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J Appl Physiol 96: 376-383, 2004. First published August 15, 2003; doi:10.1152/japplphysiol.00726.2003
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HIGHLIGHTED TOPICS
Oxygen Sensing in Health and Disease

Acetylcholine release from the carotid body by hypoxia: evidence for the involvement of autoinhibitory receptors

Dong-Kyu Kim,1 Nanduri R. Prabhakar,2 and Ganesh K. Kumar1

Departments of 1Biochemistry and 2Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106

Submitted 14 July 2003 ; accepted in final form 1 August 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
The purpose of the present study was to investigate whether hypoxia influences acetylcholine (ACh) release from the rabbit carotid body and, if so, to determine the mechanism(s) associated with this response. ACh is expressed in the rabbit carotid body (5.6 ± 1.3 pmol/carotid body) as evidenced by electrochemical analysis. Immunocytochemical analysis of the primary cultures of the carotid body with antibody specific to ACh further showed that ACh-like immunoreactivity is localized to many glomus cells. The effect of hypoxia on ACh release was examined in ex vivo carotid bodies harvested from anesthetized rabbits. The basal release of ACh during normoxia (~150 Torr) averaged 5.9 ± 0.5 fmol·min-1·carotid body-1. Lowering the PO2 to 90 and 20 Torr progressively decreased ACh release by ~15 and ~68%, respectively. ACh release returned to the basal value on reoxygenation. Simultaneous monitoring of dopamine showed a sixfold increase in dopamine release during hypoxia. Hypercapnia (21% O2 + 10% CO2) as well as high K+ (100 mM) facilitated ACh release from the carotid body, suggesting that hypoxia-induced inhibition of ACh release is not due to deterioration of the carotid body. Hypoxia had no significant effect on acetylcholinesterase activity in the medium, implying that increased hydrolysis of ACh does not account for hypoxia-induced inhibition of ACh release. In the presence of either atropine (10 µM) or domperidone (10 µM), hypoxia stimulated ACh release. These results demonstrate that glomus cells of the rabbit carotid body express ACh and that hypoxia overall inhibits ACh release via activation of muscarinic and dopaminergic autoinhibitory receptors in the carotid body.

hypercapnia; muscarinic receptors; dopaminergic receptors; glomus cells

CAROTID BODIES ARE THE SENSORY organs for detecting changes in arterial oxygen, and the ensuing reflexes are critical for eliciting appropriate cardiorespiratory changes during hypoxia. The sensory complex of the carotid body consists of glomus cells derived from the neural crest origin, the afferent nerve fibers that are in synaptic contact with glomus cells, and the supporting glial-like cells (15, 22). Glomus cells, the putative oxygen-sensing cells in the carotid body, express conventional [e.g., acetylcholine (ACh), catecholamines, and neuropeptides] as well as unconventional (e.g., nitric oxide and carbon monoxide) neurotransmitters (for recent reviews, see Refs. 29 and 43). Although the mechanisms of sensory transduction are not fully understood, it is generally accepted that transmitter molecules released from the glomus cells are important for sensory transmission (22, 31, 43).

