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Carotid body I1-imidazoline receptors: Binding, visualization and modulatory function
Respiration Physiology 112 (1998) 239 – 251
Carotid body I1-imidazoline receptors: Binding, visualization and modulatory function P. Ernsberger a,*, Y.R. Kou b, N.R. Prabhakar b,c a b
Department of Nutrition, Case Western Reser6e School of Medicine, 10900 Euclid A6enue, Cle6eland, OH 44106 -4906, USA Department of Medicine, Case Western Reser6e School of Medicine, 10900 Euclid A6enue, Cle6eland, OH 44106 -4906, USA c Department of Physiology and Biophysics, Case Western Reser6e School of Medicine, 10900 Euclid A6enue, Cle6eland, OH 44106 -4970, USA Accepted 23 February 1998
Abstract The carotid body is influenced by many neurotransmitter receptors. A novel receptor specific for imidazolines has been implicated in cardiorespiratory regulation in the brain. To test for both I1-imidazoline and a2-adrenergic receptors, which also recognize imidazolines, specific [125I]p-iodoclonidine binding to carotid body membranes was characterized. The specific a2-agents epinephrine (100 mM) or SK&F 86466 (10 mM) inhibited only a portion of specific [125I]p-iodoclonidine binding in both cat and rabbit carotid bodies, indicating the presence of I1-imidazoline as well as a2-adrenergic sites. The distribution of [125I]p-iodoclonidine binding sites was visualized autoradiographically. The cat carotid body was intensely labeled by [125I]p-iodoclonidine, with both I1-imidazoline and a2-adrenergic sites expressed. The relevance of I1-imidazoline receptors in modulation of chemosensory discharge was determined in seven cats after a2-adrenergic blockade. Clonidine (100 mg/kg) facilitated chemosensory activity particularly under hypoxia. We conclude that I1-imidazoline receptors are expressed within the carotid body and may potentiate chemosensory discharge, in contrast to the inhibitory action of a2-adrenergic receptors. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Carotid body, imidazoline receptors; Control of breathing, carotid body, transmitter substances; Mammals, cat, rabbit; Receptors, a2-adrenergic, imidazoline I2
1. Introduction
* Corresponding author. Tel.: + 1 216 3684738; fax: +1 216 3684752; e-mail: [email protected]
In its function as a peripheral sensory organ detecting changes in the partial pressure of arterial oxygen, the carotid body is influenced by
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many neurotransmitters and neuromodulators (Prabhakar, 1994). Glomus cells of the carotid body resemble chromaffin cells and are of similar neural crest original (Heath et al., 1990). Chromaffin cells within the adrenal medulla have been found to express a high density of I1-imidazoline sites (Ernsberger et al., 1993, 1995b). These sites may be functional receptors, because imidazolines induce expression of catecholamine synthesizing genes in direct proportion to their binding affinity at I1-imidazoline sites (Evinger et al., 1995). Tumor cells derived from adrenal medulla, pheochromocytoma PC12 cells, also express I1-imidazoline receptors (Separovic et al., 1996). PC12 cells resemble carotid body type I cells in their response to hypoxia (Prabhakar et al., 1995; Bright et al., 1996). We hypothesized that the chromaffin-like cells of the carotid body, like adrenal chromaffin cells and the PC12 line, might express I1-imidazoline sites. In contrast to the I1-subtype, which is localized to the plasma membrane (Ernsberger and Shen, 1997), I2-imidazoline sites are localized to mitochondria and may regulate monoamine oxidase (Tesson et al., 1995). Mitochondrial I2-imidazoline sites have been localized to the carotid body (Youngson et al., 1995), but the possible association of I1-imidazoline receptors with peripheral chemoreceptors has not been investigated. In the ventral medulla oblongata, putative I1imidazoline receptors may mediate the cardiovascular actions of imidazoline drugs (Ernsberger et al., 1990b; Haxhiu et al., 1994). A specific cellular signaling pathway involving the generation of diglycerides from phosphatidylcholine appears to be involved (Separovic et al., 1996, 1997). Furthermore, in the ventral medulla these receptors may modulate the central control of breathing and airway tone (Haxhiu et al., 1995, 1996). Therefore, in the present study, we sought to determine whether I1-imidazoline sites as well as a2-adrenergic receptors are localized to the carotid body and to assess a possible modulatory role of I1imidazoline receptors in the chemosensory response to hypoxia.
