C 6onzJlez, L Almaraz, A. Obeso and R. Rigual are at

the Depa~mento de Oioquimica, Oiologia

Mo/ecu/ar y Fisiolog[a, Facultad de

Medicina, Universidad de

Valladolid, 47005 Valladolid, 5pain.

Oxygen and acid chemoreceptk n in the carotid body chemoreceptors

C. Gonz~lez, L. Almaraz,

The carotid bodies are arterial chemoreceptors that are sensitive to blood Poe, Pco2 and pH. They are the origin of reflexes that are crucial for maintaining Pco~ and pH in the internal milieu and for adjusting the 02 supply according to the metabolic needs of the organism in situations of increased demand, such as exercise and while breathing at decreased 02 partial pressures during ascent or when living at high altitude. Chemo- receptor cells of the carotid body transduce the blood- borne stimuli into a neurosecretory response that is dependent on external Ca 2+. These cells have an 02-sensitive K + current that is reversibly inhibited by low P oz It is proposed that the depolarization produced by inhibition of this K + current activates Ca 2+ channels; Ca 2+ influx and neurosecretion follow. The cells have also a potent Na+-Ca 2+ antiporter that could be responsible for the intracellular Ca 2+ rise required to trigger the release of neurotransmitters during high Pco2 or low pH stimulation.

The carotid bodies, located near the carotid artery bifurcations, are chemoreceptor organs sensitive to changes in blood Po~, Pco~ and pH. They have two types of parenchymatous cells, chemoreceptor and sustentacular cells, which form clusters surrounded by a dense net of fenestrated capillaries (Fig. 1). The proximity of the capillaries to chemoreceptor cells minimizes the diffusion pathway for bloodborne stimuli. Chemoreceptor cells are derived from the neural crest and exhibit a heterogeneous population of synaptic vesicles in their cytoplasm that contain different putative neurotransmitters. Sustentacular cells are glial in nature and lack specialized organelles. Sensory nerve fibres reach the organ via the carotid sinus nerve, ending in synaptic apposition to the chemo- receptor cells 1.

The carotid bodies are tonic receptors with low firing frequency (< 2 impulses per s per fibre) at normal blood Po~ (100 mmHg), Pco2 (40 mmHg) and pH (7.4). The action potential frequency is augmented when blood Po decreases or when Pco~ or [H +] • . 2 , .

increase. Phys~ologacally, the most effective stimulus is the decrease in blood Po~. An increase in sensory discharges is already evident at a Po~ of 70 mmHg, when arterial blood 02 content is around 94% of the normal level, and at 50 mmHg (moderate hypoxia) the sensory activity is similar to that seen at a Pco~ of 75 mmHg and a pH of 7.0, which are indications of a severe acidosis 1.

Stimulation of the carotid bodies elicit reflexes, the main target of which is the respiratory system. A hyperventilation proportional to the stimulus intensity ensues. The carotid bodies are responsible for about 90% of the hyperventilation seen in hypoxic hypoxia, and for 20-50% of that observed in respiratory and metabolic acidosis. The rest of the respiratory drive is provided by other arterial chemoreceptors when the stimulus is hypoxia and by the central chemoreceptors in the case of acidosis z'3. Throughout these respir- atory actions, the carotid bodies play crucial homeo-

A. Obeso and R. Rigual

static and adaptive roles in maintaining Pco., and pH in the internal milieu and adjusting the 02 supply according to the metabolic needs of the organism. Other targets of chemoreflexes are the heart and the resistance and capacitance vessels, although the physiological significance of these reflexes is ill defined 3.

T h e c h e m o s e n s o r s t ruc ture : ce l l s or n e r v e end ings?

In dealing with the physiology of the carotid body at the receptor level, the first aspect to consider is which element within the receptor complex is the primary sensing element. In 1928 De Castro 4 showed that chemoreceptor cells of the carotid body are innervated by sensory neurons, and postulated that the chemoreceptor cells are the primary sensors that, via the products of their metabolism, activate the sensory nerve endings. This view was reinforced by ultrastructural and biochemical findings that recog- nized the synaptic relationships between chemorecep- tor cells and sensory nerve endings, and the presence of synaptic vesicles and putative neurotransmitters in chemoreceptor cells s'6.

In the late 1960s, Biscoe and co-workers 7 repeated De Castro's degeneration experiments and reported that the nerve endings in synaptic apposition to chemoreceptor cells were efferent, and therefore secretomotor. They attributed the primary sensory function to poorly defined free nerve endings that had been described earlier but were never confirmed. In Biscoe's conception, chemoreceptor cells were glandular-like, serving either a hormonal or a para- crine function. This model of chemoreception was dismantled when several authors confirmed De Castro's findings on the sensory nature of the endings apposed to chemoreceptor cells 1. Finally, the Po~- sensing properties of chemoreceptor cells were demonstrated by showing that carotid bodies from which the sensory afferents were chronically dener- vated responded in vitro to a decrease of Po~ in the bathing solution with an increased rate of release of dopamine (DA) 8.