Among the various transmitters expressed in the carotid body, ACh has received considerable attention. On the basis of pharmacological studies, ACh has been proposed as a major excitatory transmitter for mediating sensory excitation during hypoxia (for review, see Ref. 18). Earlier studies reported an increase in the release of ACh-like substances from cat (14) and dog (34) carotid bodies during hypoxia, supporting its role as a sensory transmitter. However, the conclusion that hypoxia releases ACh from the carotid body has been questioned because the bioassay techniques employed in these earlier investigations may not be selective for ACh. Recently, Fitzgerald and coworkers (19), using HPLC coupled to an electrochemical detection system, reported an enhanced release of ACh from the cat carotid body during hypoxia. Although this study supports the notion that ACh is important for sensory transmission in the cat carotid body, it remains uncertain whether ACh is also crucial for sensory transmission of the carotid body in species other than the cat. For instance, Zhang et al. (53) have reported that, in cocultures of newborn rat glomus cells with neurons of the petrosal ganglion, hexamethonium or mecamylamine in combination with suramin inhibited the sensory excitation by hypoxia. From these observations, Zhang et al. proposed that hypoxia facilitated the corelease of ACh and ATP (53). However, Gauda (20), using an in situ hybridization histochemical technique, made an intriguing observation that mRNA for choline acetyl transferase (ChAT) and vesicular ACh transporter, the definitive markers of cholinergic traits (45, 52), are not expressed in newborn rat carotid bodies, thus questioning the importance of ACh in the sensory transmission of the carotid body. Although hypoxia augments the carotid body sensory discharge uniformly in every species studied thus far, the exogenous effects of putative neurotransmitters seem to vary in a species-dependent manner (18, 36, 39). If ACh is important for the sensory response of the carotid body to hypoxia, then hypoxia should facilitate the release of ACh in a species-independent manner. Therefore, the objective of the present study was to determine whether ACh is released during hypoxia from the carotid bodies of rabbit, a species that has been extensively used in chemoreceptor research (3, 5, 16, 21, 27, 42). In our studies, the experimental protocols used to examine ACh release were essentially the same as those reported for the cat carotid bodies (19), thus permitting comparison of the effects of hypoxia on ACh release from the rabbit and cat carotid bodies. Our results from the rabbit carotid bodies showed that hypoxia inhibits whereas hypercapnia augments ACh release. Furthermore, our data also suggest that the inhibition of ACh release by hypoxia is coupled to activation of cholinergic and dopaminergic autoinhibitory receptors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Reagents and Materials

ACh, choline, C-18 reverse-phase microbore columns, immobilized enzyme reactor, glassy carbon electrodes, peroxidase redox polymer, and Kathon CG (bactericide) were purchased from Bioanalytical Systems (West Lafayette, IN). Atropine, neostigmine bromide, domperidone, and tetra-isopropyl-pyrophosphoramide (isoOMPA) were obtained from Sigma Chemical (St. Louis, MO). Anti-ACh and anti-tyrosine hydroxylase (TH) monoclonal antibodies were obtained from Chemicon International (Temecula, CA) and Sigma Chemical, respectively. Biotinylated anti-mouse IgG (BA-2001), diaminobenzidine (DAB), and Vector VIP were purchased from Vector Laboratories (Burlingame, CA).

General Preparation of Animals

Animal experimental protocols were approved by the Case Western Reserve University Institutional Animal Care and Use Committee and are in agreement with the National Institutes of Health Guiding Principles in the Care and Use of Animals. Adult male New Zealand White rabbits (weighing 2.0–2.5 kg) were purchased from Charles River Laboratories (Wilmington, MA).

Isolation of Carotid Bodies

Rabbits were anesthetized by intraperitoneal injection of urethane (1.2 g/kg), and the bifurcations at the common carotid arteries were rapidly excised. Carotid bodies were isolated from the bifurcation under the microscope and placed in ice-cold, oxygenated, Ca2+-Mg2+-free, modified Krebs-Ringer bicarbonate (KRB) solution containing 1 mM isoOMPA (an inhibitor of nonspecific cholinesterase) and 300 µM neostigmine [an acetylcholinesterase (AChE)-specific inhibitor] until further analysis of ACh release.

Measurements of ACh and Dopamine

Tissues (4 carotid bodies per experiment) were homogenized with a sonicator (Sonics and Materials, Danbury, CT) in 300 µl of medium, containing 0.1 N HClO4 and 0.25 mM disodium EDTA, three times for 30 s at 4°C. The homogenates were centrifuged at 12,000 g for 15 min at 4°C. The clear supernatant either after neutralization with 0.1 N Na3PO4 or as such was used for the analysis of ACh and dopamine (DA), respectively, by two dedicated HPLC-electrochemical detector (ECD) systems as described below.