2. Methods
2.1. Tissue preparation In a group of nine cats anesthetized with pentobarbital (45 mg/kg) and ventilated with room air, 18 carotid bodies were dissected out and frozen in liquid nitrogen. Intact carotid bifurcations (four) were removed from another group of cats and frozen on dry ice. Carotid bodies were also removed from 20 rabbits. No adrenergic drugs were administered to the animals at any time. For purposes of comparison, in some experiments tissue from the bovine ventrolateral medulla oblongata was processed as previously described (Ernsberger et al., 1993), and assayed in parallel to the carotid body samples.
2.2. Membrane preparation Carotid bodies were thawed on ice, pooled, and incubated in 25 ml of 20 mM HEPES-Tris buffer (pH 7.4) containing 0.5 mg/ml collagenase (Sigma, type I) and the protease inhibitors EDTA (5.0 mM), 1,10-phenanthroline (0.1 mM) and phenylmethylsulfonyl fluoride (50 mM) in order to inhibit degradation of receptor protein. The digested and lysed tissue was cooled on ice and homogenized with a ground glass homogenizer. The homogenate was centrifuged at 1000× g for 5 min at 4°C to remove nuclei and debris. The pellets were resuspended in 20 ml of homogenization buffer using a polytron (Tecmar Tissuemizer, setting 60), and centrifuged again at 1000 × g for 5 min. The supernatants were combined and centrifuged at 40000× g for 20 min at 4°C, and the resulting P2 pellet was resuspended in 25 ml of 50 mM Tris–HCl buffer (pH 7.7) containing protease inhibitors. After centrifugation again at 40000× g for 20 min, the resulting membrane pellet was resuspended a final time in Tris–HCl alone, centrifuged, flash-frozen, and stored at − 70°C for up to 2 weeks.
2.3. [ 125I]p-iodoclonidine radioligand binding microassays The small mass of the carotid body demanded development of a sensitive binding assay. One
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method of increasing assay sensitivity is by using a radioligand with high specific activity, such as one bearing an 125I atom. We have recently characterized the binding properties of [125I]p-iodoclonidine, a high-affinity ligand for I1-imidazoline as well as a2-adrenergic binding sites (Kou et al., 1991; Ernsberger et al., 1993; Liedtke et al., 1993; Ernsberger et al., 1995c). Radioligand binding microassays with [125I]p-iodoclonidine for determination of specific binding to I1-imidazoline sites and a2-adrenergic receptors were performed by a modification of these methods. Membranes were slowly thawed and resuspended in Tris–HCl buffer at a concentration of 10 mg protein/ml. Assays were conducted in a total volume of 250 ml consisting of 125 ml membrane suspension, 25 ml radioligand, and 100 ml drug or vehicle. Incubations were initiated by the addition of membrane and were carried out for 40 min at 25°C. Non specific binding was defined in the presence of 10 mM phentolamine, an imidazoline adrenergic agent. Specific a2-adrenergic binding was defined by inhibition with (− )epinephrine (0.1 mM). In experiments using catecholamines, all samples contained ascorbic acid in a final concentration of 0.001%. Incubations were terminated by vacuum filtration using a cell harvester (Brandel), equipped with Teflon tubing to reduce absorption of the radioligand, over glass fiber filters (Schleicher and Schuell c 34) which were preincubated for 4 h at 4°C in 0.03% polylethylimine to reduce non specific binding to the filter. The filters were washed four times with 5 ml ice-cold Tris– HCl, placed in scintillation vials, covered with 4 ml scintillation cocktail (BioSafe II, Research Products International), and counted at 50% efficiency (Beckman LS5801). Protein was assayed by the bicinchoninic acid method (Smith et al., 1985).