C h e m o t r a n s d u c t i o n as a p r o c e s s c o u p l i n g s t i m u l a t i o n and s e c r e t i o n

Upon stimulation, sensory cells in composite recep- tors should release neurotransmitters in proportion to the intensity of the stimulus and in parallel to the action potential frequency recorded in the sensory nerve 9. Therefore, the re-definition of the carotid body as a 'composite receptor' made sensory trans- duction in this receptor equivalent to a stimulus- secretion coupling process 9. It has been shown that, among several putative neurotransmitters, chemo- receptor cells release DA in proportion to the intensity of hypoxic and acidic stimulation, and that the release response is paralleled by the electrical

8 1 0 1 1 activity in the carotid sinus nerve ' ' . The same relationship applies to other chemostimulant agents

1 4 6 © 1992, Elsevier Science Pubfishers Ltd, (UK) 0166- 2236/92/$05.00 TINS, Vol. 15, No. 4, 1992

including cyanide, dinitrophenol and 2-deoxy- glucose 12'1z. Responses of the carotid body to selected intensities of these stimuli are summarized in Fig. 2. Although the role of DA in the organ is not conclusively established, these data indicate that DA release is a reliable index of chemoreceptor cell activation and represents a final output of sensory transduction in these cells. The possible significance of the released DA in the process of chemoreception has been considered in a recent review ~4.

An aspect of the release of DA that has been systematically studied is its dependence on external Ca 2+. Table I shows that over 95% of the DA released in response to hypoxia and high concen- trations of extracellular K +, [K+]o, and about 80% of that elicited by other stimuli are abolished in Ca 2+- free medium 8'11'15-~7. The table also shows that the dihydropyridines nisoldipine and Bay K 8644, modu- lators of voltage-sensitive Ca z+ channels, modify the release of DA induced by high [K+]o (as expected by their known actions in other systems) as well as the release induced by low Po~. In contrast, these agents do not modify the basal release or that induced by a high Pco2 (low pH) stimulus, dinitrophenol or sodium propionate ~7'~8. Therefore, although the response to all the stimuli tested is highly dependent on external Ca 2+, a clear difference emerges between the two physiological stimuli: while hypoxia seems to promote the entry of Ca 2+ via voltage-sensitive Ca 2+ channels, acidic stimuli should activate alternative pathways for Ca e+ entry.

Chemotransduetion of 02 (1) A plasma membrane model. An inference from

the data described above is that hypoxia should depolarize chemoreceptor cells to activate voltage- dependent Ca 2+ channels. Therefore, a search for the mechanisms capable of producing such a depolariz- ation was initiated. In whole-cell voltage clamp exper- iments it was found that freshly dissociated chemo- receptor cells from the carotid bodies of the adult rabbit are excitable cells with Na +, K + and Ca 2+ voltage-dependent channels. In this initial study 19 it was also found that lowering P% in the bathing solution reversibly inhibited the outwardly directed K ÷ currents without affecting the inwardly directed Na + and Ca ~+ currents. It was proposed that the inhibition of the K ÷ current initiated the depolarization of chemoreceptor cells, in a way similar to that proposed for gustatory and olfactory cells exposed to appropriate stimuli.

Analysis of the whole-cell currents z°'21 indicated that the threshold for activation of the K + currents was about -30 mV and that the currents have a component that is Ca z+ dependent. Na + currents were inhibited by submicromolar concentrations of tetrodotoxin (TTX) and exhibited typical activation and inactivation properties. Ca 2+ currents were mediated by L-type channels. In all the cells tested, switching from a voltage to a current clamp produced a depolarization and large action potentials (normally just a single action potential followed by a steady depolarization, but trains of spontaneous spikes were also recorded).

An analysis 22 of the inhibition of K + currents by low Po~ indicated that only a transient CaZ+-independent component of this type of current was sensitive to the

A B

5

Fig. 1. Drawing of the carotid artery bifurcation and a cellular cluster of the carotid body. (,6,) A frontal view of the region of the nght carotid artery bifurcation in the rabbit. The common carotid artery divides (1) to give the intemal (2) and external (3) carotid arteries. The carotid body (4) is located on the internal carotid artery close to the bifurcation. Sensory fibers originating in the petrosal ganglion (5) reach the carotid body via the carotid sinus nerve (6). The superior cervical ganglion (7) also innervates the bifurcation area, including the carotid body, via the ganglioglomerular nerves (8). The nodose ganglion (9) is situated externally to the internal carotid artery. (B) A cluster of parenchymatous cells of the carotid body, which are formed by chemoreceptor cells (1) that are partially surrounded by sustentacular cells (2). The proportion of chemoreceptor to sustentacular cells is about 3-5 to one. Chemoreceptor cells have in their cytoplasm a heterogeneous population of synaptic vesicles (3), which are usually located near the contacts with the sensory nerve endings (4) that originate from branching fibers of the carotid sinus nerve (5). The clusters are surrounded by a dense net of capillaries (6).