ACh was measured with HPLC coupled with an ECD and immobilized enzyme reactor column containing AChE and choline oxidase. ACh was separated from its endogenous metabolite choline by reverse-phase HPLC and then converted to H2O2 enzymatically online in the immobilized enzyme reactor column. The amount of H2O2, a measure of ACh, was determined with the use of a peroxidase-coated glassy carbon electrode and an ECD with an oxidation potential of 100 mV vs. Ag-AgCl. For the isocratic separation of ACh from choline, a mobile phase containing 30 mM NaH2PO4, pH 8.5, and bactericide (5 ml/l Kathon) was used at a flow rate of 130 µl/min. Under the experimental condition, ACh eluted at ~13 min with a detection limit of 80 fmol. DA was determined by HPLC-ECD (Shimadzu System) as described previously (28). Briefly, catecholamines were separated on an octadecyl silane reverse-phase column (Beckman) by isocratic elution at 40°C with a mobile phase consisting of 4% (vol/vol) acetonitrile, 0.1 M sodium nitrate, 0.08 M sodium dihydrogen phosphate, 0.2 mM sodium octyl sulfate, and 0.1 mM EDTA adjusted to pH 2.7 with phosphoric acid. Under the experimental conditions, DA was eluted at 7.4 min with an average recovery of ~82% as determined via an internal standard, 3,4-dihydroxybenzylamine. The detection limit for DA was ~75 pmol. The elution profiles were recorded and analyzed with a Hitachi D-2500 Chromato-Integrator. The concentrations of ACh and DA were determined by using standard curves correlating the concentration of either ACh or DA to the corresponding integrated peak area. Transmitter content was expressed in picomoles per carotid body.

Isolation of Glomus Cells

Glomus cells were dissociated enzymatically from freshly harvested carotid bodies from adult rabbits as described previously (40). Briefly, carotid bodies were incubated at 37°C in a medium containing trypsin (type II, 2 mg/ml, Sigma Chemical) and collagenase (type IV, 2 mg/ml, Sigma Chemical). The composition of the modified Tyrode medium, used for the isolation of glomus cells, was as follows (in mM): 140 NaCl, 5 KCl, 10 HEPES, and 5 glucose, pH 7.2. The tissues were triturated with a fire-polished, glass Pasteur pipette every 10 min. After 30 min of incubation, cells were collected by centrifugation at 200 g for 5 min and further incubated in the dissociation medium containing collagenase (2 mg/ml) and BSA (5 mg/ml) for an additional 30 min. The resulting glomus cells were collected and suspended in a 50:50 mixture of DMEM and Ham's F-12 supplemented with penicillin-streptomycin (GIBCO BRL), insulin, transferrin, selenium (ITS, Sigma Chemical), and 5% heat-inactivated fetal bovine serum. Subsequently, the cells were plated onto Lab-Tek chamber slides coated with poly-D-lysine and maintained at 37°C in a CO2 incubator; these cells were used for immunocytochemical analysis within 36 h.

Immunocytochemical Studies

Unless otherwise specified, all the procedures were performed at 25°C. Cells plated onto Lab-Tek chamber slides were treated with 0.1 M phosphate buffer, pH 7.2, containing 2% (wt/vol) paraformaldehyde, 0.5% (vol/vol) glutaraldehyde, and 10 mM 2-nitrobenzyl alcohol. After the fixation, the cells were incubated in 0.5% (vol/vol) H2O2 in PBS for 15 min to reduce endogenous peroxidase activity. To block any nonspecific binding sites, cells were first incubated in PBS solution containing 1% (wt/vol) BSA, 2% normal horse serum, and 0.2% (vol/vol) Triton X-100 for 1 h. The cells were then further incubated successively with solutions of avidin D and biotin, for 15 min each.

For immunolabeling of the glomus cells for ACh or TH, an established marker of glomus cells, monoclonal anti-ACh and anti-TH antibodies at dilutions of 1:100 and 1:4,000, respectively, were used. After the nonspecific binding sites were blocked, the cells were incubated with the primary antibody for anti-ACh or anti-TH for 1 h and washed three times for 10 min each in buffer containing Triton X-100. Thereafter, they were incubated with horseradish peroxidaseconjugated anti-mouse IgG (7.5 µg/ml; Vector Laboratories) for 1 h and washed with PBS three times for 5 min each. After the washes, for the immunostaining of ACh and TH, the peroxidase reaction was initiated with Vector VIP and DAB as substrates, yielding purple and dark brown color, respectively.

