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Stagnant asphyxia in the carotid body of the cat
Respiration Physiology (1971) 12, 381-387; North-Holland Publishing Company, Amsterdam
STAGNANT ASPHYXIA
IN THE CAROTID BODY OF THE CAT
D. I. MCCLOSKEY’ AND A. M. S. BLACK University Laboratory of Physiology, Oxford, England
Abstract. Stagnant asphyxia was allowed to develop in the carotid body when blood pressure was dropped abruptly to zero by simultaneously clamping the common carotid artery and opening a tap in the external carotid artery to the atmosphere. Discharge in single chemoreceptor fibres was observed as it increased in response to the stagnant asphyxia. When discharge was maximal, blood equilibrated with various gas mixtures was allowed to flow through the bifurcation for different periods of time before again dropping the local blood pressure to zero. After such transient interruptions of stagnant asphyxia with normoxic or hyperoxic blood, the return of stagnant asphyxic discharge was delayed in proportion to the degree and duration of preceding hyperoxia -i.e. the organ established an “oxygen credit”. It is suggested that the carotid body may have a capacity to store oxygen. Arterial chemoreceptors
Control of breathing
The peripheral arterial chemoreceptors are stimulated by stagnant hypoxia (LANDGREN and NEIL, 1951) as well as by hypoxic hypoxia. We have observed the onset of chemo-
receptor discharge in response to stagnant asphyxia developing when the blood flow through the carotid body in the cat is abruptly halted. It has been demonstrated that the onset of this chemoreceptor response is delayed in proportion to the amount of oxygen supplied prior to the stoppage of flow. The existence of a capacity of the carotid body chemoreceptors to store oxygen is suggested. A preliminary account of these findings was presented gical and Pharmacological Society in May 1968.
to the Australian
Physiolo-
Methods Experiments were done on ten cats, anaesthetized with pentobarbitone 40 mg/kg, (Nembutal: Abbott) intraperitoneally. The trachea was cannulated low in the neck and the pretracheal muscles, pharynx and larynx were removed up to the level of the hyoid bone. A cannula was inserted into the lingual artery to measure blood pressures
in the region of the carotid bifurcation. Another cannula was inserted into the external carotid artery with its tip directed towards the carotid bifurcation, and was connected to a tap which could be opened to the atmosphere. Small arterial branches in the region Accepted for publication 22 April 1971. l Beit Memorial Research Fellow.
381
382
D. I. MCCLOSKEY
AND A. M. S. BLACK
of the carotid bifurcation were tied, particular care being taken to avoid interfering with the carotid body or its arteries or veins. The vago-sympathetic trunks were cut low in the neck. The carotid sinus nerve was identified where it joined the glossopharyngeal and was dissected under parafiin on a rigid, earthed stainless steel plate with a blackened upper surface. Chemoreceptor fibres were identified by their sporadic discharge, but were accepted only if they responded to changes in the oxygen content of the inspired gas. Only single-fibre preparations were used in this study. The recording electrodes were made of fine stainless steel wire. Impulses were led into a Tektronix type 122 preamplifier and then to an oscilloscope for preliminary observation or to one channel of the recording apparatus, a Mingograf Cardirex 24B, a direct-writing ink-jet recorder with galvanometers of natural frequency of 610 cps. Blood pressure in the lingual artery was measured with an Elema-Schonander Electromanometer type 460 with a variable inductance pressure transducer (type EMT 490A, O-300 mm Hg). The output was recorded on a second channel of the Mingograf. Observations were made with the cats artificially ventilated with a Starling Ideal pump. Gas mixtures were made up with flowmeters and stored in Douglas bags. Rectal temperatures were kept constant within the range 37-39°C and the paraffin pool was kept at a similar temperature. Using these preparations it was possible to stop the blood flow in the carotid bifurcation abruptly by simultaneously clamping the common carotid artery and opening the tap in the external carotid artery to the atmosphere. That the pressure in the carotid bifurcation fell to zero was confirmed by the reading of blood pressure recorded from the lingual artery. Neither clamping the venous outflow of the carotid body, nor applying a small, constant negative pressure (- 10 cm water) within the bifurcation, during the period of stoppage of flow, altered the results observed. Observations
When the blood pressure in the carotid bifurcation was dropped suddenly to zero the discharge in single-fibre chemoreceptor preparations rose. When the blood pressure and blood flow through the bifurcation were restored the discharge fell. In all the fibres investigated it was found that the discharge rose to a high, fairly steady level in response to the stoppage of flow, but that the time taken to reach this level varied, depending on the conditions of perfusion prior to the stoppage of flow. To investigate this further, the following procedure was followed : flow was abruptly stopped, and the discharge was allowed to build up to a high, fairly steady level. At this point arterial blood at systemic arterial blood pressure was allowed back into the bifurcation for various lengths of time, and then the pressure was again dropped abruptly to zero. The discharge was observed as it rose. By altering the oxygen content of the gas mixtures used to ventilate the cats the chemical composition of the blood allowed into the bifurcation could be chosen. In all, 14 single-fibre preparations were investigated in 10 cats, and while the maximal levels of asphyxic discharge varied, the time-courses of the onsets of the
STAGNANT
ASPHYXIA
IN THE CAROTID
x 30 x 25
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00
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20
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Ti M E
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lo0
IIO
120
(seconds)
Fig. 1. Single chemoreceptor fibre. Cat. The experiment involved interrupting complete stagnant hypoxia with perfusions of various durations, at a pressure of 120 mm Hg. At zero time on the figure, the perfusion pressure was returned to zero. The panels show the returns of discharge after perfusions with (a) hyperoxic, (b) normoxic, and (c) hypoxic blood, for the durations, in seconds, shown.