P% (Fig. 3). In a recent study 23 in excised membrane patches of chemoreceptor cells isolated from adult rabbit carotid bodies, it was found that the Po2- sensitive K + channels have mean conductances of 20 pS, and that their opening probability (Po) de- creased on lowering Po2 - even when the Ca 2+ concentration in the solution bathing the internal face of the membrane was maintained below I riM.

Electrophysiological data obtained from chemo- receptor cells that have been isolated from embryos or neonatal animals are different from those just described. Cells from rabbit embryos lack TTX- sensitive inward currents, but Ca 2+ and K + currents are mostly similar to those obtained in adult chemo- receptor cells, including the sensitivity of the K + current to low Poo (Ref. 24). In addition, embryonic chemoreceptor ce~s seem to exhibit an ill-defined inwardly rectifying K + current that appears to be active at negative potentials (in the range of the estimated membrane potential of -50 mV), and that is also reversibly inhibited by low Po2 (Ref. 25).

In some studies of chemoreceptor cells isolated from neonatal rats, these cells have been found to lack TTX-sensitive transient inward currents 26, while in other studies, they appear to exhibit these currents 27. Another apparent discrepancy in these studies is that while the I-V curves for the K + current showed a prominent shoulder at test potentials between + 10 and

TINS, Vol. 15, No. 4, 1992 147

(9

._> "5

.Q

O (9 Q.

.m

16

12

8

. _ _

[ - - ] CSN activity T

l DA release ',

Low Po2 Acid CO2 CN DNP 2-DG

Fig. 2. Effects of different stimufi on the release of [3H]DA and carotid sinus nerve activity in an in vitro preparation of the carotid bodies of a cat. Responses of both activities were simultaneously obtained from the same preparation. The carotid bodies were loaded with [3H]tyrosine to label DA deposits prior to the actual expenments. The data are expressed as multiples of basal release or nerve activity, respectively. Stimuli were applied for 10 min in every case by superfusing the preparations with the indicated test solutions. Basal conditions: superfusion with balanced saline equilibrated with 95-100% 02 and adjusted to pH 7.4. Experimental conditions are shown on the x axis. (It should be noted that in the slow superfusion system used in these experiments the threshold response to low Po2 was 40-50% 02 in N2.) Low Po2 is 20% 02/80% N2, the acid solution is pH 6.8, CO2 is a solution equilibrated with 20% C02/80% 02, pH 7.4, DNP is dinitrophenol at 0.25 mM, CN is sodium cyanide at 0.25 mM, and 2-DG is glucose-free saline containing 5 mM pyruvate as an energetic substrate and 4 mM 2-deoxyglucose. In all cases data are means +_ SEM of 6--12 individual values. (Taken from Refs 10-13.)

+30 mV in the studies by Peers et al. 26'2s, such a component was not obvious in the I-V curves of the study by Stea and Nurse 27. A detailed characterization of the shoulder of the I-V curves indicated that the K + current was Ca 2+ dependent, and, in addition, it was shown that low Po~ selectively inhibited this corn-

+ 28 ponent of the K current . The identity of the

TABLE I. Ca 2+ dependence and modulation by dihydropyridines of [3H]DA- induced release from carotid bodies in the rabbit a.

Stimulus Increase in the amount of DA released under certain conditions 2 m M Ca 2+ 0 mM Ca 2+ 2 m M Ca 2+ 2 mM Ca 2+

Nisoldipine b Bay K8~14 b

25mMK + 2.50_+0.15 0.20_+0.00 0.04_+0.01 12.20+1.30

Low Po2 10.25-+2.10 0.40-+0.01 2.32+0.51 30.52+4.10 (7% 02)

High Pco2 1.61 -+0.17 0.31 +0.11 * 1.50+0.21 pH = 6.8

Na+-Prop. 1.05 -+ 0.16 0.22 + 0.05 * 1.10 -+ 0.17 (15 mM)

DNP (75 W ~) 2.44 -+ 0.22 0.56 + 0.05 2.31 _+ 0.21 *

aData are expressed as multiples of basal release and are the means_+sEM of 6--10 measurements (Refs 17, 18 and Gonz~lez, C., Almaraz, L., Obeso, A. and Rigual, R., unpublished observations). b Nisoldipine (625 riM) and Bay K 8644 (1 i~) were presented 10 min prior to and during stimulus application. * Not studied. Abbreviations: DA, dopamine; DNP, dinitrophenol; Na+-Prop. sodium propionate.

component of the K + current inhibited by low P02 was not established in the study by Stea and Nurse 27.

A conclusion emerging from all of these results is that chemoreceptor cells, which are sensitive to low P o throughout the life of the animals studied, exhibit

currents that are inhibited by low Po~. There are age-specific (and species-specific) differences in the type of K + current inhibited, as well as in other basic electrical properties of the cells. Developmental differences of some kind in the basic functioning of chemoreceptor cells are expected since the carotid bodies of fetal animals exhibit stimulus-response curves, which relate Po~ to firing frequency in the carotid sinus nerve, that are displaced far to the left compared with similar curves in the adult 29. Important differences have also been observed in the content and turnover of neurotransmitters perinataUy 3°. With- in the first two weeks of postnatal life 29'31'32 all the parameters studied are reset in such a way that the chemoreceptor function tends towards that observed in the adult. The mechanisms involved in this re- setting are u n k n o w n 32.