For double immunolabeling of ACh and TH, the cells were first immunostained with anti-ACh antibody, and peroxidase reaction was performed with Vector VIP as substrate, resulting in purple reaction product. The cells were then washed and immunolabeled with anti-TH antibody, and DAB was used as substrate for peroxidase reaction, resulting in brown reaction product. After the cells were immunolabeled, they were washed three times with distilled water and dehydrated and the chamber was detached from the slide. Images of the immunolabeled cells were photographed with a Nikon Eclipse E600 microscope equipped with a computer online data-acquisition system and SPOT software (Diagnostic Instrument). For negative controls, primary antibody was omitted during immunolabeling of cells. For positive controls, we have immunolabeled PC-12 cells that are known to express both ACh and TH.

Measurement of ACh Release From Ex Vivo Carotid Body

Freshly isolated carotid bodies from two rabbits were incubated at 37°C in 100 µl of release medium containing KRB solution (in mM: 135 NaCl, 5 KCl, 1.8 CaCl2, 1 MgCl2, 11 glucose, and 22 NaHCO3, pH 7.4) and 15 µM neostigmine. Before release studies, carotid bodies were preincubated in oxygenated (95% O2-5% CO2) KRB medium containing nominal calcium (1.8 mM) and 15 µM neostigmine for up to 60 min to stabilize the basal release of ACh. Thereafter, the tissues were incubated for 15 min successively with either KRB medium alone or medium containing appropriate chemical stimulants and preequilibrated with various gas mixtures as described below. During incubation, the medium was constantly mixed by using a gentle stream of corresponding gas mixture. The PO2 of the medium was measured with a blood-gas analyzer (Radiometer ABL 5, Copenhagen, The Netherlands). For the release studies, the following experimental protocols were used.

Series 1. To determine the effect of hypoxia on ACh release, carotid bodies (n = 4 per experiment) were incubated sequentially in medium equilibrated with the following gas mixtures balanced with 5% CO2 for the duration as indicated: 1) normoxia (PO2 = 150 Torr) for 15 min (control), 2) hypoxia (PO2 of either 90 or 20 Torr) for 15 min (stimulus), and 3) normoxia for 15 min (recovery to control level) with constant mixing. After each gas challenge, suitable aliquots of the medium were analyzed simultaneously for ACh and DA by using two independent and dedicated HPLC-ECD systems as described above. DA release was monitored as a positive control for hypoxia-evoked neurotransmitter release and served to assess the viability of the carotid body preparation.

Series 2. To assess the influence of hypercapnia on ACh release, the above protocol was repeated except that the hypoxic incubation medium was substituted with KRB medium preequilibrated with a gas mixture containing 10% CO2-21% O2-balance N2. In one set of experiments, the extracellular pH was maintained at 7.4 by increasing the concentration of bicarbonate ion, , to 44 mM. In another series of experiments, the extracellular pH (pH 6.5) was not adjusted by the addition of .

Series 3. In another series of experiments, the effect of depolarizing stimulus (KCl) on ACh release from the carotid body was determined. In these experiments, the incubation medium was first preequilibrated with normoxic gas mixture balanced with 5% CO2. Carotid bodies were then incubated for 15 min successively in medium alone and then in medium containing KCl (30, 60, or 100 mM).

Series 4. In this series of experiments, the contribution of the cholinergic and dopaminergic autoreceptors in the modulation of ACh release in the carotid body was examined. The incubation medium was first preequilibrated with normoxic gas mixture balanced with 5% CO2. Carotid bodies were then incubated for 15 min successively in medium alone and then in medium containing either atropine (1 and 10 µM) or domperidone (1 and 10 µM) for 15 min. To assess the effect of receptor antagonists on ACh release during hypoxia, the above protocol was repeated except that the medium was preequilibrated with hypoxic gas mixture (PO2 = 90 Torr). The medium was collected at the end of each gas challenge, and aliquots (20 µl) of the medium were analyzed for ACh via the HPLC-ECD method as described above. ACh release was expressed in femtomoles per minute per carotid body.

