Gene expression and function of adenosine A2A receptor in the rat carotid body

Shuichi Kobayashi1, Laura Conforti2, and David E. Millhorn1

1 Department of Molecular and Cellular Physiology, and 2 Division of Nephrology and Hypertension, Department of Internal Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0576


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study was undertaken to determine whether rat carotid bodies express adenosine (Ado) A2A receptors and whether this receptor is involved in the cellular response to hypoxia. Our results demonstrate that rat carotid bodies express the A2A and A2B Ado receptor mRNAs but not the A1 or A3 receptor mRNAs as determined by reverse transcriptase-polymerase chain reaction. In situ hybridization confirmed the expression of the A2A receptor mRNA. Immunohistochemical studies further showed that the A2A receptor is expressed in the carotid body and that it is colocalized with tyrosine hydroxylase in type I cells. Whole cell voltage-clamp studies using isolated type I cells showed that Ado inhibited the voltage-dependent Ca2+ currents and that this inhibition was abolished by the selective A2A receptor antagonist ZM-241385. Ca2+ imaging studies using fura 2 revealed that exposure to severe hypoxia induced elevation of intracellular Ca2+ concentration ([Ca2+]i) in type I cells and that extracellularly applied Ado significantly attenuated the hypoxia-induced elevation of [Ca2+]i. Taken together, our findings indicate that A2A receptors are present in type I cells and that activation of A2A receptors modulates Ca2+ accumulation during hypoxia. This mechanism may play a role in regulating intracellular Ca2+ homeostasis and cellular excitability during hypoxia.

polymerase chain reaction; in situ hybridization; immunohistochemistry; patch clamp; calcium current; fura 2-calcium imaging


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CAROTID BODY, WHICH IS located bilaterally at the bifurcation of the common carotid artery, is the major O2-sensing organ that regulates the cardiopulmonary response to hypoxia (2, 18). Hypoxia causes carotid body O2-sensitive type I cells to depolarize, increase intracellular Ca2+ concentration ([Ca2+]i), and release the transmitter dopamine (11, 18). There is growing evidence that adenosine (Ado) stimulates carotid body activity in animals (21, 23, 32) and humans (20, 37). There is also evidence that Ado is produced during hypoxia in carotid bodies (4). However, the role of Ado in regulating the cellular response to hypoxia in type I cells has not been established.

Ado, a metabolite of cellular ATP, is released from cells in response to inadequate tissue oxygenation (27, 39). Ado mediates its cellular effects via specific membrane receptors (10, 25). To date, four Ado receptor subtypes have been identified (A1, A2A, A2B, and A3). The A1 and A3 receptors are coupled to the inhibitory G protein (Gi) and mediate inhibition of adenylate cyclase (10, 25). The A2 receptor family, on the other hand, is coupled to the stimulatory G protein (Gs) and activates adenylate cyclase (10, 25). In a previous study, we found that Ado attenuates membrane depolarization and the increase in [Ca2+]i induced by hypoxia via activation of A2A receptors and inhibition of Ca2+ current (ICa) in rat pheochromocytoma (PC-12) cells, an O2-sensitive cell line (15).

A recent study has shown that Ado inhibits 4-aminopyridine-sensitive K+ currents in rat carotid body type I cells (36). However, the action of Ado on ICa and intracellular free Ca2+ has not been established in type I cells. The present study was undertaken to establish the expression of Ado receptors in the rat carotid body and to determine whether stimulation of these receptors has a role in mediating Ca2+ homeostasis during hypoxia in type I cells. Briefly, we found that rat carotid bodies express both A2A and A2B receptor subtype mRNAs but not mRNA for either the A1 or A3 receptor subtypes. We also found that the A2A receptor proteins are present in type I cells. We further found that stimulation of the A2A receptor inhibits a voltage-dependent ICa and hypoxia-induced accumulation of [Ca2+]i in type I cells. To our knowledge, this is the first morphological evidence that rat carotid body type I cells express the Ado A2A receptors and that these receptors have a functional role in mediating intracellular Ca2+ homeostasis.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reverse transcriptase-polymerase chain reaction. Carotid bodies were removed from adult rats of either sex (Sprague-Dawley, 150-200 g) that had been killed by decapitation following carbon dioxide anesthesia. Total RNA was isolated from the carotid bodies using TRI-REAGENT (Molecular Research Center, Cincinnati, OH), and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using the GeneAmpli kit (Perkin-Elmer, Norwalk, CT) as previously described (15). Briefly, for the RT reaction, 1 µg of purified total RNA was incubated in the presence of 2.5 µM oligo(dT) (16-mer), 1 mM deoxynucleotide triphosphates, 1 unit RNase inhibitor, and 2.5 units of murine leukemia virus RT. The RNA was denatured by increasing the temperature to 85°C for 5 min, and reverse transcription was allowed to proceed for 15 min at 42°C. The reaction was terminated by heating to 95°C for 5 min and then cooling to 5°C for 5 min. The PCR primers for rat A1, A2A, A2B, and A3 Ado receptors were as follows: A1 receptor: 5'-CGG CAG CAC CCA GAC GAA GA-3' and 5'-CCC ACC ATG CCG CCC TAC AT-3' (the predicted length of the amplified DNA fragment is 579 bp); A2A receptor: 5'-TTC AAA GTG GGA GCC ACG CA-3' and 5'-ATG GGC TCC TCG GTG TAC ATC-3' (1,320 bp); A2B receptor: 5'-GCC TCG AGT GCT TTA CAG ACC CCC-3' and 5'-GAA AGT TGA CTG TCC CCC GGC CTG-3' (514 bp); and A3 receptor: 5'-CAC ATC CTG CTG AAG AAG CAA CAG-3' and 5'-GAG CTG GCT CTT TAT CTG TCA TGG-3' (1,045 bp).