384
D. I. MCCLOSKEY ANDA. M. S. BLACK
discharge were similar, particularly for the faster returns of discharge. Only for the slower returns after prolonged hyperoxia was there appreciable variation : the discharge recommencing between 60 and 120 set after stopping flow. In any one preparation, however, this delay time was constant. Figure 1 shows the results of one complete experiment. At zero time the blood pressure was dropped to zero following various durations of perfusion which interrupted previously stagnant asphyxial conditions. In (a) hyperoxic, in (b) normoxic, and in (c) hypoxic blood, was used. When the perfusion had been with hypoxic blood, the discharge returned rapidly at a rate independent of the duration of the perfusion. When the perfusion had been with normoxic or hyperoxic blood, and of short duration (up to 1.5 set), the discharge returned equally rapidly. However, when the perfusion had been with normoxic or hyperoxic blood, and was of longer duration (up to 60 set), the discharge returned more slowly and its speed of return depended upon the duration of the preceding perfusion. Moreover, after perfusions of comparable duration the rate of return was slower after the hyperoxic perfusion than after the normoxic perfusion. Perfusions of longer than 60 set duration did not further delay the return of discharge. Discussion
In these experiments the carotid body chemoreceptors were made to discharge in response to stagnant asphyxia caused by abruptly halting the blood flow to them. It is not suggested that this manoeuvre abruptly halted the oxygen supply to the receptors, since some oxygen must have been present in the blood trapped in the vessels of the carotid body when flow was stopped, and also in physical solution in the extravascular tissue of the carotid body. An estimate of the amount of oxygen trapped in this way can be made and is given below. When this is done it is found that there is not enough oxygen available from these sources to maintain a normally metabolizing carotid body for the periods for which the onset of discharge is delayed following prolonged hyperoxic perfusions. Moreover, when the amounts of oxygen available from these sources are similar, as after comparable durations of perfusion with normoxic and hyperoxic blood, the delay before discharge recommences is greater after hyperoxia than after normoxia. Thus the delay in response to stagnant asphyxia in proportion to the degree and duration of preceding hyperoxia (a process which might be called “incurring an oxygen credit”), cannot be explained by variations in the amount of accountable oxygen present at the onset of stagnant conditions. Calculations leading to these conclusions are given below, and we consider the possibilities that the carotid body may have a capacity to store oxygen or energy-rich compounds in proportion to its exposure to hyperoxia, or that it may be inhibited from discharging by some chemical factor produced in proportion to the degree of hyperoxia. DALY, LAMBERTSEN and SCHWEITZER (1954) gave a figure of 2 mg for the weight of the carotid body: if its density is that of water its volume is 2 ~1. LEITNERand LIAUBET (1971) stripped carotid bodies very strenuously to beyond their capsules and reported a considerably smaller weight for the organ. (We use the figures of DALYet al. as these
STAGNANT ASPHYXIA IN THECAROTIDBODY
385
are in agreement with our own unpublished measurements of the weights of carotid bodies from 10 cats.) If the solubility of oxygen in blood and carotid body tissue is similar, this 2 ~1 would contain 0.04 ~1 of oxygen in simple solution at one atmosphere. Assume that 25% of the volume of the unperfused carotid body is trapped blood: when saturated with oxygen this blood would contain a further 0.1 1.11 of oxygen in chemical combination. This is possibly overestimating the combined oxygen, as DE CASTROand RUBIO(1968) suggest that carotid body capillaries may collapse when not perfused. Thus when stagnant conditions begin after a prolonged hyperoxic perfusion, there would be approximately 0.14 ~1 of oxygen available for the carotid body’s metabolism. If no other source of oxygen or energy exists, it is this 0.14 ~1 of oxygen on which the carotid body has been maintained following prolonged hyperoxic perfusions of the type shown in fig. 1a. Using similar assumptions one can calculate that after prolonged normoxic perfusion there is 0.106 ~1 of oxygen in the blood and in physical solution in the glomus