoreceptor
H. Schulze and H. Caspers
(1973). The relation-
fibers and tissue Po, of the carotid
body of the cat
during changes in arterial PO, and blood pressure. Pfliigers Arch. 343: 287-296. Biscoe, T. J., M. J. Purves and S. R. Sampson (1970). The frequency of nerve impulses in single carotid body chemoreceptor afferent fibers recorded in uiuo with intact circulation. J. Physiol. (London) 208: 121-131. Biscoe, T. J. (1971). Carotid
body:
structure
and function.
Physiol. Reo. 51: 437495.
Chance, B. (1957). Cellular oxygen requirements. Federation Proc. 16: 671-680. Chance, B. and H. Conrad (1959). Acid-linked functions of intermediates in oxidative J. Biol. Chem. 234: 15681570. Daly, M. DeB., C. J. Lambertsen
and A. Schweitzer
(1954). Observations
phosphorylation.
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flow
and oxygen utilization of the carotid body in the cat. J. Physiol. (London) 125: 67-89. Euler, U. S. von, G. Liljestrand and Y. Zotterman (1939). The excitation mechanism of the chemoreceptors of the carotid body. Skand. Arch. Physiol. 83: 132-152. Eyzaguirre, C. and J. Lewin (1961). Chemoreceptor activity of the carotid (London)
body of the cat. J. Physiol.
159 : 222-237.
Eyzaguirre, C., K. Nishi and S. Fidone Proc. 31: 1385-1393. Fay, F. S. (1970). Oxygen
consumption
(1972). Chemoreceptor of the carotid
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Fitzgerald,
R. S. and P. Parks
CHEMORECEPTOR
(197 I). Effect of hypoxia
in cats. Respir. Physi$.
dioxide Goodman,
N. W. (1974).
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02-co
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Some observations
on the homogeneity
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E. Neil (1972).
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Leitner,
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In: Enteroceptors,
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L. M. and M. J. Liaubet
J. Physiol.
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Springer-Verlag, pp. 47780. Joels, N. and E. Neil (1960). Chemoreceptor
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Hornbein, T. F., 2. J. Griffo and A. Roos (1961). Q uantitation of hypoxia and hypercapnia. J. Neurophysiol. 24: 561-568. Howe,
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(1971). Carotid
body oxygen
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B. B. and
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D. J. C. Cunningham In: The Regulation
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Lloyd. Oxford, Blackwell, pp. 331-349. Lloyd, B. B., D. J. C. Cunningham and R. C. Goode (1968). Depression of hypoxic man by sudden inspiration of carbon monoxide. In: Arterial Chemoreceptors, Torrance.
Oxford,
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U. C. Luft. G. Thews and E. Witzleb.
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A. S. and R. L. Riley (1966). Responses of aortic chemoreceptors. A. S. (1967). Mechanism of stimulation of aortic chemoreceptors
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Microelectrode
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I. A. Silver and J.
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STIMULUS
North-Holland Publishing Company, Amsterdam
INTERACTION IN THE RESPONSES OF CAROTID CHEMORECEPTOR SINGLE AFFERENT FIBERS’
BODY
S. LAHIRI and R. G. DeLANEY Cardiovascular-Pulmonary Division, Department of Medicine and Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, U.S.A.
Abstract. The characteristics
of steady-state
to independent
changes
was maintained
within the normal
to changes
both
in arterial
in arterial
responses
of single afferent
Po, and Pco, were investigated limits (115-130
Po, and
Pco,.
torr).
fibers of carotid
in cats. The arterial
Single chemoreceptor
The relationship
between
afferent
the activity
chemoreceptors blood
pressure
fibers responded
of chemoreceptors
and changes in arterial Pc,, was linear at a constant arterial Po,. The two stimuli showed multiplicative interaction. The activity approached zero (threshold) as arterial P,,, was decreased at a These response constant arterial PO,, a decrease in arterial Po, decreased the arterial Pco, threshold. characteristics mechanism formational reversibly
of a single fiber suggest by the two stimuli. change
that
The data
the sensory fit into
in the membrane-bound
receptor
an idea that
polymeric
may
be activated
the mechanism
chromophore
group
may which
through involve reacts
a single a conwith
0,
and shows a Bohr-shift. Arterial
Pco,
Interaction
Arterial
Po,
Mechanism
Carotid
chemoreceptors
Threshold
between
O2 and COZ stimuli
of excitation
Despite several studies on the response of carotid chemoreceptor to changes in hypoxic and hypercapnic stimuli, independently of each other or in combination, (Euler et al., 1939; Eyzaguirre and Lewin, 1961; Hornbein et al., 1961; Biscoe et al., 1970; Sampson, 1970; Fitzgerald and Parks, 1971) the quantitative relationship between the effect of one stimulus on that of the other is not clear. This, in part, is due to the fact that single fiber preparations were not used in the investigation of the stimulus-response relationship. The interplay between the two stimuli might occur in a single fiber and involve recruitment of new fibers which are possibly activated at a higher level of arterial stimulus than others. If the two processes occurred simultaneously in a multi-fiber preparation quantitation of each type of response would not be possible. Therefore, the investigations of Hornbein et al. (1961) and Fitzgerald and Parks (1971) on the relationship between changes in Accepted for publication 4 April 1975. 1 Supported in part by USPHS Grant