Assay of AChE Activity

After the gas challenges, the incubation medium was separated from the carotid body by centrifugation at 500 g for 10 min. The supernatant containing the secreted form of AChE was removed and stored at -80°C until further analysis of AChE activity. The carotid body was subsequently washed twice with PBS and sonicated three times for 30 s in 0.01 M phosphate buffer, pH 7.0, containing 0.5% (vol/vol) Triton X-100, 1 M NaCl, and 0.25 mM EDTA. The homogenate was centrifuged at 10,000 g for 15 min, and the clear supernatant was used for the analysis of enzyme activity. The activity of AChE in the medium as well as in the carotid body extracts was assayed by using the procedures described below.

AChE activity was assayed by adopting the previously described procedure (13) to a 96-well plate format. The assay is based on the enzymatic conversion of acetylthiocholine by AChE to thiocholine that reacts with DTNB to produce 5-thio-2-nitrobenzoic acid. This product has an intense yellow color with a characteristic absorbance at 405 nm. Typically, to each well, 40 µl of either medium or carotid body extract, 300 µl of 0.01 M phosphate buffer, pH 8.0, 2 µl of 0.075 M acetylthiocholine, and 10 µl of 0.01 M DTNB were added. The 96-well plate was incubated at 37°C for 30 min, and the absorbance of the reaction medium was measured at 405 nm by using a Microplate reader (Molecular Devices). AChE activity was expressed in milliunits; one milliunit is defined as the amount of substrate hydrolyzed per minute at pH 8.0. Samples containing 300 µM neostigmine, a broad-spectrum inhibitor of AChE, served as controls. Under our experimental condition, the absorbance at 405 nm was linear with the amount of enzyme present in the sample and the incubation time. A minimum of ~2 mU of AChE per sample could be detected.

Data Analysis

All values are presented as means ± SE. Statistical significance was evaluated by a paired t-test or one-way ANOVA for repeated measures. P values of <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
ACh Content and Localization in the Rabbit Carotid Body

On average, ACh content of the rabbit carotid body was 5.6 ± 1.3 pmol/carotid body (Table 1), which is similar to the value reported in the cat carotid body (17). The content of choline, the precursor and degradation product of ACh, however, was ~30-fold higher than that of ACh. To demonstrate whether ACh is expressed in glomus cells, the putative oxygen-sensing elements of the carotid body, immunocytochemical analysis was performed by using anti-ACh antibody and primary cultures of the rabbit carotid body. Many glomus cells expressed ACh-like immunoreactivity (Fig. 1a). To further confirm that ACh-expressing cells were indeed glomus cells, cells were double stained with monoclonal antibody for TH, an established marker of glomus cells (32, 38) (Fig. 1b). Many glomus cells expressed immunoreactive products for both ACh and TH (Fig. 1c). As a positive control, parallel experiments were performed on PC-12 cells, a cell line that is known to express ACh and also TH (23, 48). As expected, PC-12 cells showed ACh as well as TH-like immunoreactivities (Fig. 1, g–l). No positive staining was observed in the absence of primary antibodies for either ACh or TH in glomus as well as in PC-12 cells (negative controls, Fig. 1, d–f, and j–l). Together, these results demonstrate that ACh is expressed in the glomus cells of the rabbit carotid body.


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  		Table 1. Content of ACh and its metabolite in the rabbit carotid body

 


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  		Fig. 1. Immunocytochemical analysis of acetylcholine (ACh) expression in the rabbit glomus (A) and rat PC-12 (B) cells. Single and double staining of glomus (a–f) and PC-12 (g–l) cells for ACh and tyrosine hydroxylase (TH) are shown. Controls with the omission of primary antibody (Ab) labeling for the glomus and PC-12 cells are shown in d–f and j–l, respectively. Bars: 10 µm.