With the information provided in this and the preceding section, and restricting our consideration to adult carotid body chemoreceptor cells, the following model for low Po2 transduction can be proposed. Low Po2, by reducing the Po of K + channels, produces the initial depolarization required to activate the voltage- dependent Ca 2+ channels, and entry of Ca 2+ then follows. Simultaneous activation of Na + channels provides a fast recruitment of Ca 2+ channels, poten- tiating the entry of Ca 2+ and the release of neurotrans- mitters. Ca'~+-dependent K + channels contribute to cell repolarization (Fig. 4A).

The operativity of this model rests on the premise that inhibition of the K + channel is capable of triggering the initial depolarization. Owing to the voltage dependency of the inhibited current (the threshold activation of which is - 3 0 mV), this would be possible if, at more negative potentials, in the range of the membrane potential of -50 mV (Ref. 33), the 02-sensitive K + channels are effectively contributing to the total resting K + conductance of the cells. Taking into account the small size of the cells and the high resistance of their membranes 2°, the closure of only a few K + channels can modify significantly the membrane potential of the cells and trigger action potentials 34. This behavior is consistent with the observation made in current-clamp record- ings, where an increase in the firing frequency is observed on switching to low Pox soluti°ns22-

If the proposed model for the transduction of the hypoxic stimulus is to be accepted, it must be demonstrated that the inhibition of the K + current by low Po, is fast enough to explain the changes occurring in the intact carotid body. Since in the intact animal an increase in carotid sinus nerve action potential frequency is observed within a few seconds after application of the hypoxic stimulus 1, the.inhibi- tion of the K + current by low P% levels should develop almost instantaneously. The timecourse of Na + and K + current inhibition has been studied after super- fusing the cells with a TTX-containing hypoxic solution. It was found that the tl/2 of the effect of low P% on the K + current is less than that of TTX blockade of Na + channels (L6pez-L6pez, J. R. and Gonz~lez, C., unpublished observations). Since TTX

148 TINS, Vol. 15, No. 4, 1992

A control B control

I°wl Oo2 ~ ~ low Po~ 1 nA 0.5 nA

2 ms

C control recovery

3 nA

r

D r co tro A

5 ms 50 ms

Fig. 3. Effects of low Po2 on voltage-dependent ionic currents recorded in whole-cell configurations from chemo- receptor cells of the carotid bodies in adult rabbits. (A) Inward (Na + + C ,.12+) currents recorded during voltage steps to O mV from a holding potential of - 8 0 mV in the control solution (Po2 = 150 mmHg), and3 min after exposure to low Po2 (10 mmHg). Solutions (in mM) were: 140 Na + and 2.5 Ca 2+ in the bath; 130 Cs +, 3 Mg2+-A TP and 10 EG TA in the pipette. (B) Na + currents recorded in the control solution and 5 min after switching to the low Po2 solution. The test

+ 2 + pulse was to + 10 mV with a return to a holding potential of - 8 0 mV. Solutions (in mM): 140 Na and 0.5 Cd in the bath; 130 Cs +, 3 ME2+-ATP and 10 EGTA in the pipette. (C) K + currents recorded during pulses to + 4 0 m V with a return to - 8 0 mV in the control solution and during exposure to low Po2. The recovery trace was obtained 3 min after the solution with normal Po2 was introduced again into the chamber. Solutions (in mM): 140 Na +, 5 Ca 2+ and 0.001

+ 2 + 2 + 2 + + TTXinthebath; 130K ,3 /v lg -ATPandO.OOO5ionicCa (4.44Ca and5EGTA) in thep ipet te . (D)K currents elicited by 200 ms pulses to +40 mV with a return to a holding potential of - 8 0 mV in the control solution and during

+ 2 + + + exposure to low Po2. Solutions (in mM): 140 Na , 5 Ca and 0.001 TTX in the bath; 130 K , 3 ME 2 -ATP and 10 EGTA in the pipette. (Taken, with permission, from Ref. 22.)

blockade of Na + channels develops within a few hundred milliseconds 35, this finding indicates that the inhibition of the K + current is indeed instantaneous.

A major criticism 36 concerning the significance of the inhibition of the 02-dependent K + current relates to the nature of the relationship between Po2 and the inhibition itself. In whole-cell recordings, a maximum inhibition was found at a Po~ near 85 mmHg, and the same degree of inhibition was observed at Po2 values of 10, 65 and 110 mmHg 22. In single channel record- ings from isolated patches the maximum decrease in the Po of the 02-sensitive K + channel was observed at about 100 mmHg; again, the decrease in the Po was about the same at 50 and 130 mmHg z3. There are two problems in interpreting these results.