HL-08805. 249
250
S. LAHIRI
AND R. G. DeLANEY
arterial Po, and Pco, and the average activity of the entire carotid sinus nerves in the cats provided little information as to the sensitivity of individual receptors and did not distinguish between the response of a single receptor and recruitment of receptors. Eyzaguirre and Lewin (1961) studied activity of carotid chemoreceptors in multi-fiber preparations but the interpretation of their experiments was further complicated by relating the nervous response to inspired gas concentration rather than to arterial PO,, Pco, and [H+] which provide a better basis of quantitative description of stimulus level. Biscoe er al. (1970) mvestigated the response of single carotid chemoreceptor afferent fibers to either changes in arterial Po, or to PcoZ and [H+] but did not examine the question of interplay between the two stimuli. Joels and Neil (1961) and Eyzaguirre and Lewin (1961) did address themselves to the question but did not provide a quantitative description. They merely showed that a single afferent fiber was capable of responding to both stimuli. In the present study we investigated the characteristics of response of carotid body single afferent fibers to the two stimuli while holding arterial blood pressure within normal limits. We found that each single fiber responded to changes both in arterial PO, and Pco2, and that the response to one stimulus was enhanced by the other. Although there were differences in mean frequency of discharge, there was a basic similarity in the pattern of response among the chemoreceptors. The recent report of Goodman (1974) is in accord with this observation. Also, the receptors showed a threshold for stimulation by either arterial Pco2 or [H+]. A decrease in arterial PO, decreased arterial Pc,,/[H+] threshold. Materials and methods Experiments were done in 55 adult cats (2.5-3.0 kg) anesthetized with a-chloralose (60 mg . kg- r, intravenously). Supplemental doses of the anesthetic were administered as needed. Arterial vessels were prepared for monitoring blood pressure, sampling blood, and a brachial vein for the administration of fluids. The rectal temperature was maintained between 38-39 “C. The trachea was intubated and the larynx and esophagus were removed to expose both carotid bifurcations. PREPARATION
OF CHEMORECEPTOR
AFFERENTS
The left carotid sinus nerve was exposed ventromedially, a small strand of the sinus nerve was snipped off at the glossopharyngeal junction, and single afferent units were obtained by conventional methods. RECORDING
SYSTEM
Saline wick electrodes were employed to record carotid chemoreceptor activity. Action potentials were preamplitied in two steps, and the output was then split and fed to: (1) a four-trace amplifier (Tektronix 3A74) in the dual-beam oscilloscope set to trigger in the alternate mode on the rising phase of the chemoreceptor action potential so that the size, shape and duration of single spikes could be characterized
CAROTID
CHEMORECEPTOR
02-co2
INTERACTION
251
and recorded on moving film (Kymograph Camera, C4N, Grass Instrument Co., Quincy, Mass.); (2) a galvanometer (flat response curve to 1650 Hz) in a recording system (Honeywell 1508A Visicorder, Denver, Colo.); (3) a Grass AM8 audiometer; and (4) a pulse height discriminator and an integrator (Frederick Haer & Co., Ann Arbor, Mich.) which summed impulses per second. For analytical use, the impulses were also manually counted over a 60-second period and expressed as a mean activity per second. TESTS FOR CHEMORECEPTOR
ACTIVITY
Action potentials were considered to arise from a chemoreceptor if their activity changed promptly upon changing inspired P,,. The chemoreceptor fiber also responded promptly and briskly to a breath of CO,-enriched air. In some experiments, the cats were paralyzed with gallamine triethiodide (Flaxedil: American Cyanamid Co.), 10 mg.kg-‘, intravenously. As ventilation finally stopped, the discharge rate of the fiber increased. This activity decreased upon ventilating the animal by a respiratory pump. INSPIRED
AND END-TIDAL
GAS
The inspired gas mixtures were administered to the cat through a T-tube connected to the pneumotachograph when breathing spontaneously or through a respiratory pump(Harvard Apparatus, Model 607) when paralyzed. The tracheal concentrations of 0, and CO;? were continuously monitored by a Pulmonary Function Oxygen Monitor (Westinghouse Model 211, Pittsburgh, Pa.) and an infrared capnograph (Godart, Statham Instruments, Oxnard, Calif.) respectively. The O2 and CO, sensors were calibrated. SYSTEMIC
ARTERIAL
BLOOD
PRESSURE
The systemic blood pressure was measured by a strain gauge transducer-amplifier system which was calibrated using a mercury manometer. The mean blood pressure was maintained within f 10 torr of the initial value by the administration of 0.9% NaCl or blood from a donor cat by slow intravenous drip throughout the experiment. ARTERIAL
Blood
BLOOD
samples
GASES AND
pH
(1.5 ml) from the brachial
artery
were withdrawn
at appropriate
times, and their P,,, Pco2 and pH were immediately determined at 38 “C using a Radiometer system (Radiometer, Copenhagen). The 0, and CO* electrodes were calibrated with blood equilibrated with gas mixtures of two different compositions. The pH electrode was calibrated with buffers of two different pH. PROCEDURE
The relationship between steady-state arterial PO,, PcoZ and pH and single afferent fiber activity was determined in two types of animal preparations: either in a spontaneously breathing cat or in a paralyzed cat respired by a pump. In the
252
S.