 

Effect of Hypoxia on ACh Release

The effect of hypoxia on ACh release was determined in ex vivo carotid bodies harvested from anesthetized rabbits. Basal release of ACh during normoxia (PO2 = 150 Torr) averaged 5.9 ± 0.5 fmol·min-1·carotid body-1 (Fig. 2). Decreasing the PO2 of the medium to 90 and 20 Torr resulted in a progressive decrease in ACh release (Fig. 2). On average, ACh release decreased by 15% (P < 0.05, n = 4) and 68% (P < 0.01, n = 4) in response to PO2 of 90 and 20 Torr, respectively. ACh levels returned to basal values on return to normoxic medium (Fig. 3).



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  		Fig. 2. Effects of PO2 in the incubation medium on ACh release from the carotid body (CB) as measured by HPLC-electrochemical detection (ECD). Data are means ± SE from 4 independent experiments along with triplicate measurements. The level of oxygen in the medium is achieved by balancing it with nitrogen. *P < 0.05 and **P < 0.01, significant effects of lowering PO2 on ACh release.

 


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  		Fig. 3. Normoxic recovery of ACh release after exposure of the carotid body to hypoxia. A: mild hypoxia (PO2 = 90 Torr). B: severe hypoxia (PO2 = 20 Torr). Data are means ± SE from 4 independent experiments. *P < 0.05 and **P < 0.01, significant effects of lowering PO2 on ACh release.

 

To determine whether the hypoxia-induced decrease in ACh release was not due to impaired carotid body sensitivity to hypoxia, we simultaneously monitored DA release in the same samples. In sharp contrast to ACh, there was an approximately sixfold increase in DA release during hypoxia (PO2 = 20 Torr).

Effects of Hypercapnia and Depolarizing Stimulus on ACh Release

The effect of hypercapnia and depolarizing stimulus (100 mM K+) on ACh release was assessed to determine whether inhibition of ACh release is confined only to hypoxia or extends to other stimuli as well. The results are summarized in Table 2. Hypercapnia (10% CO2 + 21% O2, pH 7.4) increased ACh release by ~44% (P < 0.01, n = 4) compared with the normoxic control value. Acidic stimulus (extracellular pH 6.5) enhanced ACh release by 29% (P < 0.05, n = 3). Challenging the carotid body with 30 or 60 mM KCl had no effect on ACh release. However, increasing KCl concentration to 100 mM while maintaining the osmolarity resulted in a marked augmentation of ACh release (~223%; P < 0.01, n = 3).


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  		Table 2. Stimulus-evoked ACh release in the rabbit carotid body

 

Effect of Hypoxia on AChE Activity

The extracellular concentration of ACh critically depends on the activity of AChE, the enzyme that hydrolyzes ACh. AChE activity was determined in the medium as well as in cell extracts of the carotid body exposed to hypoxia to assess whether increased AChE contributes to decreases in ACh release. AChE activity in the medium, during normoxia, was 2.6 ± 0.2 mU/carotid body and was unaffected by hypoxia (Table 3). Likewise, hypoxia had no discernible effect on AChE activity in the carotid body extracts.


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  		Table 3. Effect of hypoxia on AChE activity in the carotid body extracts and in the release medium

 

Effect of Muscarinic and Dopaminergic Receptor Antagonists on ACh Release During Hypoxia

Muscarinic receptor agonists inhibit, whereas antagonists facilitate, ACh release in the central nervous system (9, 11, 37, 44, 49, 54). Rabbit carotid bodies express muscarinic receptors on type I cells (10, 24). To test whether hypoxia-induced inhibition of ACh release is coupled to activation of autoinhibitory muscarinic receptors, ACh release during hypoxia was monitored in the presence of atropine, a well-established blocker of muscarinic receptors. The results are summarized in Fig. 4. Atropine, at 1 µM, had no significant effect on ACh release during normoxia. However, there was a modest but significant increase in the basal ACh release with 10 µM of atropine (normoxia = 4.8 ± 0.1 pmol·min-1·carotid body-1 vs. normoxia + 10 µM atropine = 6.2 ± 0.3 pmol·min-1·carotid body-1; P < 0.05, n = 4; Fig. 4A). More importantly, atropine augmented ACh release during hypoxia in a dose-dependent manner, and at 10 µM concentration it caused a marked facilitation of ACh release (~200%; P < 0.01, n = 4; Fig. 4B).



