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Research on arterial chemoreceptors, particularly on the carotid body, has been fruitful in the last fifty years, to which this review is addressed. The functional anatomy of the organ appears to be well established. The biophysical bases by which glomus cells transduce chemical changes in the milieu intérieur (hypoxia, hypercapnia, acidosis) into electrical and biochemical changes in glomus cells have received much attention. Physical changes (in temperature, flow and osmolarity) are also detected by the carotid body. Electrical coupling between glomus cells themselves appears as very extensive. Sustentacular cells classically considered as ensheathing glia for glomus cells and nerve endings now appear to behave as stem cells precursors for glomus cells under chronic hypoxic conditions. Many papers have been devoted to transmitters released from glomus cells (acetylcholine, dopamine, ATP) and well as to their effects upon chemosensory nerve activity. Chemosensory neurons have been explored from generation of action potentials at peripheral nerve endings, passing to properties of perikarya at petrosal ganglia and finally at characterization of synaptic transmission at solitary tract nuclei. There is abundant literature on ventilatory and cardiovascular reflexes elicited from arterial chemoreceptors. The transient effects of sudden and brief withdrawal of chemosensory discharges by hyperoxia also provide clues on the role played by carotid bodies in the homeostasis of full organisms.

The carotid body is a major arterial chemoreceptor that senses low O2 tension, high CO2 tension and low pH in the arterial blood. It is generally believed that neurotransmitters, including acetylcholine (ACh), participate in the genesis of afferent neural output from the carotid body and modulate the function of chemoreceptor cells (glomus cells). Previous pharmacological studies suggest that M1 and M2 muscarinic ACh receptors (mAChRs) are involved in these processes. This study was designed to demonstrate the presence and localization of M1 and M2 mAChRs in the carotid body and in the petrosal ganglion of the cat. Since DNA sequences of the cat M1 and M2 mAChRs were not known, we first determined partial DNA sequences. These sequences and deduced amino acid sequences highly resembled those of human and the rat. Subsequent reverse transcription-polymerase chain reaction (RT-PCR)analysis has demonstrated that mRNAs for M1 and M2 mAChRs are present in the carotid body and the petrosal ganglion of the cat. Immunohistochemistry has indicated that the localization of these receptors appears different. Immunoreactivity for M1 mAChR was strong in nerves in the carotid body. Nerve endings positively stained for M1 mAChR appear to innervate glomus cells. Weak staining for M1 mAChRs was seen in glomus cells. On the other hand, M2 receptor protein seems to be present in glomus cells but not on nerve endings. One third of the neurons in the petrosal ganglion showed immunoreactivity for M1 mAChR. Many neurons and nerve fibers in the petrosal ganglion expressed M2 mAChR immunoreactivity. The results were consistent with previous pharmacological studies. Thus, activation of M1 mAChRs on afferent nerve endings may be linked to the increase in neural output during hypoxia. Further, M1 and M2 mAChRs on glomus cells modulate the release of neurotransmitters.

Oxygen can be said to be the most fundamentally necessary substrate for life. In those organisms having a cardiopulmonary system for delivering it in blood to the tissues the carotid body functions as the principal detector of decreases in arterial oxygen. Such a decrease stimulates an increase in neural output from the carotid body to the nucleus tractus solitarii, and this can precipitate a wide array of systemic reflex responses. The neural mechanisms involved in the genesis of increased signal from the carotid body remain unclear. But a current model of carotid body chemotransduction postulates that transmitter-laden glomus cells initiate the neural activity by being depolarized by hypoxemia and releasing an excitatory transmitter which binds to postsynaptic receptors of the adjacent sensory afferent fibers as well as to presynaptic glomus cell autoreceptors. This Frontiers Review evaluates anew the data supporting the hypothesis that acetylcholine (ACh) is an (the) essential excitatory transmitter in this process by examining ACh's fulfillment of criteria required to establish a substance as a synaptic transmitter. All eight criteria are fulfilled in the case of ACh. Indeed, additional data further support the Cholinergic Hypothesis.