2.4. Receptor autoradiography The carotid bifurcation was mounted on a cryostat chuck with mounting medium (M-1, Lipshaw) and sectioned on a Hacker cryostat ( − 18°C) at 8 mm thickness. Sections were collected by thaw-mounting on acid-cleaned, gelatin-subbed glass slides, dried in a vacuum
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desiccator for 1 h and stored at − 20°C for 24–48 h to insure adherence of the tissue to the slide before transfer to a − 70°C freezer for longer storage (up to 6 months). Adjacent sections were fixed in formalin without drying and stained with hematoxylin and eosin for histological reference. Autoradiography of I1-imidazoline and a2adrenergic receptor sites were conducted using [125I]p-iodoclonidine as the radioligand according to methods described previously (Ernsberger et al., 1995b; Haxhiu et al., 1995). Iso-osmotic buffers containing sucrose were used throughout in order to preserve the integrity of the tissue. Immediately prior to assay, slide-mounted sections were gradually warmed to room temperature in a vacuum desiccator and pre-incubated in Coplin jars in 20 mM Tris–HCl buffer (pH 7.4) containing 280 mM sucrose and 5.0 mM EDTA for 1 h and then washed in the same buffer without EDTA for 15 min to allow dissociation of any endogenous ligand bound to receptors within the tissue. Non-specific binding was determined in parallel incubations containing the imidazoline BDF-6143 (10 mM). a2-Adrenergic binding was defined in the presence of 10 mM epinephrine. To prevent uptake and degradation of epinephrine, the uptake blocker desipramine (10 mM), the monoamine oxidase inhibitor pargyline (10 mM), and the antioxidants ascorbic acid (10 mM) and dimethylsulfoxide (1% v/v) were included in the assay medium. I1-Imidazoline binding was defined in the presence of the selective inhibitor cimetidine (10 mM). After the incubation and wash periods, the slides were dipped in ice-cold distilled water to remove buffer salts which can cause chemography. Finally, sections were rapidly dried in a closed container by a stream of air passed through a column containing CaSO4 pellets and 5 ˚ molecular sieves and through a trap immersed A in a mixture of dry ice and ethanol, and then stored in a vacuum desiccator for 24–48 h at 4°C. The dried slides were arranged in X-ray cassettes and exposed to tritium-sensitive film (Hyperfilm, Amersham) for 96 h. The films were developed (D-19) for 4 min at 17°C, rinsed briefly, and fixed (Rapid Kodak Fixer) for 10 min. The sections
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were then fixed in formalin and stained with hematoxylin and eosin for histological reference.
given for 5 min and terminated by switching back to 100% O2.
2.5. In 6i6o studies
2.6. Data analysis
Seven adult cats (2 – 3 kg) were anesthetized with pentobarbital (45 mg/kg, i.p.) and femoral artery and vein cannulated for measuring arterial pressure and for intravenous administration of drugs. Additional doses of anesthetic (2 – 3 mg/kg i.v.) were given to maintain an absence of corneal and pain reflexes. Animals were tethered in a supine position and intubated caudal to the larynx. The carotid bifurcation was exposed by ligating and retracting the trachea and esophagus. Intracarotid infusion of drugs was performed in two cats through a catheter placed in the external carotid artery with its tip close to the carotid body. To maintain arterial blood gases constant, the anesthetized animals were paralyzed with gallamine (4 mg/kg, i.v.) and ventilated with a Harvard respirator. End-tidal CO2 was continuously monitored with an infrared CO2 analyzer (Beckman). Arterial blood samples were collected for measurements of PO2, PCO2 and pH in a blood gas analyzer (IL model 1303). An infusion of 8.4% NaHCO3 was given i.v. as necessary to maintain blood gases and pH. The body temperature of the animals was kept at 389 1°C by means of a servo-controlled heating blanket. Chemoreceptor activity was recorded as described in detail elsewhere (Prabhakar et al., 1989). Briefly, either the left or the right carotid sinus nerve was sectioned, desheathed, and successively split until activity from one to three fibers was obtained. Action potentials were amplified and displayed on an oscilloscope. The impulses were then converted to standard pulses (Winston Rad II), which were passed on to a rate meter (Frederick Haer). Chemoreceptor fibers were identified by their: (1) spontaneous sporadic discharge; (2) increase in activity when switched from room air to hypoxia (10% O2 in N2); and (3) decrease in activity when switched form room air to hyperoxia (100% O2). Isocapnic hypoxia was induced by switching the inspiratory port of the respirator from 100% O2 to a reservoir bag containing 10% O2 in N2. The hypoxic challenge was
Radioligand binding data were obtained as dpm, transformed to percent of total specific binding, and compared by analysis of variance. For in vivo experiments, arterial blood pressure (mmHg), chemosensory activity (impulses/sec) and arterial blood gasses were measured. Chemoreceptor activity was measured 1 min prior to the hypoxic challenge, and during the final min of each challenge. All data were presented as mean9 SEM. Statistical comparisons were made by analysis of variance (ANOVA) followed by comparison of group means with the Newman– Keuls test.
2.7. Materials [125I]p-iodoclonidine (2200 Ci/mmol) was obtained from New England Nuclear (Boston, MA), stored at − 70°C in ethanol and diluted in water prior to assay. Imidazole-4-acetic acid, epinephrine, phentolamine, clonidine and guanabenz were all purchased from Sigma (St. Louis, MO).