PCR amplification of the transcribed DNA was accomplished in the presence of 1.5 mM MgCl2, 1× reaction buffer, and 2.5 U AmpliTaq DNA polymerase. The PCR conditions were as follows: 2-min denaturation at 94°C, followed by 35 cycles consisting of 90 s at 94°C, 1 min at 50°C (for the A1 and A2A receptors) or at 60°C (for the A2B and A3 receptors), and then 2 min (for the A2A receptor) at 72°C or 90 s at 72°C (for A1, A2B, and A3 receptors). The samples were then kept at 72°C for 7 min. The products of RT-PCR were analyzed by electrophoresis on 1% agarose gels stained with ethidium bromide. The sequences of each PCR product were verified by sequence analysis and shown to be 100% homologous with the reported cDNA sequences of each Ado receptor mRNA.

In situ hybridization. Rats (Sprague-Dawley, 150-200 g) were decapitated under carbon dioxide anesthesia, and the carotid bodies were removed, fixed in 4% paraformaldehyde-PBS solution for 2 h, immersed in 30% sucrose-PBS for 2 h, and then embedded in optimal cutting temperature compound (Miles, Elkhart, IN). The tissues were cut (10- to 12-µm sections) on a microtome in a cryostat, thaw mounted onto twice gelatin-coated slides, and then stored at -70°C.

In situ hybridization was performed as described previously (6). Briefly, frozen slide-mounted sections were warmed to room temperature and rinsed three times (5 min each) in 0.1 M PBS (1× PBS; pH 7.4). The sections were immersed in 0.2 N HCl for 2 min and then treated with proteinase K (1 µg/ml in 0.1 M PBS) at 37°C for 15 min. The sections were then rinsed with 1× PBS and lightly fixed by immersion in 4% paraformaldehyde-1× PBS for 5 min. The carotid body sections were rinsed in 1× PBS twice for 5 min each, placed in PBS-glycine (2 mg/ml) for 5 min, and then washed again through PBS twice for 5 min each. The tissues were placed in 0.25% acetic anhydrate in 0.1 M triethanolamine-0.9% NaCl (pH 8.0) for 10 min at room temperature. The sections were again washed twice in PBS, dehydrated, and delipidated by transferring them through 50% ethanol (EtOH; 1 min), 70% EtOH (2 min), 95% EtOH (2 min), 100% EtOH (2 min), chloroform (5 min), 100% EtOH (1 min), and 95% EtOH (1 min), and air-dried.

The synthetic oligodeoxyribonucleotides probes were labeled at the 3'-end with alpha -[35S]thio-dATP (Dupont NEN, Boston, MA), and terminal deoxynucleotidyltransferase (GIBCO BRL, Gaithersburg, MD) to specific activities of 8-12 × 106 counts · min-1 · pmol-1. Labeled probes (1 × 107 counts · min-1 · ml-1) were suspended in hybridization buffer containing 50% formamide, 4× saline-sodium citrate (SSC), 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 10% dextran sulfate, 250 µg/ml of yeast tRNA, 500 µg/ml of salmon sperm DNA, and 100 mM dithiothreitol. After overnight hybridization (37°C) with the labeled probe, the sections were washed in 1× SSC containing 10 mM sodium thiosulfate at 55°C (4 × 15 min) and at room temperature (1 × 60 min). Each slide was air-dried and dipped in autographic emulsion (Kodak NTB-2, 2:1 with 0.6 M ammonium acetate). The slides were developed and fixed after 1 wk and counterstained with toluidine blue (0.25%). The carotid body sections were analyzed using a microscope equipped with light-field and dark-field condensors.

Probe sequences were taken from published rat A2A receptor cDNA sequences (9). The oligo probe (CGC CGC AGG TCT TCG TGG AGT TCC CGT CTT TCT) does not recognize other members of the Ado receptor families (GenBank Blast search). A sense probe was constructed and used as a negative control. The ability of the antisense probe to recognize a mRNA for the Ado A2A receptor was verified by Northern blot analysis (16). Hybridization was also performed with a fragment of the full-length cDNA probe encoding the A2A receptor (9). The oligonucleotide probe was labeled at the 5'-end with [gamma -32P]ATP using T4 polynucleotide kinase (Promega, Madison, WI). The A2A cDNA was excised with Xho I and Xba I (1,577 kb) and labeled using a random-primed DNA labeling kit (Prime-A-Gene; Promega) and 2-[alpha -32P]dCTP (Dupont NEN). No difference in hybridization was observed with the oligo and cDNA probes.