LAHIRI
AND R. G. DeLANEY
spontaneously breathing cats arterial blood gas changes were made by changing the inspired gas mixture alone. In this series the lower range of arterial PcoZ was limited to a few torr below normal, e.g. about 24 torr. In the pump-assisted preparation the arterial blood gas changes were made both by changing the rate and stroke volume of the pump and by varying the inspired gas composition. In these animals arterial Pco2 was lowered to values of the order of 10 torr, and thus investigation of chemoreceptor response over a wide range of stimulus was possible. A change in end-tidal gas tensions to pre-determined levels was associated with a change in the chemoreceptor activity which stabilized in less than 5 min. At this time an arterial sample was taken in order to relate mean activity of the afferent fiber to arterial Po2, Pco2 and [H+]. The agreement between end-tidal and arterial Pco2 values was usually very close but the arterial Po2’s were lower than the end-tidal Po2’s. The end-tidal values were used as a guide to arterial values. For defining the stimulus-response relationship over a wide range, arterial Po, was changed while arterial Pcoz was held constant. Subsequently, the arterial Pco2 was changed to another level where it was held constant while arterial Po, was again varied. For analytical purpose, the mean activity was then related to arterial Pco, at constant arterial Po,. A complete study required about 2 hours of recording. STABILITY
OF THE PREPARATION
The stability of response of the chemoreceptor preparation to a given stimulus, and the stability of acid-base of arterial blood need mention. Some of the afferent fibers failed after an initial response to hypoxia, and the study on these fibers could not be completed. To assess any development of instability during the progression of the experiment, arterial Po, was changed from one level to another maintaining arterial Pco2 constant. These transient responses also formed a part of another experimental design. Those chemoreceptor responses which were not reproducible were not used. There were occassions when the response to the same stimulus changed with time. The acid-base status of arterial blood was checked frequently by using Pro* and pH data. Within the test period the arterial acid-base did not change appreciably. However, after severe hypoxia ( < 30 torr) metabolic acidosis developed. Since this acidosis distorted the relationship between arterial Pco2 and chemoreceptor activity, the particular experiment was terminated as soon as it appeared. Results IDENTIFICATION
OF SINGLE
FIBER
ACTIVITY
The single fiber spikes were determined by the identity of their size, shape and duration. Figure 1 illustrates activity of two single fibers. The spikes never superimposed in our single fiber preparation. Figure 2 illustrates the response of a single fiber (large spikes) to changes in arterial Po, and PcoI. The effect of a decrease in arterial Po, at two levels of arterial Pco2 (28 and 34.5 torr, respectively) under steady-state conditions are shown. The
CAROTID
Fig. 1. Identification different
CHEMORECEPTOR
of single afferent
preparations.
The large
spikes
because
0 &o
chemoreceptor
fiber discharge.
A and B depict
in each case were considered
of their identity
253
2 INTERACTION
to originate
spikes from two
from a single fiber
of shape, size and duration.
paC02
Pa
A
02 tarr 490
9
95
1 C
43
tarr
28
I
II
IfI
II
I
1I
I
‘I
I
D
495
E
97
F
42
35
I G
., _., . .__
._.
-.
.
..
.
I . . .._.. _ 25 loop
I SEC.
Fig. 2. Response changes
in activity
of a single fiber (large as arterial
spikes)
PO, decreased
to changes
in arterial
PO, and Pc,,.
from 490 to 43 torr at arterial and G from 495 to 24.5 torr at arterial Pro2 of 34.5 torr (see table
A, B and C show
P,, of 28 torr, and D, E, F 1 and text for details).
relevant blood gas data and the mean activities of the single fiber are given in table 1. It is clear that as arterial P,, decreased the rate of activity in the single fiber increased (at a constant arterial Pc,,): A, B and C at arterial Pc,,=28 torr; D, E, F and G at arterial P co2 = 34.5 torr; the rate of increase was greater at lower arterial P,,. Hypercapnia potentiated the effect of hypoxia. Of interest is the observation that at at arterial P,, of about 500 torr only one unit was active (compare A and D in fig. 2), its response increasing with the level of hypercapnic stimulus. A decrease in arterial P,, was not only associated with a similar increase in the activity of the unit (compare B and D in fig. 2) but was also accompanied by evidence of discharge from a new unit.
254
S. LAHIRI
AND R. G. DeLANEy
TABLE Stimulus
response
Serial
Impulses
no.