Current modelling of carotid body (CB) chemotransduction postulates an essential role for neurotransmitters, including dopamine (DA). Catecholamines (CA) released from incubated/superfused cat CBs has often been reported to diminish rapidly over the course of the exposure. The purpose of the first set of experiments was to determine the effects of including L-dihydroxyphenylalanine (L-DOPA), the immediate precursor to DA, in the incubation medium. CBs were removed from deeply anesthetized cats, cleaned of connective tissue, and placed in separate incubation tubes containing Krebs Ringer Bicarbonate solution (KRB) at 37 degrees C. One tube contained 40 microM L-DOPA. Both tubes were bubbled for 2 hr with a normoxic gas mixture (21% O2/6% CO2). This was followed immediately by a 30-minute exposure to a hypoxic gas mixture (4% O2/5% CO2). The mean amounts of DA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and norepinephrine (NE) released during 30 min exposures were always greater when L-DOPA was present. The use of gas mixture like the above normoxic gas mixture in incubation studies has often been considered quasi-hypoxic. Hence, in a second set of experiments we tested the effect of high oxygen mixture (95% O2/5% CO2). All other features of these experiments were the same as the above. The high oxygen environment correlated with lower DA release suggesting a reduced excitation/inhibition. The subsequent exposure to hypoxia, however, provoked a much larger release of DA and NE. The data demonstrate the substantial effect of oxygen on the release of CAs and the apparent need of a DA precursor like L-DOPA to allow detection of the changes in CA release from the CBs upon exposure to a hypoxic stimulus.

Previous pharmacological, immunocytochemical, electrophysiological, and microfluorometric studies have suggested that acetylcholine (ACh) is a critically important excitatory transmitter in the chemotransduction of hypoxia by the cat carotid body (CB). With the use of HPLC this study shows that the in vitro cat CB releases ACh under normoxic conditions; this release is increased when the CB is challenged with hypoxia. The preliminary observation that greater amounts of ACh are liberated in the presence of gallamine and AFDX116 suggests the presence of functioning M2 muscarinic receptors on the glomus cells of the CB.

Hypoxia, hypercapnia and acidosis stimulate the carotid body (CB) sending increased neural activity via a branch of the glossopharyngeal nerve to nucleus tractus solitarius; this precipitates an impressive array of cardiopulmonary, endocrine and renal reflex responses. However, the cellular mechanisms by which these stimuli generate the increased CB neural output are only poorly understood. Central to the understanding of these mechanisms is the determination of which agents are released within the CB in response to hypoxia, and serve as the stimulating transmitter(s) for chemosensory nerve endings. Acetylcholine (ACh) has been proposed as such an agent from the outset, but this proposal has been, and remains, controversial. The present study tests two hypotheses: (1) The CB releases ACh under normoxic/normocapnic conditions; and (2) The amount released increases during hypoxia and other conditions known to increase neural output from the CB. These hypotheses were tested in 12 experiments in which both CBs were removed from the anesthetized cat and incubated at 37 degrees C in a physiological salt solution while the solution was bubbled with four different concentrations of oxygen and carbon dioxide. The incubation medium was exchanged at 10 min intervals for 30 min (three periods of incubation). The medium was analyzed with high performance liquid chromatography-electrochemical detection for ACh content. Normoxic/normocapnic conditions (21% O2/6% CO2) produced a total of 0.639 +/- 0.106 pmol/150 microl (mean +/- S.E.M.; n = 12). All stimulating conditions produced larger total outputs: 4% O2/2% CO2 produced 1.773 +/- 0.46 pmol/150 microl; 0% O2/5% CO2, 0.868 +/- 0.13 pmol/150 microl; 4% O2/10% CO2, 1.077 +/- 0.21 pmol/150 microl. These three amounts were significantly greater than the normoxic/normocapnic condition, but indistinguishable among themselves. Further, the amount of ACh released did not diminish over the 30 min of stimulation. These data support the concept that during hypoxia ACh functions as a stimulating transmitter in the CB, and are consistent with the earlier reports of cholinergic enzymes and receptors found in the CB.

With immunocytochemical techniques using a monoclonal antibody for alpha7 subunits of neuronal nicotinic acetylcholine receptors, we have found these subunits to be exclusively expressed in nerve fibers in the carotid body. Double-immunostaining showed that alpha7 subunit-positive nerve endings enveloped tyrosine hydroxylase-positive glomus cells. Some carotid sinus nerve fibers and tyrosine hydroxylase-positive petrosal ganglion neurons also expressed alpha7 subunits. These data support a role for acetylcholine in carotid body neurotransmission., (Copyright 1998 Elsevier Science B.V.)