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  		Fig. 4. Atropine augments ACh release from the carotid body. A: ACh release during normoxia (PO2 = 150 Torr). B: ACh release during hypoxia (PO2 = 90 Torr). Data are means ± SE from 4 independent experiments. *P < 0.05 and **P < 0.01, significant effects of lowering PO2 on ACh release. ns, Not significant.

 

Glomus cells of the carotid body express dopaminergic D2 receptors that are involved in the feedback regulation of DA release in the carotid body (4, 5). The finding that hypoxia increased DA release by sixfold prompted us to test whether D2 receptors also contribute to hypoxia-induced inhibition of ACh release. Domperidone (10 µM), a D2 receptor antagonist, had no significant effect on the basal ACh release (data not shown) but significantly facilitated ACh release during hypoxia (~55%; P < 0.05, n = 4; Fig. 5).



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  		Fig. 5. Effect of domperidone on ACh release from the carotid body during normoxia (PO2 = 150 Torr) and hypoxia (PO2 = 90 Torr). Data are means ± SE from 4 independent experiments. **P < 0.01, significant effects of lowering PO2 on ACh release.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGMENTS
 REFERENCES
 
Our biochemical analysis of the whole carotid bodies showed that ACh is expressed in low picomole levels in the rabbit carotid body. ACh may be localized in the nerve fibers and/or glomus cells. Glomus cells are presently believed to be the primary sites of sensory transduction for the hypoxic stimulus. Therefore, it seems necessary to determine whether ACh is expressed in glomus cells. This was accomplished by immunocytochemical analysis of ACh in primary cultures of the rabbit carotid body. We have used TH immunoreactivity as a marker for glomus cells. In the present study, for the localization of ACh, we used a monoclonal antibody specific to ACh that allowed direct visualization of ACh rather than the antibody for ChAT, the rate-limiting enzyme for ACh synthesis as used in previous studies as a marker for ACh (50, 51). The ACh antibody has been successfully used previously to demonstrate ACh localization in the rat central nervous system (7). The validity of our method for the localization of ACh in the glomus cell was further confirmed with PC-12 cells, which are known to express both TH and ACh (23) as positive controls (Fig. 1, g–i). Our data showed that many TH-containing carotid body cells also express ACh-like immunoreactivity. These findings are consistent with ChAT expression previously demonstrated in rabbit glomus cells (50). Thus our results establish that ACh is not only present in the chemoreceptor tissue but more importantly also expressed in many glomus cells of the rabbit carotid body.

A major and an intriguing finding of the present study is that hypoxia inhibits ACh release from the rabbit carotid body and that the magnitude of the inhibition depends on the severity of hypoxia. These observations are in sharp contrast to those reported in the cat carotid body (19). Methodological differences per se cannot account for the observed differences because the protocols used for release studies as well as the method for ACh measurement employed in the present study were essentially the same as those reported in the study on the cat carotid body (19). The following observations suggest that the inhibition of ACh release during hypoxia is not due to deterioration of the ex vivo carotid body preparation. First, inhibition of ACh release was reversed when the hypoxic stimulus was terminated. Second, DA release was markedly facilitated during hypoxia within the same samples wherein ACh release was measured, a finding that is consistent with previous reports in the rabbit carotid body (3, 16, 21). Third, unlike hypoxia, hypercapnia and K+ (depolarizing stimulus) stimulated ACh release. A similar hypoxia-evoked inhibition of ACh release was also reported in the rat striatum (8).