Immunohistochemistry for Ado A2A receptor and tyrosine hydroxylase in rat carotid body. Immunohistochemistry was performed as previously reported (22). Carotid body sections were prepared as described for the in situ hybridization study. The slides were washed twice for 5 min each in 1× PBS and incubated for 20 min in 1× PBS containing 0.2% Triton X and 10% goat serum to permeabilize and block nonspecific binding.

For simultaneous detection of both Ado A2A receptor and tyrosine hydroxylase (TH), a double-labeling technique was used in which sections were incubated in a mixture of the rabbit polyclonal anti-Ado A2A receptor antiserum (10 µg/ml; Chemicon, Temecula, CA) and the mouse monoclonal anti-TH antibody (1:200; Chemicon). The antibody was diluted in 1× PBS containing 1% goat serum and incubated overnight at room temperature. The sections were washed three times for 5 min each with 1× PBS and then incubated with the secondary antibodies for 1 h at room temperature. The secondary antisera consisted of fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin (IgG) (1:800; Chemicon) and tetramethylrhodamine isothiocyanate-labeled goat anti-mouse IgG (1:200; Chemicon). The sections were then rinsed three times for 5 min each in 1× PBS, covered with a coverslip in glycerol-PBS (3:1), and examined with a standard fluorescence microscope equipped with proper filter combinations. Nonspecific staining was assessed by following the same protocol but without the primary antibody.

Carotid body primary cultures and immunocytochemistry for TH. Carotid bodies were removed from adult rats decapitated under carbon dioxide anesthesia and were incubated for 40 min at 37°C in 5 ml of Ca2+/Mg2+-free medium (in mM: 140 NaCl, 0.5 KCl, 2 Na2HPO4, 0.5 NaH2PO4, and 1 glucose) containing 0.15% trypsin and 0.2% collagenase. During the incubation period, the isolated carotid bodies were triturated every 10 min with a fire-polished glass pipette. The isolated cells were then centrifuged at 500 g for 5 min; the pellet was resuspended in DMEM-F-12 medium supplemented with 10% fetal bovine serum, 100 U/ml of sodium penicillin G, and 100 µg/ml of streptomycin sulfate and plated on coverslips. Cells were left undisturbed overnight in a humidified incubator with an environment of 21% O2-5% CO2 balanced with N2. Selected cells were used for patch-clamp and Ca2+-imaging studies the next day.

Type I cells were identified by the presence of TH immunoreactivity. The type I cells were plated on coverslips coated with poly-L-lysine and placed on 35-mm culture dishes covered with culture medium. The cells were incubated overnight at 37°C in 21% O2 and then rinsed in 1× PBS and fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. Next, the cells were washed three times (5 min each) in PBS and incubated for 15 min in 0.2% Triton X-100 and 1% bovine serum albumin for the purpose of cell permeabilization and for blocking of nonspecific binding. The blocking serum was replaced with 2 ml/dish of an anti-TH antibody (Protos Biotech, New York, NY) diluted 1:1,000 in 1× PBS and incubated overnight at room temperature. The cells were washed three times (5 min each) with PBS and then incubated with biotinylated anti-rabbit secondary antibody for 15 min. Cells were then washed three times and incubated in an avidin and biotinylated horseradish peroxidase macromolecular complex for 30 min. The cells were washed three additional times with PBS and then incubated with 3,3'-diaminobenzidine (2 ml/dish). The development of chromagen staining was observed with a microscope for 2 min. Cells were then washed three times with PBS, covered with a coverslip in glycerol-PBS (3:1), and analyzed with a light microscope.

Patch-clamp recordings. The methods for patch-clamp recordings were described previously by Kobayashi et al. (15). Briefly, the cells were plated on coverslips and placed in a perfusion chamber (volume 200-400 µl) that was mounted on the stage of an inverted interference microscope (ITM-2, Olympus, Japan). The cells were constantly perfused with the recording solution at a flow rate of 2-3 ml/min. ICa was recorded in the conventional whole cell voltage-clamp mode by using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) (12). The patch pipettes had a resistance of 4-5 MOmega when filled with an internal solution. All experiments were performed at room temperature (25°C).

ICa was recorded using Ba2+ as charge carrier. The pipette solution for ICa included (in mM) 140 cesium gluconate, 1 CaCl2, 2 MgCl2, 10 EGTA, 10 HEPES, 3 ATP sodium, and 0.2 GTP (pH adjusted to 7.2 with Tris base), whereas the external solution included (in mM) 122 N-methylglucamine glutamate, 20 BaCl2, 2 MgCl2, 2.8 CsCl, 10 HEPES, and 10 glucose (pH adjusted to 7.4 with Tris base). Ba2+ was used for several reasons. First, Ba2+ blocks K+ channels that could potentially mask ICa. Ba2+ also enhances the resolution of Ca2+ channels because the magnitude of currents is larger with Ba2+ as a charge carrier. In addition, the use of Ba2+ avoids Ca2+-induced inactivation of Ca2+ channels.