Acetylcholine increases intracellular calcium of arterial chemoreceptor cells of adult cats. J. Neurophysiol. 78: 2388-2395, 1997. Several neurotransmitters have been reported to play important roles in the chemoreception of the carotid body. Among them acetylcholine (ACh) appears to be involved in excitatory processes in the cat carotid body. As one of the steps to elucidate possible roles of ACh in carotid body chemoreception in the cat, we examined the effect of ACh on intracellular calcium concentration ([Ca2+]i) of cultured carotid body cells. The carotid body from adult cats was dissociated and cultured for up to 2 wk. [Ca2+]i was measured from clusters of cells with a microfluorometric technique using Indo-1 AM. Experiments were performed at 37 degrees C, and cells were continuously superfused with modified Krebs solutions equilibrated with 5% CO2-16% O2-79% N2. ACh (100 mu M) caused a marked increase in [Ca2+]i in approximately 70% of clusters, and the responses to 1-300 mu M of ACh were concentration dependent. The magnitude and kinetics of the ACh response were mimicked by the application of nicotine, whereas muscarinic agonists, pilocarpine, and muscarine failed to evoke a similar response. ACh-induced increase in [Ca2+]i was dependent on extracellular Ca2+: it was greatly reduced or completely abolished by a transient removal of extracellular Ca2+. The response was consistently but only partially reduced by caffeine (5 mM) or nifedipine (10 mu M). The effect of mecamylamine (100 mu M) was inhibitory but small. Moreover, the increase in [Ca2+]i in response to ACh was also observed in some clusters that did not respond to high K (100 mM) Krebs. These results suggest that ACh increases [Ca2+]i of cultured carotid body cells by activating neuronal nicotinic ACh receptors, leading to Ca2+ influx via nicotinic channels. In addition, other pathways such as Ca2+ influx through L-type calcium channels, perhaps secondary to membrane depolarization, and Ca2+ release from intracellular stores may participate in increasing [Ca2+]i in response to ACh. Muscarinic receptors appear to play only a small role, if any.

From the 1930s into the 1970s, the role of acetylcholine (ACh) in the carotid body's chemotransduction of hypoxia was debated. Since the late 1970s, the issue has been pursued only intermittently or not at all. The purpose of this study was to test again with a new preparation the hypothesis that ACh is an excitatory neurotransmitter in the cat carotid body's chemotransduction of hypoxia. We tested the effect of the specific nicotinic blocker mecamylamine and the muscarinic blocker of all five muscarinic receptors, atropine. We further tested the effects of M1 and M2 muscarinic-receptor blockers. The carotid body region was selectively perfused with hypoxic Krebs-Ringer bicarbonate (KRB) solutions that were blocker free or contained varying doses of the blockers. Both mecamylamine and atropine reduced the response to hypoxic KRB in a dose-related manner. The M2 muscarinic-receptor blockers gallamine and AFDX 116 increased the response to hypoxic KRB, whereas the M1 muscarinic-receptor blocker pirenzepine reduced the response to hypoxic KRB. These data are consistent with an excitatory role for ACh in the carotid body chemotransduction of hypoxia in the cat.

Though exogenously delivered acetylcholine excites the carotid body, past evidence has been considered as unsupportive in assigning acetylcholine an excitatory role during hypoxia or hypercapnia. With ganglionic transmission used as the model, data is presented which aims at blocking the postsynaptic cholinergic receptors, at preventing the presynaptic release of acetylcholine, and at quantitating its release under stimulating conditions. The data support an excitatory role for acetylcholine during hypoxia.

It has been postulated that the weak carotid chemoreceptor responses of neonatal mammals may be due to inhibition produced by high levels of endogenous dopamine release or exaggerated sensitivity to dopaminergic inhibition. This was studied by measuring the effect of domperidone, a selective dopamine D2-receptor antagonist, on the carotid chemoreceptor response to O2 and CO2 in anesthetized neonatal and adult cats. The animals were exposed to four levels of isocapnic O2 (arterial PO2 of approximately 35-45, 55-65, 80-90, > 300 Torr) and four levels of isoxic CO2 (end-tidal PCO2 of approximately 21, 40, 58, and 78 Torr) before and after D2-receptor blockade. Whole nerve activity was recorded from the carotid sinus nerve (CSN). Both neonatal and adult cats increase CSN activity during hypoxia and hypercapnia (P < 0.001). Domperidone caused an increase in CSN activity at all O2 levels in adults (P < 0.01) but only during hypoxia in neonates (P < 0.001). Domperidone caused an increase in CSN activity during normo- and hypercapnia in adults but only during hypercapnia in neonates (P < 0.001). Domperidone approximately doubled an index of hypoxic sensitivity in the normoxia-hypoxia range (100 to 40 Torr) in the neonatal group but had little effect on sensitivity to hypoxia in adults. We conclude that the inhibitory role of endogenous dopamine in the carotid chemoreceptors changes with postnatal development.