(set-‘)
of single afferent Arterial
fiber of carotid
1
body in the cat (These data correspond Arterial
Brachial
PH
pressure
Arterial
(mean 0.2
490
28.0
7.385
115
2.1
95
28.3
7.380
113
5.3
43
28.0
7.390
119
1.9 4.4
495 97 42
35.0 34.5
7.310
115
34.5
7.320 7.315
114 120
34.0
7.310
105
15.0 24.1
24.5
25r--------
to fig. 2).
arterial torr)
p"O, = 35 torr
/
20-
15IMPULSES S-SC-'
IO-
20
30 PaCO,,
Fig. 3. Effect on single fiber activity of changes in arterial level of arterial PO,, an increase in arterial Pc,, increased at lower arterial
torr P,,? at four levels of arterial P,,. At any the rate of activity, this effect being greater P,,.
CAROTID
QUANTITATIVE
EFFECT
CHEMORECEPTOR
OF CHANGE
0 2-co
IN ARTERIAL
Pcol
255
2 INTERACTION
AND
PO,
To examine the effect of hypocapnia and alkalosis on the activity in a single unit, the animals were paralyzed and hyperventilated by a respiratory pump. One example of this relationship between arterial Pco2 and the mean activity of a single unit at four levels of arterial Po, is shown in fig. 3. It is clear that at all levels of arterial P,, with a decrease in arterial Pco2 a decrease in the mean activity of the chemoreceptor was approximately linear. At an arterial P,, of 400 torr, the activity approached zero as arterial PcoZ was decreased to about 13-14 torr (threshold stimulus). At lower levels of arterial PoZ, activity of the chemoreceptor unit also decreased towards zero when arterial Pco2 was decreased to values lower than 13 torr. Hypoxia also modified the effect of arterial Pco2 in the following conspicuous ways: at any level of arterial Pco2, the activity was greater at a lower arterial P,? and the activity for a given change of arterial Pco2 was also greater during hypoxia. Thus, there was a clear multiplicative interaction between the two stimuli. It is interesting to note that at Pao2 of 35 torr during hypercapnia above 30 torr the increase in response was less than linear. Figure 4 shows the effect of change of arterial Pco2 and PO2 over a wide range. At arterial PCoZof the order of 10 torr the response to moderate hypoxia (48 torr) practically disappeared. The response characteristics appeared similar to those seen in fig. 3. The receptor showed a strong multiplicative stimulus interaction. Also, the
Pa = 02
CA T# x-x
25
6/
torr
IMPULSES set“
0
40
20
60
80
100
Fig. 4. Effects on single fiber activity of wide changes in arterial Pc,, and Po,. In addition to the effects described in fig. 3 these data show that at low levels of arterial Pco, the rate of activity approaches zero indicating
threshold
stimuli.
At arterial
Po,=25
torr, the activity
P co1 increased.
appeared
to level off as arterial
256
S. LAHIRI
AND R. G. DeLANEY
15PCQ = 24 forr
I
IOIMPULSES
set-’ 5-
CAT # 67
0
30
20
IO
40
ARTERIAL
Fig. 5. Relationship Increases
between
in the mean activity arterial
rate of single fiber activity are associated
PCo2. This relationship
[H+],
50
nmoles,
and arterial
with the increases is enhanced
[H+]
in arterial
by decreases
3
I-’
at five levels of arterial [H+] induced
in arterial
by increases
Po,. in
P,,.
receptor showed Pace? threshold which was lower with lower Pao2. The experimental observations above arterial Pcoz of 50 torr were only a few, and therefore the description of the linear response over this range of arterial Pco2 may not be justifiable. Clearly, there was a plateau in the response to PaLco2at a Paoz of 25 torr. Although we did not explore the extent to which the effect of arterial Pco2 was due to the CO, molecule per se or its effect on arterial [H +I, we can examine the response of the chemoreceptor unit to arterial [H+] that occurred concomitantly with the change of arterial Pco,. The relationship between chemoreceptor response to arterial [H+] at five levels of arterial P,, is shown in fig. 5. At each level of arterial Po, the relationship, beginning from near zero activity, appeared linear. It is also apparent that at the level of [H ‘1 of 15-30 nmoles.ll’ the chemoreceptor activity decreased nearly to zero. This arterial [H ‘1 threshold appeared to depend on arterial Po2, the threshold [H+] being lower at lower arterial Po, (see later). VARIATION
OF COMBINATION
EFFECTS OF ARTERIAL
P,,
AND PcoJ
An apparent variation of the interactive effects of arterial Po, and arterial Pco2 was found among several of the units investigated. There was one unit which responded feebly to a rise in arterial PC-.,. Increases of arterial Pco2 from 35 to 50 torr increased the activity by less than 1 impulse. set- ’ at arterial Po, of 89 and 515 torr. At an arterial P,, of 40 torr, the impulse frequency went up by 3 set-’ for a similar increase in arterial Pcol from 35 to 50 torr. This unit, however, responded vigorously to a change in arterial Po, from 515 to 26 torr at Paco2 of 35 torr by increasing the activity from 0.6 to 18 impulses. set ’ . Thus, there appeared to be little multiplicative
CAROTID
CHEMORFKEPTOR
0 2&o
257
2 INTERACTION