Spherical cells 10-15 µm in diameter were voltage clamped using the tight seal whole cell recording method. Type I cells were selected by morphological criteria based on the finding of the immunocytochemical study. Seventy to seventy-five percent of the series resistance (<9 MOmega ) was compensated for electronically. The ICa was measured from a holding potential (HP) of -80 mV with depolarizing steps to +20 mV (160 ms in duration). Leak and capacitance currents were subtracted using small hyperpolarizing pulses (P/4 protocol). The current-voltage relationship of ICa was measured with 100-ms-long test pulses from a HP of -80 mV to test potentials ranging from -50 to +60 mV (10-mV increments). All solutions containing drugs were applied extracellularly after stable recordings of ICa had been obtained (usually 3 min after establishing the whole cell configuration). Signals were electronically filtered at 1 kHz and digitally sampled. The digitized signals were analyzed on a computer using pCLAMP 5.5 analysis programs (Axon Instruments).

Ca2+ imaging. [Ca2+]i was evaluated using the fluorescent indicator fura 2 (Molecular Probes, Eugene, OR) and a Ca2+-imaging system (Intracellular Imaging, Cincinnati, OH). The rat carotid body cells were isolated as described above and incubated under a normoxic condition overnight. The cells were then incubated with serum-free DMEM-F-12 medium containing fura 2-acetoxymethyl ester (5 µM) and pluronic F-127 (0.01%) for 40 min at 37°C. Next, the cells were rinsed twice with the same medium without fura 2 and left at 37°C for 15 min to allow for hydrolysis of the ester. Coverslips were placed in a chamber that was mounted on the stage of a Nikon TMS microscope. Light from a 300-W xenon arc illuminator passed through a computer-controlled filter changer and shutter unit that contained 340- and 380-nm filters. Light from selected cells was collected with an integrating charge-coupled device video camera. The ratio of light intensity at the two wavelengths was calculated on-line and stored on the computer. The 340- to 380-nm fluorescence ratio (F340/F380) was used as an indicator of [Ca2+]i. Experimental solutions were perfused by a peristaltic pump at a flow rate of 5 ml/min. Type I cells were identified by morphological criteria based on the findings of the immunocytochemical study. The response to acute severe hypoxia was evaluated by perfusing cells with the hypoxic medium that had been saturated with 100% N2 gas and then added along with 1 mM sodium dithionate (Na2S2O4), an O2 chelator. The PO2 of the perfusate in the chamber on the stage of the microscope was 10.2 ± 0.1 mmHg (n = 3) in our experimental settings.

Data analysis. The results are expressed as means ± SE; n is the number of observations. Analysis of variance followed by Student's t-test was used to evaluate the significance of the obtained data. Statistical significance was accepted at the conventional P < 0.05 level by two-tailed evaluation.

Materials. Ado and sodium dithionate were obtained from Sigma (St. Louis, MO). ZM-241385 was purchased from Tocris Cookson (Ballwin, MO). 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) was purchased from RBI (Natick, MA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Rat carotid bodies express Ado A2A and A2B receptor mRNAs. RT-PCR studies were performed to identify the expression of Ado receptors in rat carotid bodies. Results from these experiments revealed that both the A2A and A2B receptors are expressed in the rat carotid body (Fig. 1). We failed to detect expression of either the A1 or A3 receptors in the carotid body, but we did detect mRNAs for these receptors in rat brain (A1) and lung (A3). Sequence analysis of the PCR products revealed 100% homology with the cDNA sequences for both the A2A and A2B receptors (GenBank). These findings indicate that rat carotid bodies express the A2A and A2B receptor mRNAs but not the A1 or A3 receptors.


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Fig. 1.   Expression of adenosine (Ado) receptor mRNA in carotid bodies as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Ethidium bromide visualization of products obtained by RT-PCR of total RNA from carotid bodies for Ado A1 (A), A2A (B), A2B (C), and A3 (D) receptors is shown. A pair of oligonucleotides were used as primers to specifically amplify the cDNA fragments of these receptors (see MATERIALS AND METHODS). The PCR products were analyzed by ethidium bromide-1% agarose gel electrophoresis. The left lanes correspond to PCR or pGEM marker; size of the DNA fragments for each marker is indicated on the left. RT-PCR studies revealed that carotid body cells express both A2A and A2B Ado receptors (the product predicted size 1,320 and 544 bp, respectively). On the other hand, neither the A1 nor the A3 Ado receptor was detected (579 and 1,045 bp, respectively). Total RNAs from rat whole brain and rat lung were used as positive controls for A1 and A3 receptors. False amplification of the genomic DNA was ruled out by performing RT-PCR without RT in reaction mixtures as negative controls (shown as RT-)

In situ hybridization detection of Ado A2A receptor mRNA in the rat carotid body. We next performed in situ hybridization studies to determine the localization of Ado A2A receptor mRNA in carotid body tissue sections. The synthetic oligodeoxynucleotide antisense probe used in these experiments recognized a single band of the appropriate size for the Ado A2A receptor in PC-12 cells (Fig. 2).


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Fig. 2.   Specificity of antisense Ado A2A receptor oligonucleotide probe used for in situ hybridization. We constructed synthetic oligodeoxyribonucleotide probe for in situ hybridization. We verified the specificity of this probe by Northern blot analysis. Total RNA from PC-12 cells was electrophoresed using RNA denaturating gels and then blotted onto nylon membrane. The membrane was hybridized with either oligonucleotide probes (antisense and sense) or cDNA probe for Ado A2A receptor mRNA. The membrane hybridized with antisense oligo probe (left) shows a single signal, the position of which is the same as that on the membrane hybridized with cDNA probe (middle). No signals were identified on the membrane hybridized with sense oligo probe (right).