On the one hand, the resting Po,, in carotid body tissue is about 75 mmHg, accordifig to Whalen et al. 37'38 (lower according to other authors, see below), and yet maximum inhibition of the K + current is obtained at 85 mmHg in whole-ceU recordings. On the other hand, carotid sinus nerve action potential frequency starts to rise at a tissue Po2 of about 50 mmHg and continues to increase as tissue Po~ decreases 1, while the inhibition of the K + current by low Po follows the complex pattern described above. Thus, t~he physiological significance of the K + current inhibition as an initial event in the hypoxic transductive cascade is questionable, unless a lack of modulation of the K + channels, due to the recording conditions, is responsible for the high sensitivity of the channel and the peculiar shape of the relationship between Po~ and the K + current inhibition.

Working with an intact carotid body preparation in vitro, it has been found 39 that the relationship between P02 and the increase in cyclic AMP (cAMP) observed during hypoxic stimulation is similar in shape to that for the K + current inhibition, but is displaced to the left by about 40 mmHg. In preliminary whole-cell recordings it has been .observed that addition of dibutyryl-cAMP (a permanent analogue of the natural nucleotide) to the bathing solution also inhibits the transient component of the K + current (L6pez-L6pez, J. R., unpublished observations). Therefore, it is con- ceivable that, in physiological conditions, cAMP (and probably other second messengers) modulate the 02- sensitive K + channel to make it sensitive to the effective 02 pressures. In this context is should also be mentioned that addition of forskolin (an activator of adenylate cyclase), isobutylmethyLxantine (an inhibitor of phosphodiesterase) or dibutyryl-cAMP to the bathing solution potentiates the release of DA in response to different stimuli, the potentiation being maximum with the hypoxic stimulus 4°.

Another important aspect to be considered in the membrane model of P02 transduction is the identity of the 02 sensor. Although it has been suggested many times that it is a hemoprotein 1, nothing is known about its chemical nature. Whatever its chemical identity, it seems to be located in the plasma membrane, because low Po~ decreases the eo of K + channels in excised membrane patches. In fact, it has been postulated that the channel itself may be the sensor ~3. Recently the suggestion has been made 41'42 that a heine-linked NADPH oxidase could be the 02

TINS, Vol. 15, No. 4, 1992 149

A K + 02

C a 2 + , f ~ ~__

! ~ 3 Ca 2+

_ \ \ \ \ \ \

cAMP /

5 / /

//+ 1

ATP

8 DA

1 AH co: I

DNP H + ~ /

Cl-

\ Ca 2+ - cI-

Fig. 4. Models for sensory transduction in chemoreceptor cells of carotid bodies. (A) The proposed cascade for low Poe transduction. On lowering P02 (I) a signal originates at the 02 sensor, inhibiting the K + current and activating adenylate cyclase (2). A drop in membrane potential follows the inhibition of the K + current, which in turn activates voltage-dependent Ca 2+ channels leading to Ca 2+ influx and an increase in the intracellular concentration of Ca 2+, [Ca2+]i (3). Increased [Ca2+]i results in the activation of the exocytotic machinery (4). Increased cyclic AMP levels modulate the K + current and the exocytotic machinery, making low Po2 stimulation more effective (5). The model does not exclude the participation of other second messengers systems

2+ 2+ as well as an interplay between influx of extracellular Ca and Ca from intracellular stores. (B) Proposed model for the transduction of acidic stimuli. Some possible ways of increasing the intracellular H + concentration [H+]~ are indicated (I), such as by diffusion of protonated weak acids (AH), C02 or H + ions, or by the operation of protonophores like dinitrophenol (DNP). The increase in [H+]i stimulates (dashed arrows) the operation of Na +-coupled, H+-extruding systems that bring Na + into the cell. Two of these systems are represented: the Na +--H + exchanger (2) and a Na +-dependent HC03--CI- exchanger (3). The increase in [Na +]i drives the entry of Ca 2+ through the Na+-Ca 2+ exchanger (4), resulting in the activation of the exocytotic machinery (5). Evidence for the presence of a Na +-independent HC03 - -C I - exchanger has recently been provided 64. Since experimental data show that 20% of the acid-evoked release of DA is not dependent on external Ca 2+, this part of the response could be attributed to Ca 2+ derived from intracellular stores. [Part (B) taken, with permission, from Ref. 17.]

sensor in chemoreceptor cells. According to this proposal, a reduction in hydrogen peroxide produced by the oxidase during low Po2 stimulation changes the glutathione redox state, which in turn alters the configuration of membrane proteins and hence the conductivity in ionic channels. The participation of this system in the modulation of ionic conductances during hypoxic stimulation is conceivable, but it remains to be demonstrated which conductances are affected. On the other hand, the isolated membrane patch record- ings show that low Po~ is able to modify K + conductance even when this mechanism is not oper- ative.

(2) The metabolic hypothesis: a mitochondrial model. A different view of low Po~ transduction in the chemoreceptor cells of the carotid body has evolved in recent years from the metabolic hypothesis of chemo- reception put forward in 1963 by Anichkov and Belenkii 43. They proposed that hypoxia, as well as metabolic poisons, activate the chemoreceptors by decreasing ATP levels. No link was provided between the presumed decrease in ATP and the release of the 'hypoxic' neurotransmitter.