tissue. This is not much less than the amount present after hyperoxia since the oxygen combined with haemoglobin is approximately the same in both cases because of the shape of the haemoglobin dissociation curve. Figure la shows that the delay before discharge starts after hyperoxia is 100 set, whereas after normoxia (fig. lb) the delay is only 20 sec. The difference in accountable available oxygen between these two is 0.04 ~1. Is 0.04 ~1 enough to maintain the metabolism of the glomus for the 80 set difference between the two? Is the 0.14 ~1 present after hyperoxia enough to maintain the metabolism for 100 set? DALY et al. (1954) found an oxygen consumption of 9 ml/100 g/min in the blood perfused carotid body of the cat. PURVES(1970) gave a similar figure for the denervated carotid body (the carotid bodies in our experiments were denervated). He reported a dependence of oxygen consumption upon arterial oxygen and carbon dioxide tensions and upon blood pressure in the innervated, but not in the denervated carotid body, so this is not relevant here. In contrast to these results are the figures given by FAY(1970): in a saline perfused preparation he found an oxygen consumption of 1.5 ml/l00 g/min, less than 20% of the value originally found by DALY et al. Because JOELSand NEIL (1968) have reported that saline perfusion considerably reduces the responsiveness and vascular tone of the carotid body, our further calculations rely more on the figures of the other workers. LEITNERand LIAUBET(1971) measured oxygen consumption of the carotid body in vitro, and reported absolute values of the same order as those reported by FAY (1970), when their preparations were exposed to an outside oxygen tension of 680 torr. However, an unoxygenated core of tissue can exist in the in vitro preparation, and the figures given by FORSTER(1968) indicate that an external oxygen tension of 2000 torr is required to supply the whole of a sphere of the dimensions of the carotid body. It is difficult to estimate what fraction of the carotid body’s metabolism Leitner and Liaubet were observing. This is particularly so because the organs they used were exposed for some time during dissection to an oxygen tension equal only to that of room air before being investigated, so that an inner core of cells may have died before they commenced their experiments.
386
D. I.MCCLOSKEY AND A.M. S.BLACK
If the figures of Daly et al. and Purves are correct, the glomus would consume 0.3 ~1 of oxygen in 100 set, or 0.24 ,~lin 80 sec. But it has already been shown that one can account for only 0.14 ~1 of oxygen after prolonged hyperoxia, when discharge is delayed for 100 set, and it is extremely unlikely that all of this available oxygen would be consumed before discharge began. It has also been shown that discharge begins some 80 set later after hyperoxia than after normoxia, when the difference in accountable available oxygen is only 0.04 ~1. Clearly, in neither case can we account for enough oxygen to maintain carotid body metabolism for the observed periods. On FAY'S (1970) figures for oxygen consumption, however, the glomus would consume only 0.05 ,~l in 100 set, and 0.04 ~1 in 80 set: in both cases considered the accountable available oxygen would suffice to maintain glomus metabolism. Accepting the oxygen consumption given by Daly et al. and Purves, instead of Fay’s, means, in he light of the above calculations, that a separate oxygen storage system in the carotid body itself can be postulated. It is clear from fig. la that a large part of such a store is filled only in hyperoxic conditions, and that in these conditions it takes a considerable time to fill. The only oxygen available to fill such a store in hyperoxic conditions must come from physical solution in the perfusing blood, which could explain the long filling time. The same oxygen could be removed from a store by hypoxic blood flowing through much faster than by stagnant conditions because hypoxic blood has reduced haemoglobin with which to take it away. In perfused preparations the sudden replacement of hyperoxic by hypoxic perfusing blood gives a chemoreceptor response in a few seconds (MC CLOSKEY, 1968; MCCLOSKEY and BLACK, unpublished observations), suggesting that the oxygen credit we have described here is a store of oxygenper se. If the store is not oxygen, but energy-rich compounds, then its rapid dissipation in hypoxic (perfusion) hypoxia but not in stagnant hypoxia is difficult to explain. Moreover, it would