stimulus response in this fiber. However, it is possible, though unlikely, that a plateau was already reached in its response to Pacoz. Unfortunately, this fiber was not studied at Paco2 lower than 35 torr. Other types of responses were encountered in fibers which showed inherently a low frequency of discharge. In one multi-fiber strand the submaximal activity of a single fiber in steady-state seldom exceeded 3 set-‘. In the same strand, there was another fiber which responded with a frequency of 18 see-’ at the same stimulus level. However, both of them showed the same pattern of multiplicative stimulus interaction. In summary, all 38 single afferent fibers showed an increase in the rate of discharge as arterial PO, was decreased from 500 to 30 torr at the normocapnic level. All except three of these fibers showed considerable multiplicative stimulus interaction, and their sensitivities to any change in arterial stimulus were very similar, although there were differences in mean frequency of impulses. THRESHOLD
STIMULI
Most of the single fibers examined showed an arterial Pco2 or [H+] threshold, i.e. a value of arterial Pco2 and [H+] above which chemoreceptor activity began to increase and below which the activity approached zero in the steady state. In the artificially ventilated animals these values were actually reached at several levels of arterial Po,. The dependence of arterial PcoZ and [H+] threshold on arterial P,, was obvious. The results from one experiment are shown in a threshold Paco2-Pao2 diagram in fig. 6. Each point in the diagram represents a pair of Paco2 and Pao2 at which the chemoreceptor was practically silent. Clearly, Paco2 threshold decreased with a decrease of Paoz. The decrease in the threshold Paco2 was greater for a given decrease in Pao, as the intensity of hypoxia increased. In the same preparation
20
1
Pace, XA
torr IO -
_-x-+-
x/ x CdTX6,
0 0 Fig. 6. Threshold sents a combination
I 20
stimulus
I 40 for carotid
of Pa,,,
I 60
I 80
chemoreceptors
and Pa,,
I 100
1 120
Pao2,
torr
shown
, 140
in Pa co,-Pa,,
I , 180 j,,
I 160
diagram.
at which the receptor was just active. was lower at a lower Pa,,.
Clearly
Each point Pa,,,
repre-
threshold
258
S.
another
fiber showed
pattern
of the threshold
LAHIRI
a higher Pa,,,-Pa,,
AND R. G. DeLANEY
Paco2 threshold
at the same
Pao,,
although
the
curve was similar.
Discussion MULTIPLICATIVE
STIMULUS
INTERACTION
This study showed response characteristics of single afferent fibers from the carotid chemoreceptors in the cat. Apparently, there was a spectrum of stimulus-response relations for the carotid chemoreceptors. There were variations in multiplicative stimulus interaction, arterial P co2-PoZ threshold and in mean frequency of discharge. However, if differences in mean frequency of discharge were taken into account, the similarities between the response curves of most of the receptors were quite clear. This finding is in conformity with the claim of homogeneity of response of chemoreceptor fibers (Goodman, 1974). Occasional deviation from a homogeneity indicated that the basic phenomenon might be influenced by many factors, such as variations of innervation, size and types (medullated us. nonmedullated) of fibers, number of steps involved in the excitation process, etc. In general, the observed stimulus-response curves resembled those reported by Hornbein et al. (1961) Eyzaguirre and Lewin (1961) and Fitzgerald and Parks (1971) in multi-fiber or whole nerve preparations. None of the fibers showed any inhibition of activity . with increase in arterial Pco2, unlike the observation of Paintal and Riley (1966) on aortic chemoreceptor fiber. However, Sampson and Hainsworth (1972) and Fitzgerald( R. S. Fitzgerald, personal communication) found that increases in arterial activity. P car increased aortic chemoreceptor An explanation of the phenomenon of multiplicative stimulus interaction at the level of carotid body chemoreceptors requires an examination of the effect of the stimuli on carotid body blood flow and oxygen uptake. Purves (1970a) reported small changes in blood flow with changes in the stimulus level but it has been reported that a considerable change in blood flow did not change carotid body tissue P,, (Acker ef al., 1973) nor the activity of carotid chemoreceptors (see Biscoe, 1971). Thus it is unlikely that any change in blood flow associated with the experimental procedure materially contributed to the chemoreceptor response reported in this paper. According to Purves (1970a) both hypoxia and hypercapnia decreased carotid body oxygen uptake, and he supported the previously postulated contention (see Howe and Neil, 1972) that this decreased oxygen uptake might be the mechanism of chemoreceptor stimulation. But it is not clear if his data on 0, uptake corresponded to the hypoxic-hypercapnic multiplicative interaction we observed in the activity of chemoreceptors. Also, oxygen uptake of the carotid body after sympathectomy was found to be independent of Pao, (Purves, 1970b), although it is known that the pattern of response of chemoreceptors is not changed by sympathectomy (see Biscoe, 1971; Torrance, 1974). Thus, oxygen uptake need not be crucial to receptor excitation.