Criticisms of the metabolic hypothesis came from the consideration that cytochrome oxidase has a high affinity for Oz, while the carotid body chemoreceptors have a very high blood flow and a hypoxic threshold at arterial blood Po~ of about 70 mmHg 44. The descrip- tion of a cytochrome oxidase with a very low 02 affinity (Km~90 mmHg) within the carotid body tissue 45, in addition to the normal high-affinity one (Km~ 5 mmHg), seemed to provide one of the missing pieces in the metabolic hypothesis. The existence of such a cytochrome oxidase has been questioned on the basis of direct spectrophotometric measurements 46, but supported on the basis of good fitting of O2-consumption data to a simulated model comprising two cytochrome oxidases 37'3s. More re- cently, however, Wilson et al. 47, using a new optical method for measuring 02 concentrations, found that the O2 dependence of oxidative phosphorylation in isolated liver mitochondria extends well into the physiological range of Po~. They concluded that in tissues such as the carotid body there is no need for a low-affinity cytochrome oxidase to generate a de- crease in the [ATP]/[ADP][Pi] quotient (phosphate potential), even at moderate hypoxic stimulation.

The oxygen partial pressures reported for the carotid body tissue are not helpful in making any inference on the existence in chemoreceptor cells of a cytochrome oxidase with a low affinity for oxygen. With the mean oxygen partial pressures reported by

70 Acker and co-workers in normal resting conditions 25 and 7 mmHg for the cat and the rabbit carotid body, respectively), the chemoreceptors of the carotid body would be firing at an almost maximal rate ff it is assumed that the Po~ sensor is a low-affinity cytochrome oxidase. On the contrary, the tissue PQ

37 38 values reported by Whalen and co-workers ' would in fact require the existence of a low-affinity enzyme for the mitochondria to be able to trigger chemorecep- tion in the first place. No carotid body tissue Po2 values were reported by Wilson.

Whatever the case regarding the cytochrome oxidases, a different question concerns the nature of the signal for chemoreception that results from the detection of low Pox at the mitochondria. According to

150 TINS, VoL 15, No. 4, 1992

some authors 48 (see below and Refs 36 and 49), the signal is a decrease in the mitochondrial electrochemi- cal gradient, which then results in an impairment of the mitochondrial uptake of Ca 2+, a rise in the intra- cellular Ca 2+ concentration [Ca2+]i, and the release of neurotransmitters. According to other authors, the important signal is a decrease in the phosphate poten- tial in the cell cytoplasm 47'5°'~1. Although a decrease in the mitochondrial electrochemical gradient also pro- duces a decrease in the phosphate potential, the latest evidence argues that the intrinsic mitochondrial electro- chemical gradient is not paramount to chemoreception, because oligomycin (a blocker of the ATP-synthesizing mitochondrial ATPase) preserves such a gradient and excites the chemoreceptors, while hypoxia and cyan- ide, which decrease the gradient, also excite the chemo- receptors. The signal triggering the chemorecep- tion would be, according to these authors 47,s°,5~, the decrease in the phosphate potential, although no ex- planations of how it might occur have been put forward. As pointed out by Acker et al. 41, it is difficult to en- vision a decrease in the phosphate potential being a sig- nal for low Po~ chemoreception, since the carotid body is in fact requtred to function maximally at low P%.

A restatement of the Ca 2 ÷ version of the metabolic hypothesis has been put forward quite recently 36'49. According to this new theory, low Po slows the

• , 2

electron transfer in the resptratory chain of chemo- receptor cells, leading to a decrease in the mitochon- drial proton electrochemical gradient. As a conse- quence, mitochondria release Ca 2÷ and increase [Ca2+]i, which then triggers neurotransmitter re- lease. The presence of a low-affinity cytochrome oxidase is included in the model to explain the full range of carotid body response to hypoxia, although the experimental data presented do not support the premise (see Ref. 49). The assumption is made that mitochondria have high enough levels of Ca 2÷ to produce significant rises in [Ca2+]i. In the model, the voltage-operated Ca 2÷ channels are activated in normal conditions to provide the Ca 2+ needed to load the mitochondria. The crucial experimental observa- tion for the model is that the rise in [Ca2+]i observed both during anoxia (achieved by N2 equilibration and addition of dithionite) or cyanide application persists, although reduced in amplitude, in bathing solution that is virtually free of Ca 2+ (Refs 52, 53), and it is not modified by caffein (an agent that releases Ca 2+ from endoplasmic reticulum). The effects of dithionite, a very powerful and unspecific reducing agent, on intra- cellular Ca 2÷ deposits are unknown (see below).

It is difficult to reconcile all the mitochondrial Ca 2+ versions of the metabolic hypothesis with some well- established facts such as the dependence on extra- cellular Ca 2+ and the dihydropyridine sensitivity of the low P%-induced release of DA 8' 16,18 (see Table I). It is also difficult to reconcile these proposals with the current ideas about the Ca 2÷ content of mitochondria in healthy resting cells ~4's5. Mitochondria have levels of Ca 2+ that are not dissimilar to those found in the cell cytosol; therefore, they would play a minor role, or no role at all, in the transient elevation of cytosolic Ca 2+ (Refs 54, 55). Contrary to the proposals of Biscoe and Duchen 36'49, if mitochondria accumulate Ca 2+ it must be during enhanced physiological activity, not at rest, because it is only during cell activation that cytoplasmic Ca 2+ would reach the concentrations

required for operation of the low-affinity, Ca2+-uptake systems in mitochondria S4'55.