mean there is a metabolism in the carotid body tissue in which the balance of an ATP:ADP or similar system is shifted towards the energy-rich moiety over a high Po, range. MILLS and JOBSIS (1970) have described a carotid body endoxidase with an unusually high critical Po2, but this peculiarity alone would not provide an energy store. On the argument we have used oxygen may be regarded as an inhibitor of discharge. Our results are satisfactorily explained if its level builds up slowly in the carotid body during hyperoxic perfusion, can be slowly dissipated in stagnant hypoxic conditions, and more rapidly discharged in hypoxic conditions (i.e. hypoxic perfusion). EYZAGUIRRE and ZAPATA (1968) have suggested that an inhibitory transmitter may be released in the carotid body in some circumstances. Such a transmitter would explain our results if it satisfied the above criteria given for oxygen as an inhibitor: slow accumulation during hyperoxic perfusion, slow dissipation in stagnant hypoxia, and faster dissipation in perfusion hypoxia. An inhibitory chemical produced at a rate dependent on arterial oxygen tension, and able to be either washed away in perfusing blood or gradually overcome in a stagnant asphyxic situation, would fill this role. Nevertheless, as PURVES (1970) has shown no dependence of oxygen consumption upon oxygen
STAGNANT ASPHYXIA IN THE CAROTIDBODY
387
tension in the denervated carotid body, the previous calculations on oxygen gesau remain applicable. An inhibitory chemical substance would, to explain our results, have to be powerful enough to suppress discharge long after all available oxygen has been used. We cannot exclude this alternative explanation of our results, although we do not find it attractive. If no inhibitor of discharge apart from oxygen itself is involved in our experiments, then our results can be taken to suggest the presence of a specialized carotid body tissue oxygen store. It is of interest that CHALAZONITIS (1969) has reported sensitivity to hypoxia in certain giant neurones of Aplysia and Helix in which oxygen can bind to a haemoglobin-like pigment. Alternatively our results might be taken to show that if there is no capacity of the glomus to store oxygen, then the oxygen consumption figures of FAY (1970) are more likely to be correct than those given by DALY et al. (1954) and PURVES(1970). Whatever the explanation of our observations, the stagnant preparation is of interest in that it is particularly suited to experiments in which it is desired to distinguish a direct chemoreceptor effect from an effect occurring secondarily to vasomotor changes within the carotid body. Acknowledgement
We are grateful to Dr R. W. TORRANCE for his advice on some aspects of this work. References DE CASTRO,F. and M. RUBIO (1968). In: Arterial Chemoreceptors. Proceedings of the Wates Symposium, ed. by R. W. Torrance. Oxford, Blackwell, pp. 267-278. CHALAZONITIS,N. (1969). Stimulation and depression of neurones by changes in gas partial pressures and in pH. J. Physiol. (London) 202: 2-3 p. DALY, M. de B., C. J. LAMBERTSEN and A. SCHWEITZER(1954). Observations on the volume of blood flow and oxygen utilization of the carotid body in the cat. J. Physiol. (London) 125: 67-89. EYZAGUIRRE,C. and P. ZAPATA (1968). In: Arterial Chemoreceptors. Proceedings of the Wates Symposium, ed. by R. W. Torrance. Oxford, Blackwell, pp. 213-252. FAY, F. S. (1970). Oxygen consumption of the carotid body. Am. J. Physiol. 218: 518-523. FORSTER,R. E. (1968). In: Arterial Chemoreceptors. Proceedings of the Wates Symposium, ed. by R. W. Torrance. Oxford, Blackwell, pp. 115-l 32. JOELS,N. and E. NEIL (1968). In: Arterial Chemoreceptors. Proceedings of the Wates Symposium, ed. by R. W. Torrance. Oxford, Blackwell, pp. 153-178. LANDGREN,S. and E. NEIL (1951). Chemoreceptor impulse activity following haemorrhage. Acru Physiol. &and. 23: 158-167. LEITNER, L.-M. and M.-J. LIAUBET(1971). Carotid body oxygen consumption of the cat in vitro. Pfliigers Arch. ges. Physiol. 323: 315-322. MCCLOSKEY,D. I. (1968). In: Arterial Chemoreceptors. Proceedings of the Wates Symposium, ed. by R. W. Torrance. Oxford, Blackwell, pp. 279-296. MILLS, E. and F. F. JOBSIS (1970). Simultaneous measurement of cytochrome A3 reduction and chemoreceptor afferent activity in the carotid body. Nuture 225: 1147-l 148. PURVES,M. J. (1970). The effect of hypoxia, hypercapnia, and hypotension upon carotid body flow and oxygen consumption in the cat. J. Physiol. (London) 209: 417432.