The carotid body is an oval-shaped organ that is located at the bifurcation of internal and external carotid arteries (Fig. 3A). Figure 3B shows the higher magnification of the carotid body. It can be seen that clusters of cells (glomi) are separated from each other by walls of connective tissues. The clusters consist of two types of cells: cells that have a clear round nucleus (type I cells) and cells that are located in the periphery of the clusters and have a disk-shaped nucleus with dense-chromatin (type II cells). Figure 3C is a dark-field photomicrograph showing the localization of Ado A2A mRNA. The silver grains indicating the presence of A2A receptor mRNA are arranged as clusters. Figure 3D shows a dark-field photomicrograph of the carotid body hybridized with sense strand oligonucleotide A2A probe. Note the absence of hybridization. These results indicate that rat carotid bodies express Ado A2A receptor mRNA.


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Fig. 3.   In situ hybridization for Ado A2A receptor mRNA in rat carotid body. A: localization of the carotid body. The carotid body is an oval-shaped organ that can be seen at the bifurcation of internal and external carotid arteries (arrow). B: higher magnification of rat carotid body. Clusters of cells (glomi) consist of 2 types of cells: cells in the center that have a clear round nucleus (type I cells) and cells in the periphery that have a disk-shaped nucleus with dense chromatin (type II cells). The glomi are separated from each other by walls of connective tissue. C: dark-field photomicrograph of the same section shown in B. Silver grains reflecting Ado A2A receptor mRNA (white dots) are arranged as clusters. D: dark-field photomicrograph of negative control section hybridized with the sense strand oligo probe for Ado A2A receptors. No significant signals are observed.

Colocalization of Ado A2A receptor and TH proteins in the rat carotid body. We next performed immunohistochemical studies for localization of the Ado A2A receptor and TH in the carotid body. A double-labeling immunofluorescence technique was used to show the colocalization of A2A receptors and TH proteins. Immunoreactivities for Ado A2A receptors and TH were detected in numerous cells that were arranged in clusters (Fig. 4, A and B). Importantly, we found that the immunoreactivity for A2A receptors was colocalized in individual cells with TH immunoreactivity. The TH immunoreactivity is an established marker for type I cells (19). Immunoreactivity was not observed in carotid body sections that had been identically processed except for omission of the primary antibodies against the Ado A2A receptor (Fig. 4C) or TH (Fig. 4D). To our knowledge, this is the first evidence that rat carotid body type I cells, which are TH immunoreactivity positive, express Ado A2A receptor proteins.


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Fig. 4.   Double-labeling immunohistochemistry for Ado A2A receptor and tyrosine hydroxylase (TH) in rat carotid body. Carotid bodies were dissected from adult Sprague-Dawley rats, and a double-labeling technique was performed in which sections were incubated in a mixture of the rabbit polyclonal anti-Ado A2A receptor antiserum and the mouse monoclonal anti-TH antibody (see MATERIALS AND METHODS). A: immunoreactivity for Ado A2A receptor in rat carotid body. The presence of A2A receptor protein was visualized using immunofluorescence with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulins (IgG). FITC-positive cells (green) are arranged in clusters. B: immunoreactivity for TH in rat carotid body. The presence of TH protein was visualized using tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse IgG. TRITC-positive cells (red) are also arranged in clusters. Note that FITC and TRITC immunoreactivities are colocalized in the carotid body. Insets show higher magnification of the same area visualized by either FITC (inset in A) or TRITC (inset in B). C: absence of immunoreactivity for A2A receptor in a negative control section. FITC-fluorescent signals are not seen in the control section, which was processed with the same protocol with the omission of the primary antibody. D: absence of immunofluorescence for TH in a negative control section. TRITC fluorescence was negative in the section that was processed without the primary antibody.

Modulation of voltage-dependent ICa by A2A receptor stimulation in isolated rat carotid body type I cells. We could distinguish type I cells from non-type I cells by both morphological and immunocytochemical criteria. The majority of cells were round and TH positive (Fig. 5). TH positive cells could be easily distinguished from the smaller spindlelike TH-negative cells.


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Fig. 5.   Immunocytochemical identification of type I cells in isolated carotid body cells. Cells were enzymatically isolated from adult rat carotid bodies. Immunocytochemical staining for TH, a marker for adrenergic cells, was performed using enzyme-mediated detection (horseradish peroxidase, HRP). Isolated cells were labeled with rabbit anti-TH antibodies. The presence of TH protein was visualized using enzymatic reaction between HRP and diaminobenzidine with avidin-biotin intensification method. Isolated cells essentially consist of 2 types of cells. The majority of cells (80% or more) were TH positive (solid arrow). These cells were round and some had sizes of ~10 µm in diameter. TH-positive cells could be easily distinguished from the spindlelike TH-negative cells (open arrow).