CAROTID
THRESHOLD
CHEMORECEPTOR
02&oz
INTERACTION
259
STIMULUS
Various changes
authors who investigated the response of peripheral chemoreceptors to in arterial blood gas tension addressed themselves to the question of arterial Po, threshold. Defining threshold as the Po, at which the rate of discharge begins to change with the changes of Po, Biscoe et al. (1970) assigned a range of arterial PO, of 140-440 torr (presumably at normal arterial Pco, and pH) for carotid chemoreceptors, and Paintal and Riley (1966) at inspired P,, of 8&160 torr for aortic chemoreceptors. Our results are generally in conformity with those of Biscoe et values of Paintal and Riley could al. (1970). The unusually low P,, threshold be due to low arterial Pco2 and pH in their preparation. However, according to those authors the effect of arterial CO,-H+ on aortic chemoreceptors was not stimulatory, and therefore any low arterial CO*-H f could not have influenced arterial P,, threshold. On the other hand, in the absence of a potentiating effect of C02-H+, arterial P,, threshold for the aortic chemoreceptors could be lower than that of the carotid chemoreceptors. The variation of arterial Po, threshold for different receptors at a constant arterial P co2 and pH observed in this work could be due to quantitative differences in the mechanistic component(s) ultimately responsible for the initiation of impulse. However, an explanation might be found in the relation between arterial PO, and receptor Paz. The POr at different receptors may not be the same at the same arterial Po2, although it may bear a constant relation with arterial PO,. Unfortunately, we do not know the site of the receptor, nor its PO,. There are, however, two reports of measurements of carotid body tissue PO, using 0, electrodes (Whalen and Nair, 1973; Acker et al., 1973). But the meaning of their findings is not clear. Whalen and Nair (1973) reported that, in a cat breathing air, tissue PO, of carotid body decreased from 80 to 40 torr as the electrode was driven from the outer to inner zone. But Acker et al. (1973) found opposite results; that is, O-10 torr at the periphery and 60-70 torr at the center. However, they seem to agree that tissue PoL, although it may bear a constant relation with arterial PO,. Unfortunately, we do not know the site of the receptor, nor its PO,. There are however, two reports of could vary from one location to another. This variation of receptor PO2 might account for the observed differences in the apparent Pa,,-Pa,,, threshold. We do not know if carotid body tissue PO, is altered by changes in arterial Pco, and pH but we found that arterial PO, threshold was dependent on arterial Pco2 and [H+]. Low enough arterial Pco2 (about 10 torr) and arterial [H+] (about 12-22 nmoles.l-‘) either abolished or nearly abolished the response of most of the chemoreceptors to moderate hypoxia. Joels and Neil (1960) briefly reported that perfusate pH of 7.75 or [H+] of 17.8 nmoles.ll’ nearly inactivated carotid chemoreceptors. We also observed that chemoreceptor stimulation by hemorrhagic hypotension was considerably diminished by alkalosis (unpublished). At a constant normal blood pressure a multiplicative stimulus interaction determined receptor threshold (see fig. 6). It is unlikely that this effect of CO,-H+ on Pao2 threshold is
S. LAHIRI
260
AND R. G. DeLANEY
due to its direct effect on the receptor Po:. Fitzgerald and Parks ( 197 1) found an arterial Pco2 and [H+] threshold in their whole nerve preparation in the cat at about 21 tot-r and 27.5 nmoles.ll’, respectively, but they apparently did not consider the effect of arterial PoZ on these threshold values. In considering the chemoreceptor threshold, we have not made a strict distinction between near silence and absolute silence of receptors. This distinction is difficult to make because of the random nature of chemoreceptor impulse. If it can be made, one may find that the threshold response is a continuous function of the receptor p,, as defined by l/( PoZ -C) (Lloyd and Cunningham, 1963). MECHANISM
OF RECEPTOR
EXCITATION
Most of the proposed mechanisms of carotid body chemoreceptor activation by hypoxia have been linked, in one way or another, to changes in oxidative metabolism by hypoxia. Many metabolic inhibitors have been found to stimulate the carotid body (see Howe and Neil, 1972). It has also been reported that oxygen uptake is decreased with a decrease of Po, of arterial blood, or of perfusing or bathing fluids (Fay, 1970; Purves, 1970a; Leitner and Liaubet, 1971). The observations on the relationship between arterial P,, and oxygen uptake by other tissues have shown that arterial Po, has to be decreased to a considerably lower value (less than 40 torr) before oxygen uptake is compromised (e.g. Lubbers, 1968; Tuteur and Lahiri, 197 1; Whalen et al., 1973). Also, oxygen uptake of man is not diminished as a result of tolerable hypoxia (e.g. Pugh et ul., 1964). The critical Po, of isolated mitochondria from tissues