This section on low P% (hypoxic hypoxia) trans- duction should not be concluded without some con- siderations on the mechanism(s) of action of cyanide, a metabolic poison that produces histotoxic hypoxia and is a powerful chemostimulant t. Because it is easy to work with, cyanide has often been used in arterial chemoreception experiments with the tacit or explicit assumption that it excites the arterial chemoreceptors in the same way that low P02 does. However, in 1968 Coxon $6 had already pointed out that cyanide inhibits, in addition to cytochromes, some 40 different enzymes (see also Ref. 57); that is, cyanide not only blocks 02 consumption to produce histotoxic hypoxia but also affects many other cellular processes. Therefore, it would be an oversimplification to ascribe the chemo- stimulant power of cyanide solely to its capability to produce histotoxic hypoxia. In fact, it has been re- ported 41'46 that cyanide (10 mM) and anoxia induce different light absorbance spectra in the carotid body tissue, indicating that different pigments have been affected by both stimuli.

In recent electrophysiological experiments 5s in chemoreceptor cells of carotid body tissue that were isolated from adult rabbits, but were not exposed to culture medium, it was found that cyanide (2 mM) increased a Ca2+-dependent component of the whole K + currents elicited by brief depolarizing steps, and at the same time decreased Ca 2+ currents. At holding potentials more positive than -30 mV and in the presence of extracellular Ca 2+, prolonged exposures to cyanide also produced an increase in outward currents. This was interpreted to be due to a steady increase in Ca2+-dependent K + currents produced by a steady increase in cytoplasmic [Ca2+] 5s. These observations, added to the fact that the inclusion of ATP in the recording pipette did not modify the electrical responses to the poison 58, led to the proposal that the presumed increase in cytoplasmic Ca 2+ must be due to the release of Ca 2+ from mitochondria 36'49"58. It was assumed that cyanide acted by the same mechanisms as low Po~, and so these findings were pivotal for the restatement of the metabolic hypothesis36, 49.

The-~ta of Biscoe and Duchen 58, however, can be interpreted in another way. On the one hand, it is questionable that mitochondria can release Ca 2+ (see above) and in fact Biscoe et aL 52 found that 'in only six out of eleven preparations, a response (an increase in [Ca2+]i) was still seen soon after removal of [Ca2+]o, but was smaller than the control. In the remainder, [Ca2+]i fell rapidly on removal of [Ca2+]o, and no increase could be seen in response to cyanide'. These findings evoke two considerations. (1) If the previously mentioned electrical response to prolonged exposure to cyanide was in fact due to an increase in [Ca2+]i, the intracellular origin of this ion is uncertain. It seems plausible that at holding potentials more positive than -30 mV the Na+-Ca 2+ antiporter is producing a net Ca 2+ influx 59. (2) The contention in subsequent papers 36'49 that cyanide increases [Ca2+]i in the absence of extracellular Ca 2+ seems questionable, unless [Ca2+]i deposits involved in the response are depleted over an unusually fast timecourse.

On the other hand, the net effects of cyanide on chemoreceptor cells from the carotid bodies of adult

TINS, VoL 15, No. 4, 1992 151

rabbits contrast with those of low Po... The natural , . 2 ,

stimulus produces a reduction m a transient Ca -~-- independent component of the K + current 19'22'2a and does not modify Ca 2+ currents 19'22. However, it should be noted that in these latest studies chemo- receptor cells were exposed to culture medium for between 4 and 48 hours, and, although unlikely (the same responses were obtained in all the cells, independent of their age), some differences between preparations cannot be excluded. In chemoreceptor cells from carotid bodies of rabbit embryos that had been cultured for three days, low Pot reduced K ÷ currents and did not affect Ca 2÷ currents, while cyanide (1 mM) reduced Ca 2+ currents 24. In chemo- receptor cells from carotid bodies of neonatal rats, cyanide (2 mM) reduced an uncharacterized compon- ent of the whole K ÷ current and low P% reduced the Ca2+-dependent component of the K ÷ current 28'57. Even when the apparent differences in the electro- physiological responses to cyanide between prep- arations of chemoreceptor cells are taken into account, we would suggest, in agreement with other authors 24'41'46'56'57, great caution in equating the mechanisms of action of low P% and cyanide.

Transduction of acidic stimuli The Ca 2+ dependence of the release of DA induced

by acidic stimuli and the insensitivity of DA release to dihydropyridines (see Table I) prompted a search for Ca z÷ influx pathways into chemoreceptor cells other than voltage-dependent Ca 2+ channels. Since the parameter sensed by these cells is phi 11'6°'61, the increase in [H+]i and the influx of Ca 2+ must be coupled in some way. On intracellular acidification, cells export protons in exchange for Na +, leading to an increase in [Na÷]i. If the cells have a Na+-Ca 2÷ antiporter in their plasma membrane, an increase in [Na+]i would reverse the normal functioning of this antiporter, producing net Ca ~+ influx and activation of Ca2+-dependent responses 62. These predictions have been demonstrated in mammalian cardiac muscle 6a.