We performed electrophysiological studies to examine the effect of A2A receptor stimulation on modulating voltage-dependent ICa in the TH-positive type I cells. Whole cell voltage-clamp recordings were performed in which Ba2+ (20 mM) was used as a charge carrier. ICa values were elicited from a HP of -80 mV with depolarizing steps to +20 mV (pulses of 160 ms in duration). The current density for ICa in type I cells was 11.8 ± 1.2 pA/pF (n = 17). We found that the application of Ado induced an inhibition of ICa in a concentration-dependent manner (Fig. 6A). The effect of Ado on ICa was determined as percent change by comparing the peak amplitude of ICa in the presence of Ado to that in the absence of Ado. Maximal inhibition of ICa was observed at the concentration of 50 µM (32.5 ± 4.8%, n = 6). A higher concentration of Ado (100 µM) failed to induce further inhibition (33.3 ± 3.5%, n = 3). Figure 6B shows the peak current-voltage relationship before and after the application of Ado (50 µM). ICa values were measured with test pulses of 100-ms duration from a HP of -80 mV to test potentials from -50 to +60 mV. Ado decreased ICa at the voltage range examined (n = 3). The inhibitory effect of Ado on ICa was blocked in a dose-dependent manner by the A2A receptor antagonist ZM-241385 (28) (Fig. 6C). The action of Ado (50 µM) on ICa was 17.2 ± 3.4% (n = 3) and 3.5 ± 2.9% (n = 5) in the presence of 1 and 10 nM ZM-241385, respectively. On the other hand, the action of Ado on ICa was not affected by the A1 receptor antagonist DPCPX (10 µM, 29.5 ± 5.1%, n = 4). These results indicate that Ado inhibits voltage-dependent ICa in type I cells, and this effect is mediated via activation of A2A receptors.


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Fig. 6.   Effect of Ado on voltage-dependent Ca2+ current (ICa) in carotid body type I cells. A, bottom: concentration-response relationship of the effect of Ado on ICa. ICa was measured at room temperature every 30 s by 160-ms test pulses from a holding potential (HP) of -80 mV to +20 mV. Peak current amplitude was measured for evaluation. The charge carrier was Ba2+ (20 mM). The response was evaluated as percent decrease from baseline inward currents. The numbers in parentheses indicate the number of cells for each data point. Top: superimposed current traces recorded before, during, and after application of Ado (10 µM or 50 µM). Ado (50 µM) elicited a decrease in the amplitude of ICa, which fully returned to baseline level after washing. B: peak current-voltage relationship before and during the application of Ado (50 µM). ICa was measured with 100-ms-long test pulses from a HP of -80 mV to test potentials ranging from -50 to +60 mV (10-mV increment). Ado decreased ICa at the voltage range examined (n = 3). C: effect of Ado A2A receptor antagonist ZM-241385 on the response of ICa to Ado. The inhibitory effect of Ado on ICa was blocked dose dependently by the A2A receptor antagonist ZM-241385. The action of Ado (50 µM) on ICa was abolished in the presence of ZM-241385 (10 nM). On the other hand, the action of Ado was not affected by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 10 µM; n = 4). The numbers in parentheses indicate the number of cells for each group. Values are means ± SE.

Effect of Ado receptor activation on intracellular free Ca2+ during hypoxia in rat carotid body type I cells. We reported previously that Ado attenuates the hypoxia-induced elevation of [Ca2+]i in the O2-sensitive PC-12 cell line (15). In the current study, we performed experiments to determine whether Ado mediates this response in type I cells. We found this to be the case. The type I cells were first exposed to hypoxic perfusate (severe hypoxia: 100% N2 and 1 mM sodium dithionate, PO2 10 mmHg in the chamber) and then to either hypoxia alone or hypoxia with Ado (50 µM). These two protocols were conducted in separate groups of cells (n = 14 and 18, respectively). The response was evaluated as a relative change in F340/F380 following exposure to hypoxia. Figure 7A shows a representative recording of [Ca2+]i during repeated exposure to severe hypoxia in a type I cell. Hypoxia induced a gradual rise in [Ca2+]i, which reached a peak within 3 min and returned to baseline levels following reoxygenation. Note that the peak levels of [Ca2+]i were similar during both the first and second exposures to hypoxia. Figure 7B shows that the hypoxia-induced elevation of [Ca2+]i was markedly reduced in the presence of Ado (50 µM). On washout of Ado, an increase in [Ca2+]i was once again elicited by exposure to hypoxia. The averaged data from these experiments revealed that there was no significant difference in the peak levels of [Ca2+]i between two repeated exposures to hypoxia alone (1st, 1.63 ± 0.05; 2nd, 1.61 ± 0.07; n = 14). However, the hypoxia-induced elevation of [Ca2+]i was significantly reduced in the presence of Ado (50 µM; P < 0.05; n = 18; Fig. 7C). It is important to note that Ado alone did not alter [Ca2+]i in cells exposed to normoxia (data not shown). Our findings indicate that the A2A receptor plays a role in modulating the hypoxia-induced change in [Ca2+]i in carotid body type I cells.