It is likely that hypoxia increases the release of AChE, the enzyme responsible for the hydrolysis of ACh in the medium. Enhanced degradation of ACh in the extracellular medium by AChE would decrease ACh content in the medium, thus giving rise to a decrease in ACh release during hypoxia. However, the finding that the AChE level in the extracellular medium is unaffected during hypoxia precludes such a possibility. It has been previously shown that muscarinic receptors (9, 49), especially the M2 subtype (2, 6, 12, 47), exert inhibitory effects, whereas nicotinic receptors facilitate ACh release (33). Notably, the ratio of nicotinic and muscarinic receptors was ~1:12 in the rabbit carotid body. Therefore, it is conceivable that hypoxia activates the autoinhibitory muscarinic receptors that are localized on the glomus cells (10, 24), resulting in decreased ACh release during hypoxia. Consistent with this notion is the finding that atropine, an antagonist of muscarinic receptors, markedly facilitated ACh release during hypoxia. How are the muscarinic receptors stimulated in the carotid body during hypoxia? Although HPLC-ECD combined with microbore column chromatography is sensitive to detect low femtomole levels of ACh, it was inadequate to monitor ACh release within 1–2 min of hypoxic exposure. A minimum 15-min incubation period is required to reliably monitor ACh release from the pooled carotid bodies. It is likely that hypoxia indeed facilitates ACh release in the initial period of hypoxia, which in turn, by activating the muscarinic receptors, further inhibits ACh release. At the end of 15 min of incubation, the net effect of hypoxia was, therefore, inhibition of ACh release from the carotid body. On the other hand, blockade of muscarinic receptors by atropine during hypoxia relieved this autoinhibition, resulting in the sustained facilitation of ACh release. Not only atropine, but domperidone, a dopaminergic D2 receptor antagonist, also facilitated ACh release during hypoxia, albeit of lower magnitude than that found with atropine. This finding suggests that dopaminergic D2 receptors, in addition to muscarinic receptors, also contribute to the modulation of ACh release during hypoxia. Simultaneous monitoring of DA showed a sixfold increase in DA release during hypoxia. It is, therefore, conceivable that DA released during hypoxia could activate D2 receptors located on the glomus cells of the carotid body and thus influence ACh release. In this scenario, it appears that, in the rabbit carotid body, hypoxia has a biphasic effect on ACh release, i.e., an initial facilitation followed by a sustained inhibition mediated by the action of muscarinic as well as dopaminergic receptors.

Unlike hypoxia, hypercapnia, acidosis, and depolarizing stimulus (K+) stimulated ACh release from the rabbit carotid body. Several reports suggest that the intracellular acidification associated with hypercapnia could affect the agonist affinity of the muscarinic receptors (1, 26, 30). In addition, depolarization has been shown to reduce the agonist affinity of the presynaptic autoinhibitory M2 receptor in frog neuromuscular junctions (46) and in rat cortical synaptosomes (25, 35). Therefore, it is likely that hypercapnia and depolarizing stimulus cause a sustained ACh release by preventing the action of muscarinic receptors. It is interesting to note that the magnitude of ACh release by acidosis is lower than the release observed during isoprotonic hypercapnia (Table 2). This difference in ACh release could be due to low pH-induced modification of nicotinic receptors (41). Although hypoxia is the major stimulus to the carotid body, increases in the arterial PCO2 also augment the sensory activity. Our finding that hypercapnia facilitates ACh release suggests a transmitter role for ACh in mediating the sensory excitation of the carotid body during high CO2.

In conclusion, our results indicate that hypoxia has a biphasic effect on ACh release from the carotid body, i.e., an initial facilitation followed by sustained inhibition. Furthermore, our data show that muscarinic and dopaminergic receptors contribute to the inhibitory effect of hypoxia on ACh release. These observations suggest that complex interactions among transmitters markedly influence hypoxia-induced transmitter release from the carotid body.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Niranjana Natarajan for technical help in the analysis of AChE activity.

GRANTS

This study was supported by National Heart, Lung, and Blood Institute Grant HL-25830.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. K. Kumar, Dept. of Biochemistry, School of Medicine, Case Western Reserve Univ., 10900 Euclid Ave., Cleveland, OH 44106-4935 (E-mail: kgk{at}po.cwru.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 

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