like kidney has been found to be of the order of 1 torr (Stolp et al., 1971). The oxygen affinity for the usual terminal oxidase cytochrome a, has been reported to be less than 0.1 torr (e.g. Chance, 1957). Thus, if the foregoing relationship is applicable to the carotid body its 0, uptake would not be expected to be impaired with the high blood by a small decrease of arterial Po, from 100 torr particularly flow that the carotid body is known to have (Daly et al., 1954). To satisfy the hypothesis of metabolic change as a trigger one could postulate that PO2 at the receptor site is very low in the face of high arterial and venous Paz, or the mitochondrial oxidative system is modified in such a way that metabolism is reduced even at a high PO2 or an appropriate combination of both. It is possible that the high oxygen uptake is not directly linked with the excitation process. The component of 0, uptake which is sensitive to small changes in arterial Po, might be concerned with the metabolism, for example, of catecholamines. This process in turn may lower receptor Po, leading to a decreased oxygen uptake and excitation of the receptor. On the other hand, it could be that diminished 0, uptake has little to do with the initiation of impulses, because the sympathectomized carotid body which retains a normal pattern of stimulus response shows an oxygen uptake independent of a considerable change in Pa,, (Purves, 1970b). Recently, Mills and Jijbsis (1972) produced spectrophotometric evidence that there are two components of cytochrome a3 in the carotid body in the cat - one with high 0, affinity, the usual component, and another, with low O2 affinity. According
CAROTID
CHEMORECEPTOR
02-co,
INTERACTION
261
to Mills and Jobsis, the cytochrome a3 with low 0, affinity in the carotid body functions as the O2 sensor. They reported that with a decrease of perfusate PO2 from the normal value this oxidase was reduced and this was associated with the increase in the activity of carotid chemoreceptors. But there is no certainty that the receptor Po, was close to the perfusate Po2, as the authors presumed. On the other hand, the finding that oxygen uptake of the saline perfused carotid body was lower (Fay, 1970) than that in vivo suggests that there might have been a large extravascular Po, gradient in some areas of the perfused carotid body of Mills and Jobsis ( 1972). The Po, at the receptor whose activity was recorded could, in effect, have been very low. Mills and Jobsis (1972) did not take the effect of CO,-H+ on chemoreceptor activity into account. If CO,-H + were to act through the sensor cytochrome a3 it would be expected that O2 equilibria of the cytochrome would be influenced by CO,-H+. No such data on this component of cytochrome a3 are available but the usual cytochrome a3 does not show any measurable effect of [H+] in the physiological range (Wald and Allen, 1957; Chance and Conrad, 1959). It is possible, however, that CO,-H+ stimulates the same chemoreceptor by a completely different mechanism (see Fitzgerald and Parks, 1971). But there is no convincing evidence showing that the effect of CO,-Hf could be dissociated from the effect of hypoxia (see .Howe and Neil, 1972). Goodman (1974) who studied single carotid chemoreceptors simultaneously in pairs, concluded that the mechanism of impulse generation was common to both hypoxia and hypercapnia. In this context it is interesting to note that Paintal (1967) speculated that the Po, sensor of chemoreceptors functioned as a mechanoreceptor but his hypothesis does not take the effect of CO,-H+ and the multiplicative stimulus interaction into account. In a long series of papers Eyzaguirre and his colleagues (cf. Eyzaguirre et al., 1972) produced evidence that acetylcholine may be involved in generating the spike potential - a view not accepted generally (see Torrance, 1974). However, the question how hypoxia initiates the release of acetylcholine is not clear. Several authors have reviewed the subject recently (Biscoe, 1971; Howe and Neil, 1972; Torrance, 1974). None of the existing theories seems to explain adequately the results of the present study. We, therefore, propose a hypothesis in order to take into account the phenomenon of multiplicative 02C02 interaction as seen in the threshold and sensitivity of the chemoreceptors. An attempt is also made to explain the stimulatory effect of CO,-H+ on the activity of chemoreceptors at a nonhypoxic arterial Po2, and the plateau in the response to CO,-H+ during hypoxia. In proposing the hypothesis an assumption is made that a series of events occurs between chemoreception and final triggering of a spike potential. The hypothesis concerns the crucial question of how the stimulus is sensed by the receptor. The observations showing a multiplicative effect of one stimulus upon another suggest a mechanism common to both hypoxia and CO,-H+. This common mechanism may involve a single step or more than one step in the series of events leading to a discharge.