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Fig. 7.   Effect of Ado on hypoxia-induced enhancement of intracellular free Ca2+ in carotid type I cells. A: a representative recording of cytosolic free Ca2+ concentration ([Ca2+]i) during hypoxia in a carotid body type I cell. [Ca2+]i was estimated by using fluorescent indicator fura 2. The ratio of 340- to 380-nm fluorescence (F340/F380) was used to evaluate [Ca2+]i. Hypoxia was induced by saturating the 1 mM sodium dithionate-containing perfusate with 100% N2. Hypoxia induced the elevation of [Ca2+]i, which returned to the baseline level on reoxygenation. The response to hypoxia was similar between 2 repeated exposures. B: a recording of [Ca2+]i during hypoxia in a type I cell in the presence and absence of Ado. The cells were first exposed to hypoxia alone and then to hypoxia with Ado (50 µM). The hypoxia-induced elevation of [Ca2+]i was smaller in the presence of Ado. On removal of Ado, an increase in [Ca2+]i was elicited when the cells were reexposed to hypoxia alone. C: the averaged data from Ca2+-imaging study. The elevation of [Ca2+]i during hypoxia was significantly inhibited in the presence of Ado (50 µM; * P < 0.05). The two protocols were performed in separate group of cells. The number of cells tested is shown in the parentheses. Values are means ± SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of the present study was to establish evidence for Ado A2A receptors in carotid body type I cells. Our RT-PCR studies showed that rat carotid bodies do indeed express Ado A2A and A2B receptor mRNAs. We found no evidence for either Ado A1 or A3 receptor mRNA in rat carotid bodies. We and others showed previously that the O2-sensitive PC-12 cells express the A2A and A2B receptors but not the A1 or A3 subtypes (13, 15, 35). Therefore, the Ado receptor subtypes expressed in carotid bodies are the same as those expressed in the PC-12 cells. The RT-PCR experiments were performed using total RNA extracted from carotid body tissues. Thus it is possible that the RNA samples were contaminated with RNA from cells other than type I cells. Results from our in situ hybridization study suggest that Ado A2A receptor mRNA in rat carotid bodies is localized to the type I cells. Our studies focused on the A2A receptors because our previous paper showed that the A2A receptor plays an important role in modulating [Ca2+]i and therefore the cellular response to hypoxia in PC-12 cells (15). Although previous studies reported the presence of A2A receptor mRNA in carotid bodies of developing (38) and adult rats (14), these reports lacked a detailed description of the localization of A2A receptor mRNA in the carotid body. Our findings show clearly that the expression for A2A receptor mRNA in rat carotid bodies has clusterlike patterns of distribution.

The carotid body is organized in clusters of cells (glomi), which are separated from each other by walls of connective tissue (19). The clusters are formed by two cell types, O2-sensitive type I cells and sustentacular type II cells. Type I cells are more numerous than type II cells and have a clear round nucleus and a distinctly granular cytoplasm. Type II cells are located in the periphery of the cell clusters and have a disk-shaped nucleus with dense chromatin, and their cytoplasm lacks the granular appearance common in type I cells. It is thought that the ratio of type I to type II cells is ~5:1 (19). Our findings suggest that Ado A2A receptors are present predominantly in type I cells. We found that numerous cells in carotid bodies contained immunoreactivity for A2A receptors. The A2A receptor immunoreactive cells were arranged in clusters. Importantly, we found that immunoreactivity for A2A receptors was colocalized with that of TH, which is a marker for type I cells (19). This is perhaps the most convincing evidence that the A2A receptors are present in type I cells.

Isolated type I cells from rat carotid bodies were used for patch-clamp and Ca2+-imaging studies. The majority of cells in our preparation were round, had sizes of ~10-15 µm in diameter, and were immunopositive for TH. These spherical cells were selected and used to examine the effect of Ado receptor stimulation on the voltage-dependent ICa in type I cells. We found that activation of the Ado receptor mediates inhibition of ICa in type I cells. This effect was concentration dependent and was abolished by the selective A2A receptor antagonist ZM-241385 but not by a selective A1 receptor antagonist, DPCPX (17). The effect of Ado receptor stimulation on ICa was similar to that measured in PC-12 cells (15). To our knowledge, the present study is the first evidence that voltage-gated ICa in carotid body type I cells is modulated by the A2A receptor. It was reported previously that Ado had no effect on voltage-dependent ICa in rat carotid body type I cells (7). These investigators only tried a small concentration of Ado (10 µM) in their study. We found that a relatively high concentration of Ado is required to induce the significant change in ICa in type I cells. There is evidence that Ado is produced during hypoxia in carotid bodies (4). Because type I cells are compactly packed into a small volume under in vivo conditions, it may be possible that Ado around type I cells reaches a relatively high concentration during hypoxic exposure. It also was reported recently that Ado inhibits L-type ICa and catecholamine release in the rabbit carotid body type I cells via activation of A1 receptors (31). These investigators failed to detect an effect of A2A receptor activity. This finding contrasts with our results that the effect of Ado on ICa was blocked by ZM-241385, a selective A2A receptor antagonist, but not by the A1 receptor antagonist DPCPX. Moreover, the A1 receptor mRNA was not detected in the rat carotid body by our RT-PCR study. Therefore, it is possible that the expression of different Ado receptor subtypes in type I cells varies among different species.