STAGNANT ASPHYXIA
IN THE CAROTID BODY OF THE CAT
D. I. MCCLOSKEY’ AND A. M. S. BLACK University Laboratory of Physiology, Oxford, England
Abstract. Stagnant asphyxia was allowed to develop in the carotid body when blood pressure was dropped abruptly to zero by simultaneously clamping the common carotid artery and opening a tap in the external carotid artery to the atmosphere. Discharge in single chemoreceptor fibres was observed as it increased in response to the stagnant asphyxia. When discharge was maximal, blood equilibrated with various gas mixtures was allowed to flow through the bifurcation for different periods of time before again dropping the local blood pressure to zero. After such transient interruptions of stagnant asphyxia with normoxic or hyperoxic blood, the return of stagnant asphyxic discharge was delayed in proportion to the degree and duration of preceding hyperoxia -i.e. the organ established an “oxygen credit”. It is suggested that the carotid body may have a capacity to store oxygen. Arterial chemoreceptors
Control of breathing
The peripheral arterial chemoreceptors are stimulated by stagnant hypoxia (LANDGREN and NEIL, 1951) as well as by hypoxic hypoxia. We have observed the onset of chemo-
receptor discharge in response to stagnant asphyxia developing when the blood flow through the carotid body in the cat is abruptly halted. It has been demonstrated that the onset of this chemoreceptor response is delayed in proportion to the amount of oxygen supplied prior to the stoppage of flow. The existence of a capacity of the carotid body chemoreceptors to store oxygen is suggested. A preliminary account of these findings was presented gical and Pharmacological Society in May 1968.
to the Australian
Physiolo-
Methods Experiments were done on ten cats, anaesthetized with pentobarbitone 40 mg/kg, (Nembutal: Abbott) intraperitoneally. The trachea was cannulated low in the neck and the pretracheal muscles, pharynx and larynx were removed up to the level of the hyoid bone. A cannula was inserted into the lingual artery to measure blood pressures
in the region of the carotid bifurcation. Another cannula was inserted into the external carotid artery with its tip directed towards the carotid bifurcation, and was connected to a tap which could be opened to the atmosphere. Small arterial branches in the region Accepted for publication 22 April 1971. l Beit Memorial Research Fellow.
381
382
D. I. MCCLOSKEY
AND A. M. S. BLACK
of the carotid bifurcation were tied, particular care being taken to avoid interfering with the carotid body or its arteries or veins. The vago-sympathetic trunks were cut low in the neck. The carotid sinus nerve was identified where it joined the glossopharyngeal and was dissected under parafiin on a rigid, earthed stainless steel plate with a blackened upper surface. Chemoreceptor fibres were identified by their sporadic discharge, but were accepted only if they responded to changes in the oxygen content of the inspired gas. Only single-fibre preparations were used in this study. The recording electrodes were made of fine stainless steel wire. Impulses were led into a Tektronix type 122 preamplifier and then to an oscilloscope for preliminary observation or to one channel of the recording apparatus, a Mingograf Cardirex 24B, a direct-writing ink-jet recorder with galvanometers of natural frequency of 610 cps. Blood pressure in the lingual artery was measured with an Elema-Schonander Electromanometer type 460 with a variable inductance pressure transducer (type EMT 490A, O-300 mm Hg). The output was recorded on a second channel of the Mingograf. Observations were made with the cats artificially ventilated with a Starling Ideal pump. Gas mixtures were made up with flowmeters and stored in Douglas bags. Rectal temperatures were kept constant within the range 37-39°C and the paraffin pool was kept at a similar temperature. Using these preparations it was possible to stop the blood flow in the carotid bifurcation abruptly by simultaneously clamping the common carotid artery and opening the tap in the external carotid artery to the atmosphere. That the pressure in the carotid bifurcation fell to zero was confirmed by the reading of blood pressure recorded from the lingual artery. Neither clamping the venous outflow of the carotid body, nor applying a small, constant negative pressure (- 10 cm water) within the bifurcation, during the period of stoppage of flow, altered the results observed. Observations
When the blood pressure in the carotid bifurcation was dropped suddenly to zero the discharge in single-fibre chemoreceptor preparations rose. When the blood pressure and blood flow through the bifurcation were restored the discharge fell. In all the fibres investigated it was found that the discharge rose to a high, fairly steady level in response to the stoppage of flow, but that the time taken to reach this level varied, depending on the conditions of perfusion prior to the stoppage of flow. To investigate this further, the following procedure was followed : flow was abruptly stopped, and the discharge was allowed to build up to a high, fairly steady level. At this point arterial blood at systemic arterial blood pressure was allowed back into the bifurcation for various lengths of time, and then the pressure was again dropped abruptly to zero. The discharge was observed as it rose. By altering the oxygen content of the gas mixtures used to ventilate the cats the chemical composition of the blood allowed into the bifurcation could be chosen. In all, 14 single-fibre preparations were investigated in 10 cats, and while the maximal levels of asphyxic discharge varied, the time-courses of the onsets of the
STAGNANT
ASPHYXIA
IN THE CAROTID
x 30 x 25
x
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x
x
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BODY
P 0
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.**e
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.