262
S. LAHIRI
AND R. G. DeLANEY
Because of the common nature, it could be argued that the sensor is primarily an oxygen receptor and the effect of CO,-H+ is secondary to it, or the sensor is a COzpHf receptor and oxygen regulates the stimulus strength at the receptor site. We postulate that the receptor unit is presumably located in the membrane of glomus cells. The unit is a chromophore group which reversibly binds with 0, and which may play a pivotal role in chemoreception. The receptor is sensitive to arterial Po, because arterial Po, determines oxygenation of the receptor unit, and thus its conformation. This structural change may then, for example, facilitate release of a transmitter which eventually activates sensory fibers. It is presumed that the receptor is situated close to arterial blood, and the receptor Po, bears a close relation with arterial Po, rather than Po, of the rest of the carotid body tissue. The observation that the steady-state tissue Po, of the carotid body was often autoregulated when arterial Po, was varied but the activity of chemoreceptors varied with arterial Po, (Acker et al., 1973; H. Acker, personal communication) lends support to such a notion. An increase in acidity at the receptor site decreases its affinity for 0, (analogous to Bohr effect), thereby decreasing the concentration of O,-bound receptor. This decrease in O,-bound receptor causes a conformational change initiating or altering activity of sensory fibers even though the receptor P,, may remain unchanged. The relationship betwgcn activity of a single unit and arterial Po, at different levels of arterial Pco, or pH could be analyzed in terms of 0, equilibrium curves. It is assumed that the maximum activity in steady states represents maximum steady-state O2 desaturation of the receptors during severe arterial hypoxia. The observed activity of the receptor at various intermediate levels of arterial Po, expressed as percent of the maximum activity is equivalent to desaturated active receptors. From these data O2 equilibrium curves could be constructed. The raw data from fig. 4 were used to construct these curves at three levels of arterial pH. The curves are shown in fig. 7. Application of the Hill empirical equation to these curves provides values of n which lie between 2.7 and 3.8. This result indicates that the number of binding sites interacting with ligand is greater than 3. These preliminary data suggest that the receptor unit could be a hemoglobin like polymeric chromophore molecule. It is interesting to note that the arterial Po, at 1000,;; 0, desaturation of the receptor at pH 7.25 was about 25 tort-. The corresponding receptor Po, might have been very low which excited the receptor maximally. However, the position of the curves would vary depending on the Pao, at which the maximum activity is observed. This example also illustrates that 100’yO0, saturation which silenced the receptor (threshold) was dependent on arterial pH as well. The foregoing hypothesis thus appears to account for the 02C02 multiplicative interaction which determines threshold and activity of the receptors. A similar hypothesis invoking a possible role of an extravascular pigment has been considered previously (Lloyd et al., 1968). The maximum plateau in COZ response during hypoxia suggests a maximal steady-state response at a maximal desaturated state of the receptors. Also, an
CAROTID
CHEMORECEPTOR
0 2-co
263
2 INTERACTION
Arterial
p-2
PH
60 RECEPTOR sop Ye 40
20
0 0
20
40
Fig. 7. Calculated arterial
O2 equilibrium
60
60 PO02
curves corresponding
to activity
pH. The data from fig. 4 were used for the calculation.
7.25 and Po, of 25 torr was taken as 100% 0s which is presumably
located
pH 7.25 were expressed
in a cellular
as percent
desaturation
membrane.
of the maximum
loo
(mmlig)
equivalent
The observed activity.
of chemoreceptors
The maximal
From
activity
at three levels of at arterial
of a hypothetical
activities
at higher
pH of
chromophore levels of Paol at
these data at an arterial
pH of 7.25
and similar data at other pH values the curves were constructed. These curves correspond receptor activity but closely resemble 0, equilibrium curves of a polymeric chromophore
to chemogroup with
a Bohr shift (see text for details).
apparent plateau at other levels of PO, may correspond to 0, equilibrium curves of a polymeric chromophore if plotted in terms of O2 desaturation against Paco2 at a constant Pao,. A change from one CO2 plateau to another can be achieved by changing the level of Pa,,. The plateau observed, for example, by Fitzgerald and Parks (1971) can thus be explained. The effect of CO,-H+ on the activity of chemoreceptors in the absence of arterial hypoxia is not easily understood in terms of this hypothesis unless it is assumed that CO,-H+ lowers O2 content of the receptors. It is possible that the phenomenon is analogous to Root shift (Root, 1931) found in 0, equilibration of hemoglobin in some marine fish. According to the Bohr shift the receptor is expected to show a reciprocal Haldane effect (see Kilmartin and Rossi-Bernardi, 1973). That is, oxygenation of the receptor would increase the local [H+] which in turn would tend to desaturate and excite it. The net result is a balance of these two opposite effects which illustrates an example of biological feedback mechanism at a molecular level.
S. LAHIRI
264
AND R. G. DeLANEY
As mentioned, the applicability of these data to the proposed hypothesis is not necessarily unique. Torrance (1975) has advanced a hypothesis which explains multiplicative C02-O2 interaction in the response of carotid chemoreceptors. He has proposed that the afferent nerve endings in the space between Type I and Type II cells are sensitive only to H ‘, and [H+] in the space is maintained stable by a pump which is PO2 sensitive. The pump raises (HCO;) in the space more than in arterial blood during hypercapnia, and hypoxia decreases the effectiveness of the pump. This system would give rise to [H+] in the space with changes in Pa,-.oz at different levels of Pao2 resembling the fan of CO2 response curves of chemoreceptors. According to the author this hypothesis also predicts a multiplicative O,-CO, interaction at the threshold because of the variation in the local [H+] with arterial PO2 for any arterial [H+]. The crucial part of this attractive scheme is a P,, sensitive bicarbonate pump. Our hypothesis focuses on the Po2-sensitive mechanism for the initiation of events, release of a transmitter, secretion of bicarbonate or whatever, leading to an impulse. Our working hypothesis is that a polymeric chromophore at a membrane site may function as an internal probe for 0, and initiate the process of excitation. Its reaction with 0, is reversible, unlike other proposed mechanisms in which 0, is irreversibly consumed. This distinction of reversibility is a unique feature of the hypothesis and is amenable to experimental verification. Acknowledgments
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