The purpose of this study was to test the hypothesis that acetylcholine (ACh) is an excitatory neurotransmitter during the hypoxic stimulation of the carotid body. Cats were anesthetized, paralyzed, and artificially ventilated. The common carotid artery was fitted with a loop containing a stopcock for selectively perfusing the carotid body. Neural activity was recorded from the whole carotid sinus nerve. After the cats had been ventilated on 10% O2 for 3 min with the carotid body being normally perfused with its own hypoxic arterial blood, the stopcock was turned, and either equally hypoxic Krebs-Ringer bicarbonate solution (KRB) containing alpha-bungarotoxin, mecamylamine, and atropine or hypoxic blocker-free KRB perfused the carotid body for 2 min. The stopcock was returned to its original position, allowing blocker-free hypoxic blood to perfuse the carotid body once again. With this protocol we found 1) the cholinergic blockers reduced the carotid body response to hypoxic KRB in a dose-dependent manner; 2) carotid baroreceptor activity was not reduced by the blockers, suggesting that the action of the blockers was not nonspecific (whereas lidocaine rapidly reduced both chemoreceptor and baroreceptor activity); 3) inclusion of the blockers in perfused hypoxic blood also reduced neural output from the carotid body; and 4) the blockers reduced the carotid body's neural response to hypoxic KRB containing substance P (20 micrograms/100 ml), suggesting that substance P may be linked to ACh in the carotid body. We conclude that these data provide good evidence supportive of an excitatory role for ACh in carotid body hypoxic excitation.

This study aimed to characterize neural responses of the carotid chemoreceptors of the maturing cat to natural stimuli and to determine the time course of carotid chemoreceptor development from the neonatal period to adulthood. Carotid sinus nerve (CNS) responses to O2 and CO2 were studied in cats at 1, 4, and 8 wk of age and in adult cats (n = 6 at each age). Pentobarbital sodium-anesthetized cats were exposed to three levels of O2 (arterial PO2 = 40-45, 80-90, and > 300 Torr) at five levels of arterial PCO2 (22, 35, 48, 63, and 75 Torr) while the moving average of whole nerve output from the CSN was recorded. Ganglioglomerular nerves were sectioned. All cats at every age increased CSN activity during hypoxia. However, the CSN response to hypoxia was not sustained in some immature cats. Of the cats that sustained CSN activity during hypoxia, four of the six 1-wk-old cats showed a biphasic pattern of response, with an initial overshoot followed by a steady level of discharge. Older cats did not exhibit this pattern. CNS sensitivity to hypoxia was weakest in 1-wk-old kittens but increased to nearly adult levels by 4 wk of age. Carotid chemoreceptor responses to CO2 were also smallest in 1-wk-old kittens and increased with maturation. However, unlike hypoxia responses, CO2 sensitivity during hypoxia continued to develop between 8 wk and adulthood. O2-CO2 interaction did not become significant until after 4 wk of age. Thus, carotid chemoreceptor responses to both O2 and CO2 are weak in newborn cats and increase during postnatal development.(ABSTRACT TRUNCATED AT 250 WORDS)

The hypothesis that the entry of extracellular calcium ions into some compartment, quite possibly the type I cells, through voltage-gated calcium channels (VGCC) is essential for hypoxic chemotransduction in the cat carotid body was tested using an in situ perfusion technique. The neural output of the carotid body of anesthetized, paralyzed, and artificially ventilated cats in response to perfusions with Krebs-Ringer bicarbonate solution (KRB), calcium-free KRB, KRB containing calcium channel blockers, or KRB containing BAY K 8644 was recorded. Selective perfusion of the carotid body with hypoxic calcium-free KRB significantly decreased carotid chemoreceptor activity, suggesting that extracellular calcium is essential for hypoxic chemotransduction. Selective perfusion of the carotid body with hypoxic KRB containing verapamil (10-100 microM), diltiazem (10-100 microM), or nifedipine (10-100 microM) dose dependently attenuated the increase in chemoreceptor activity produced by hypoxia, suggesting that VGCC need to be activated for hypoxic chemotransduction. The carotid body response to hyperoxic KRB containing the calcium channel agonist BAY K 8644 (10 microM) was 267 +/- 87% of hyperoxic control KRB, suggesting that an enhanced influx of calcium ions through VGCC stimulates carotid chemoreceptor activity. Selective perfusion of the carotid body with severely hypoxic KRB containing BAY K 8644 did not increase chemoreceptor activity above that produced by severe hypoxia alone. This suggests that severe hypoxia increases intracellular calcium in some compartment of the carotid body to achieve stimulatory maximum response and that further increase in intracellular calcium does not produce further elevation of neural activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Carotid chemoreceptor activity was increased by the perfusion of the carotid body in vivo with hypoxic HEPES-buffered solution (HBS) containing CO2/HCO3- (HBA+), but not with hypoxic HBS without CO2/HCO3- (HBS-). When the perfusate was switched to hypoxic HBS+ during hypoxic HBS-perfusions, chemoreceptor activity increased immediately. Thus, CO2/HCO3- played a critical role in the hypoxic chemotransduction of the in vivo perfused carotid body.