Relatively little is known about how A2A receptors couple to Ca2+ channels. The A2A receptors stimulate protein kinase A (PKA) activity via Gs protein and adenylate cyclase (25). It has also been shown that the activity of cardiac and skeletal Ca2+ channels is enhanced by PKA-dependent phosphorylation (30, 34). Our results show that A2A receptors inhibit Ca2+ channels in rat carotid body type I cells. We also have reported that stimulation of A2A receptors inhibits voltage-dependent ICa via activation of PKA in rat PC-12 cells (15). This action of Ado was abolished by a specific PKA inhibitor and was absent in a PKA mutant PC-12 cell line (15). In support of our results, Park et al. (26) showed that both L-type and N-type Ca2+ channels are inhibited by activation of A2A receptors in PC-12 cells. L-type Ca2+ channels are subdivided into alpha 1S, alpha 1C, and alpha 1D depending on their molecular structure (1). It has been shown that the alpha 1D L-type channels are inhibited by PKA in rat pinealocytes (5). Rat carotid body type I cells predominantly express the L- and N-type Ca2+ channels (7). It is therefore possible that the alpha 1D type Ca2+ channels are the targets for the A2A receptor-mediated inhibition in type I cells. It is important to note that A2A receptors are broadly distributed through various organs and exert a wide range of actions. Thus it is entirely possible that Ado may modulate Ca2+ channel activity by different mechanisms in different tissues and cell types.

We also examined the effect of Ado receptor stimulation on Ca2+ influx in type I cells during hypoxia. It was shown previously that the increase in [Ca2+]i in O2-sensitive cells during hypoxia occurs as the result of membrane depolarization and activation of voltage-dependent Ca2+ channels (11, 18, 40). The hypoxia-induced elevation of [Ca2+]i in type I cells is inhibited by pharmacological blockade of voltage-dependent Ca2+ channels (2). We found that the hypoxia-induced elevation of [Ca2+]i was significantly attenuated in the presence of Ado. The mechanism by which Ado attenuates the hypoxia-evoked increase of [Ca2+]i is unknown but could be due to inhibition of voltage-dependent Ca2+ channels via the A2A receptor. This could be potentially an important mechanism for regulation of neurotransmitter release and gene expression during hypoxia. It has been shown that the voltage-sensitive Ca2+ channels are involved in the secretion of dopamine during hypoxia in type I cells (8, 24) and also in PC-12 cells (33). In addition, our laboratory has reported that the hypoxia-induced elevation of [Ca2+]i is essential for regulation of gene expression for TH, the rate-limiting enzyme for the synthesis of dopamine (29). Therefore, it is possible that activation of A2A receptors modulates the release of dopamine and regulation of O2-sensitive genes in carotid body type I cells during hypoxia.

It was reported recently that Ado inhibits 4-aminopyridine-sensitive outward K+ currents in rat type I cells; the subtype of Ado receptors involved in this action was not identified (36). This effect of Ado on K+ currents is likely to be important for maintaining cellular depolarization during hypoxia. In the present study, we found that Ado inhibits voltage-dependent ICa and reduces the increase in cytosolic free Ca2+ during hypoxia in type I cells. We presented evidence that these responses are mediated by the A2A receptor. This modulatory effect on intracellular free Ca2+ via A2A receptors may play an important role in feedback modulation of the cellular response to hypoxia, since it has been shown that Ado is produced during hypoxia in carotid bodies (4). We propose that modulation of K+ currents by Ado maintains membrane excitability of type I cells during hypoxia, whereas inhibition of ICa by A2A receptor stimulation regulates the levels of intracellular free Ca2+. Thus the cellular response to hypoxia in type I cells appears to involve Ado-mediated inhibition of both K+ and Ca2+ currents.

It has been reported previously that Ado has a stimulatory effect on the carotid body activities via A2 receptors in several animal species (21, 23, 32). Most of these previous studies were performed in an in vivo model or in isolated whole carotid body preparations. It should be noted that these preparations include cells other than type I cells (vasculature, afferent nerve endings of carotid sinus nerves), which might also express A2 receptors and affect the Ado-induced changes in intact carotid body activity. A recent paper showed, using an in vitro single nerve fiber recording, that Ado stimulates carotid sinus nerve afferent discharge in rats, supporting the stimulatory effect of Ado via nonvascular mechanisms (36). However, this result does not exclude a direct excitatory effect of Ado on afferent nerve endings primarily via activation of postsynaptic A2 receptors. It also has been reported that activation of A2 receptors has an excitatory effect on other sensory afferent nerve endings (3). Nevertheless, it is possible that Ado mediates various physiological responses, including inhibition of Ca2+ channels in type I cells and excitation of sensory terminals of the carotid sinus nerve.

In summary, we found that rat carotid body type I cells express Ado A2A mRNA and proteins. We also found that stimulation of the A2A receptor inhibits the voltage-dependent ICa and hypoxia-induced elevation of intracellular Ca2+ in type I cells. It is clear that intracellular free Ca2+ is important for the regulation of cellular functions during hypoxia. We propose that the A2A receptors may play a role in modulating these functions by regulating the level of intracellular free Ca2+ during hypoxia.


    ACKNOWLEDGEMENTS

This study was supported by grants to D. E. Millhorn from the National Heart, Lung, and Blood Institute (R37-HL-33831 and HL-59945) and the United States Army (DAMD179919544).


    FOOTNOTES

Address for reprint requests and other correspondence: D. E. Millhorn, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati, 231 Bethesda Ave., PO Box 670576, Cincinnati OH 45267-0576 (E-mail: David.Millhorn{at}uc.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. §1734 solely to indicate this fact.

Received 6 December 1999; accepted in final form 27 March 2000.


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RESULTS
DISCUSSION
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