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no0 =.:
00
o
IO
20
30
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50
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Ti M E
70
80
90
lo0
IIO
120
(seconds)
Fig. 1. Single chemoreceptor fibre. Cat. The experiment involved interrupting complete stagnant hypoxia with perfusions of various durations, at a pressure of 120 mm Hg. At zero time on the figure, the perfusion pressure was returned to zero. The panels show the returns of discharge after perfusions with (a) hyperoxic, (b) normoxic, and (c) hypoxic blood, for the durations, in seconds, shown.
384
D. I. MCCLOSKEY ANDA. M. S. BLACK
discharge were similar, particularly for the faster returns of discharge. Only for the slower returns after prolonged hyperoxia was there appreciable variation : the discharge recommencing between 60 and 120 set after stopping flow. In any one preparation, however, this delay time was constant. Figure 1 shows the results of one complete experiment. At zero time the blood pressure was dropped to zero following various durations of perfusion which interrupted previously stagnant asphyxial conditions. In (a) hyperoxic, in (b) normoxic, and in (c) hypoxic blood, was used. When the perfusion had been with hypoxic blood, the discharge returned rapidly at a rate independent of the duration of the perfusion. When the perfusion had been with normoxic or hyperoxic blood, and of short duration (up to 1.5 set), the discharge returned equally rapidly. However, when the perfusion had been with normoxic or hyperoxic blood, and was of longer duration (up to 60 set), the discharge returned more slowly and its speed of return depended upon the duration of the preceding perfusion. Moreover, after perfusions of comparable duration the rate of return was slower after the hyperoxic perfusion than after the normoxic perfusion. Perfusions of longer than 60 set duration did not further delay the return of discharge. Discussion
In these experiments the carotid body chemoreceptors were made to discharge in response to stagnant asphyxia caused by abruptly halting the blood flow to them. It is not suggested that this manoeuvre abruptly halted the oxygen supply to the receptors, since some oxygen must have been present in the blood trapped in the vessels of the carotid body when flow was stopped, and also in physical solution in the extravascular tissue of the carotid body. An estimate of the amount of oxygen trapped in this way can be made and is given below. When this is done it is found that there is not enough oxygen available from these sources to maintain a normally metabolizing carotid body for the periods for which the onset of discharge is delayed following prolonged hyperoxic perfusions. Moreover, when the amounts of oxygen available from these sources are similar, as after comparable durations of perfusion with normoxic and hyperoxic blood, the delay before discharge recommences is greater after hyperoxia than after normoxia. Thus the delay in response to stagnant asphyxia in proportion to the degree and duration of preceding hyperoxia (a process which might be called “incurring an oxygen credit”), cannot be explained by variations in the amount of accountable oxygen present at the onset of stagnant conditions. Calculations leading to these conclusions are given below, and we consider the possibilities that the carotid body may have a capacity to store oxygen or energy-rich compounds in proportion to its exposure to hyperoxia, or that it may be inhibited from discharging by some chemical factor produced in proportion to the degree of hyperoxia. DALY, LAMBERTSEN and SCHWEITZER (1954) gave a figure of 2 mg for the weight of the carotid body: if its density is that of water its volume is 2 ~1. LEITNERand LIAUBET (1971) stripped carotid bodies very strenuously to beyond their capsules and reported a considerably smaller weight for the organ. (We use the figures of DALYet al. as these
STAGNANT ASPHYXIA IN THECAROTIDBODY
385