The sodium-proton (Na(+)-H+) antiporter has been found in virtually every tissue where its presence has been investigated. Its principal physiological role is to regulate intracellular pH (pHi). Amiloride (10(-3)-10(-4) M) is a known blocker of the antiporter when Na is present in normal physiological concentrations (130-140 x 10(-3) M). In order to determine if the Na(+)-H+ antiporter participated in the chemoreception of hypercapnia or hypoxia anesthetized, paralyzed, artificially ventilated cats were fitted with a loop in the right common carotid artery for the selective perfusion of the carotid body. Neural activity (imp/10 sec) was recorded from single or few fiber preparations during hypercapnia (PaCO2 = 48-64 Torr) while the carotid body was perfused with Krebs-Ringer bicarbonate solution for 2.5 min, then with its own hypercapnic arterial blood (4 min), then with Krebs-Ringer bicarbonate solution containing 0.6-0.8 x 10(-3) M amiloride (2.5 min), then with its own hypercapnic blood (4 min). After 20 min of rest the protocol was repeated during hypoxia (PaO2 = 35-45 Torr). The carotid body response to hypercapnic blood was unaffected by a preceding perfusion of the amiloride-containing solution but the response to hypoxic blood was decreased by 25% by the amiloride-containing solution. The data suggest the possibility of different mechanisms being involved in the chemoreception of hypercapnia and hypoxia.

The response (imp . s-1) of single- or few-fiber preparations from the carotid body (10 experiments) and the aortic body (5 experiments) to various levels of hypercapnia on different backgrounds of hypoxia were analyzed by two statistical techniques--analysis of variance and the Duncan's new multiple-range test. These analyses showed an initial statistically significant increase in the slope of the response to increasing arterial pressure of CO2 (PaCO2) as PaO2 fell. But the slope of the response to carbon dioxide later showed a clear tendency to become less; i.e., no significant increase in imp . s-1 when a PaCO2 rose (substantially) with normoxic (carotid body) and hypoxic (carotid and aortic bodies) backgrounds. The response of the aortic body to hypercapnia showed no statistically significant increase if the background was hyperoxia or normoxia. The characteristic of the chemoreceptor to become saturated in its response to carbon dioxide while still retaining its ability to respond to hypoxia suggests the possibility that at least some of the mechanisms involved in the chemoreception of hypoxia differ from those involved in the chemoreception of hypercapnia.

The purpose of this study was to determine the content of catecholamines (CA) in the cat carotid body before and after 0.5 h exposures to normoxic normocapnia [arterial O2 partial pressure (Pao2) 126 +/- 28 Torr, arterial CO2 partial pressure (Paco2) 36.4 +/- 1.5 Torr], hypoxic normocapnia (Pao2 25 +/- 3 Torr, Paco2 36.7 +/- 3.3 Torr), and normoxic hypercapnia (Pao2 132 +/- 13 Torr, Paco2 = 98.2 +/- 7.6 Torr). CA synthesis was blocked using alpha-methylparatyrosine methyl ester (AMPT) prior to alterations in the inspired air. There was a significant decrease in carotid body content of dopamine (DA), norepinephrine (NE), and epinephrine (E) 1 h after AMPT administration. Analysis of variance and Duncan new multiple range procedures revealed that during the subsequent 0.5-h exposures to normoxia, hypoxia, or hypercapnia, only the decrease in DA during hypoxia was significantly greater than that during normoxia; the loss during hypercapnia was not. The decreases in NE during the three exposures were indistinguishable among themselves as were the decreases in E. The decrease in CA content is probably attributable to increased release. The data reveal that the release of CAs during the chemoreception of hypoxia is different from that during the chemoreception of hypercapnia and support the concept of different mechanisms for the chemoreception of hypoxia and hypercapnia.