Evidence has been obtained indicating that chemo- receptor cells indeed possess a strong Na÷-Ca 2+ antiporter, a Na+-H + exchanger, and a Na +- dependent anion exchanger 17. For example, blockade of the Na ÷ pump with ouabain or removal of extracellular K ÷ induces DA release that is dependent on extracellular levels of Na + and Ca 2+, and that is insensitive to dihydropyridines. Release of DA in- duced by acidic stimuli was totally dependent on the presence of Na ÷ in the medium and partially inhibited by ethylisopropylamiloride, a blocker of the Na+-H + antiporter. The release was further decreased by reduction of HCO3- ions and elimination of C1- from the medium. These findings led to the proposal of the model for transduction of acidic stimuli 17 shown in Fig. 4B. With minor variations, all the mechanisms in- volved in this model have been corroborated by direct measurements of [Ca2+]i and pHi 52'64.

The interactions between hypoxic and acidic stimuli a can be explained adequately in the trans- duction models in Fig. 4. Thus, the increased chemo- receptor response in carotid bodies observed during moderate hypoxia on lowering pH or increasing Pc% could be the result of a Bohr-like effect at the O2 sensor, with the subsequent decrease in the affinity for O2 (Ref. 65) and a greater response by chemo-

receptor cells to a given hypoxic level, or, as is the case in the heart 6s, it could be due to Ca 2+ entering via the Na*-Ca 2÷ exchanger as a result of acidosis. The influx of Ca ~+ via the antiporter for a given level of acidosis would be augmented during hypoxia because the cells are depolarized for a longer period of time in this condition and would add to the Ca ~÷ that is entering the cells via the Ca 2* channels activated by the hypoxia. The relationship between [Ca2+]i and the exocytotic release of neurotransmitters for the differ- ent ranges of intracellular Ca 2+ levels 66 would deter- mine the potentiating, additive (or less than additive) interactions between acidic and hypoxic stimuli 3. Finally, with very intense hypoxia, when the release of neurotransmitter reaches high levels, it is conceivable that postsynaptic events could also contribute to the ineffectiveness of acidic stimuli to increase the hypoxic response.

The kinetics of the model in Fig. 4B need to be worked out to see if they fit the rapid response of the intact carotid body chemoreceptors to the acidic stimuli. The steep dependence of pHi on pHo seen in chemoreceptor cells 64'67 and the speed and magnitude of the increase in [Na+]i observed in other systems in response to an acidic challenge 68' 69 are compatible with the prompt response of the carotid body to acid. The model does not consider the participation of ionic channels to be relevant because, on lowering pHo, a parallel inhibition of Na ÷, K ÷ and Ca 2+ conductances was found (L6pez-L6pez, J. R. and Ganfornina, M. D., unpublished observations); also, the release of DA elicited by acidic stimuli was not sensitive to dihydro- pyridines (see Table I). However, it should be noted that in studies performed in neonatal rats 26, a Ca 2÷- dependent K ÷ current was inhibited and Ca 2÷ currents were unchanged when the cells were perfused with weak acid-containing media or when pHo was lowered. These findings seem to indicate a previously undis- covered difference between chemoreceptor cells of adult and neonatal animals and suggest that membrane potential and then probably Ca 2 ÷ channels play a role in acid transduction.

Concluding remarks The carotid body is an arterial chemoreceptor

activated by decreases in blood P% and pH or increases in blood Pco2- From the point of view of a systemic physiologist, the carotid bodies represent the origin of reflexes with homeostatic and adaptive roles, which maintain blood gases and pH within normal ranges. For cellular physiologists, the struc- tures of the carotid body that sense changes in blood gases and pH - the chemoreceptor cells - represent a sensory receptor in which the variations in blood gases and pH are transduced into a neurosecretory response. In this review, we have attempted to dissect and understand the ensemble of the basic mechanisms operating in the cells, from the arrival of the bloodborne stimuli to the exocytotic release of neurotransmitters. We have discussed current (and sometimes opposite) views on these aspects, but many basic mechanisms that surely operate in chemo- receptor cells have not yet been explored. As a consequence, the models proposed must be seen as incomplete. Further characterization of ionic channels and ion transporters, as well as their modulation by second messengers, and the definition of the nature of

152 TINS, VoL 15, No. 4, 1992

the changes that occur in the receptors in perinatal periods and during acclimatization at high altitude are, among many others, aspects of chemoreceptor cells that need to be worked out. Such knowledge will then give a more complete picture of the transductive cascade in the chemoreceptor cells of the carotid bodies.

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Acknowledgement~ The authors would like to thank Prof's B. Herreros and J. 6atria-Sancho for critical discussions in preparing the manuscript. Supported by DGICYT (PB89/0358) of Spain.

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