are in agreement with our own unpublished measurements of the weights of carotid bodies from 10 cats.) If the solubility of oxygen in blood and carotid body tissue is similar, this 2 ~1 would contain 0.04 ~1 of oxygen in simple solution at one atmosphere. Assume that 25% of the volume of the unperfused carotid body is trapped blood: when saturated with oxygen this blood would contain a further 0.1 1.11 of oxygen in chemical combination. This is possibly overestimating the combined oxygen, as DE CASTROand RUBIO(1968) suggest that carotid body capillaries may collapse when not perfused. Thus when stagnant conditions begin after a prolonged hyperoxic perfusion, there would be approximately 0.14 ~1 of oxygen available for the carotid body’s metabolism. If no other source of oxygen or energy exists, it is this 0.14 ~1 of oxygen on which the carotid body has been maintained following prolonged hyperoxic perfusions of the type shown in fig. 1a. Using similar assumptions one can calculate that after prolonged normoxic perfusion there is 0.106 ~1 of oxygen in the blood and in physical solution in the glomus tissue. This is not much less than the amount present after hyperoxia since the oxygen combined with haemoglobin is approximately the same in both cases because of the shape of the haemoglobin dissociation curve. Figure la shows that the delay before discharge starts after hyperoxia is 100 set, whereas after normoxia (fig. lb) the delay is only 20 sec. The difference in accountable available oxygen between these two is 0.04 ~1. Is 0.04 ~1 enough to maintain the metabolism of the glomus for the 80 set difference between the two? Is the 0.14 ~1 present after hyperoxia enough to maintain the metabolism for 100 set? DALY et al. (1954) found an oxygen consumption of 9 ml/100 g/min in the blood perfused carotid body of the cat. PURVES(1970) gave a similar figure for the denervated carotid body (the carotid bodies in our experiments were denervated). He reported a dependence of oxygen consumption upon arterial oxygen and carbon dioxide tensions and upon blood pressure in the innervated, but not in the denervated carotid body, so this is not relevant here. In contrast to these results are the figures given by FAY(1970): in a saline perfused preparation he found an oxygen consumption of 1.5 ml/l00 g/min, less than 20% of the value originally found by DALY et al. Because JOELSand NEIL (1968) have reported that saline perfusion considerably reduces the responsiveness and vascular tone of the carotid body, our further calculations rely more on the figures of the other workers. LEITNERand LIAUBET(1971) measured oxygen consumption of the carotid body in vitro, and reported absolute values of the same order as those reported by FAY (1970), when their preparations were exposed to an outside oxygen tension of 680 torr. However, an unoxygenated core of tissue can exist in the in vitro preparation, and the figures given by FORSTER(1968) indicate that an external oxygen tension of 2000 torr is required to supply the whole of a sphere of the dimensions of the carotid body. It is difficult to estimate what fraction of the carotid body’s metabolism Leitner and Liaubet were observing. This is particularly so because the organs they used were exposed for some time during dissection to an oxygen tension equal only to that of room air before being investigated, so that an inner core of cells may have died before they commenced their experiments.
386
D. I.MCCLOSKEY AND A.M. S.BLACK
If the figures of Daly et al. and Purves are correct, the glomus would consume 0.3 ~1 of oxygen in 100 set, or 0.24 ,~lin 80 sec. But it has already been shown that one can account for only 0.14 ~1 of oxygen after prolonged hyperoxia, when discharge is delayed for 100 set, and it is extremely unlikely that all of this available oxygen would be consumed before discharge began. It has also been shown that discharge begins some 80 set later after hyperoxia than after normoxia, when the difference in accountable available oxygen is only 0.04 ~1. Clearly, in neither case can we account for enough oxygen to maintain carotid body metabolism for the observed periods. On FAY'S (1970) figures for oxygen consumption, however, the glomus would consume only 0.05 ,~l in 100 set, and 0.04 ~1 in 80 set: in both cases considered the accountable available oxygen would suffice to maintain glomus metabolism. Accepting the oxygen consumption given by Daly et al. and Purves, instead of Fay’s, means, in he light of the above calculations, that a separate oxygen storage system in the carotid body itself can be postulated. It is clear from fig. la that a large part of such a store is filled only in hyperoxic conditions, and that in these conditions it takes a considerable time to fill. The only oxygen available to fill such a store in hyperoxic conditions must come from physical solution in the perfusing blood, which could explain the long filling time. The same oxygen could be removed from a store by hypoxic blood flowing through much faster than by stagnant conditions because hypoxic blood has reduced haemoglobin with which to take it away. In perfused preparations the sudden replacement of hyperoxic by hypoxic perfusing blood gives a chemoreceptor response in a few seconds (MC CLOSKEY, 1968; MCCLOSKEY and BLACK, unpublished observations), suggesting that the oxygen credit we have described here is a store of oxygenper se. If the store is not oxygen, but energy-rich compounds, then its rapid dissipation in hypoxic (perfusion) hypoxia but not in stagnant hypoxia is difficult to explain. Moreover, it would mean there is a metabolism in the carotid body tissue in which the balance of an ATP:ADP or similar system is shifted towards the energy-rich moiety over a high Po, range. MILLS and JOBSIS (1970) have described a carotid body endoxidase with an unusually high critical Po2, but this peculiarity alone would not provide an energy store. On the argument we have used oxygen may be regarded as an inhibitor of discharge. Our results are satisfactorily explained if its level builds up slowly in the carotid body during hyperoxic perfusion, can be slowly dissipated in stagnant hypoxic conditions, and more rapidly discharged in hypoxic conditions (i.e. hypoxic perfusion). EYZAGUIRRE and ZAPATA (1968) have suggested that an inhibitory transmitter may be released in the carotid body in some circumstances. Such a transmitter would explain our results if it satisfied the above criteria given for oxygen as an inhibitor: slow accumulation during hyperoxic perfusion, slow dissipation in stagnant hypoxia, and faster dissipation in perfusion hypoxia. An inhibitory chemical produced at a rate dependent on arterial oxygen tension, and able to be either washed away in perfusing blood or gradually overcome in a stagnant asphyxic situation, would fill this role. Nevertheless, as PURVES (1970) has shown no dependence of oxygen consumption upon oxygen
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tension in the denervated carotid body, the previous calculations on oxygen gesau remain applicable. An inhibitory chemical substance would, to explain our results, have to be powerful enough to suppress discharge long after all available oxygen has been used. We cannot exclude this alternative explanation of our results, although we do not find it attractive. If no inhibitor of discharge apart from oxygen itself is involved in our experiments, then our results can be taken to suggest the presence of a specialized carotid body tissue oxygen store. It is of interest that CHALAZONITIS (1969) has reported sensitivity to hypoxia in certain giant neurones of Aplysia and Helix in which oxygen can bind to a haemoglobin-like pigment. Alternatively our results might be taken to show that if there is no capacity of the glomus to store oxygen, then the oxygen consumption figures of FAY (1970) are more likely to be correct than those given by DALY et al. (1954) and PURVES(1970). Whatever the explanation of our observations, the stagnant preparation is of interest in that it is particularly suited to experiments in which it is desired to distinguish a direct chemoreceptor effect from an effect occurring secondarily to vasomotor changes within the carotid body. Acknowledgement
We are grateful to Dr R. W. TORRANCE for his advice on some aspects of this work. References DE CASTRO,F. and M. RUBIO (1968). In: Arterial Chemoreceptors. Proceedings of the Wates Symposium, ed. by R. W. Torrance. Oxford, Blackwell, pp. 267-278. CHALAZONITIS,N. (1969). Stimulation and depression of neurones by changes in gas partial pressures and in pH. J. Physiol. (London) 202: 2-3 p. DALY, M. de B., C. J. LAMBERTSEN and A. SCHWEITZER(1954). Observations on the volume of blood flow and oxygen utilization of the carotid body in the cat. J. Physiol. (London) 125: 67-89. EYZAGUIRRE,C. and P. ZAPATA (1968). In: Arterial Chemoreceptors. Proceedings of the Wates Symposium, ed. by R. W. Torrance. Oxford, Blackwell, pp. 213-252. FAY, F. S. (1970). Oxygen consumption of the carotid body. Am. J. Physiol. 218: 518-523. FORSTER,R. E. (1968). In: Arterial Chemoreceptors. Proceedings of the Wates Symposium, ed. by R. W. Torrance. Oxford, Blackwell, pp. 115-l 32. JOELS,N. and E. NEIL (1968). In: Arterial Chemoreceptors. Proceedings of the Wates Symposium, ed. by R. W. Torrance. Oxford, Blackwell, pp. 153-178. LANDGREN,S. and E. NEIL (1951). Chemoreceptor impulse activity following haemorrhage. Acru Physiol. &and. 23: 158-167. LEITNER, L.-M. and M.-J. LIAUBET(1971). Carotid body oxygen consumption of the cat in vitro. Pfliigers Arch. ges. Physiol. 323: 315-322. MCCLOSKEY,D. I. (1968). In: Arterial Chemoreceptors. Proceedings of the Wates Symposium, ed. by R. W. Torrance. Oxford, Blackwell, pp. 279-296. MILLS, E. and F. F. JOBSIS (1970). Simultaneous measurement of cytochrome A3 reduction and chemoreceptor afferent activity in the carotid body. Nuture 225: 1147-l 148. PURVES,M. J. (1970). The effect of hypoxia, hypercapnia, and hypotension upon carotid body flow and oxygen consumption in the cat. J. Physiol. (London) 209: 417432.