3. Results
3.1. Radioligand binding microassays in isolated membranes Specific [125I]p-iodoclonidine binding was determined in membranes from the cat carotid body. As shown in Fig. 1, [125I]p-iodoclonidine binding was only partially inhibited by a high concentration (100 mM) of the endogenous a2-agonist ( − )epinephrine (379 3% inhibition). A similar fraction of total specific [125I]p-iodoclonidine binding was blocked by the specific a2-antagonist SK&F 86466 (449 3% inhibition at 10 mM). In contrast, clonidine, which binds to both I1-imidazoline and a2-adrenergic sites, inhibited the total population of specific sites. These findings imply that only 40% of [125I]p-iodoclonidine binding
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sites in the cat carotid body are a2-adrenergic receptor sites. Imidazole-4-acetic acid, which binds to I1-imidazoline sites but not a2-adrenergic receptors (Ernsberger et al., 1987, 1990a,b), inhibited 7296% of the specific [125I]p-iodoclonidine binding sites at a concentration of 100 mM, consistent with the presence of specific I1-imidazoline binding. Significant inhibition of binding was also observed for cimetidine, a histamine H2 blocker with moderate affinity for I1-imidazoline sites (Ki :100 nM) but which does not bind to a2-receptors (Ernsberger et al., 1987, 1990a,b). A total of 1.5 fmol of specific binding sites per carotid body were labeled by 0.6 nM [125I]p-iodoclonidine, of which 0.9 fmol were insensitive to epinephrine and thus non-adrenergic. The remaining 0.6 fmol per carotid body had the characteristics of a2-adrenergic binding sites. Specific binding sites for [125I]p-iodoclonidine were also present in rabbit carotid body membranes (Fig. 2). In this species, specific [125I]piodoclonidine binding was 0.309 0.01 fmol per carotid body or 399 2 fmol per mg protein. About one-half of specific [125I]p-iodoclonidine binding could be inhibited by agents specific for a2-adrenergic receptors such as SK&F 86466 and
Fig. 1. Effects of a2-selective and I1-selective agents on [125I]piodoclonidine binding to cat carotid body membranes in a radioligand binding microassay. Inhibition of [125I]p-iodoclonidine (0.5 nM) binding by a maximal concentration of several competing drugs. Data are expressed as percent of total specific [125I]p-iodoclonidine binding as defined in the presence of 10 mM phentolamine. IAA, imidazole-4-acetic acid.
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Fig. 2. Inhibition of [125I]p-iodoclonidine binding to rabbit carotid body membranes by imidazolines and non-imidazolines. Inhibition of [125I]p-iodoclonidine (0.5 nM) binding by a maximal concentration of several competing drugs. Data are expressed as percent of total specific [125I]p-iodoclonidine binding as defined in the presence of 0.1 mM piperoxan. The non-imidazole a2-adrenergic antagonist SK&F 86466 (10 nM and 10 mM) dose-dependently inhibited slightly more than half of total specific [125I]p-iodoclonidine binding. The binding values are within the range of data obtained with full inhibition curves in brainstem membranes (Ernsberger et al., 1993). Guanabenz (0.1 mM), a non-imidazole a2-adrenergic agonist, also inhibited only slightly more than half of the specific [125I]p-iodoclonidine binding sites. Imidazole-4-acetic acid (IAA, 10 mM) inhibited a portion of specific binding. These results indicate that, as for cat carotid body, about half of the specific sites labeled by 0.5 nM [125I]p-iodoclonidine are a2adrenergic receptors while the other half are I1-imidazoline binding sites.
guanabenz (Fig. 2). Conversely, about one-half the binding sites recognized imidazole-4-acetic acid (0.1 mM). The affinity of guanabenz for carotid body a2-receptors was determined using three concentrations of guanabenz ranging from 1 to 100 nM in two separate experiments (Fig. 3). Guanabenz potently inhibited [125I]p-iodoclonidine binding in both experiments (IC50 values of 2.1 and 2.4 nM; pseudo-Hill slopes (nH) of 0.73 and 0.88). These affinity values for guanabenz at a2-adrenergic binding sites are in agreement with previous results obtained in the bovine ventrolateral medulla oblongata (79 1 nM; Ernsberger et al., 1990b), and are consistent with the potent inhibitory action of guanabenz on chemoreceptor activity previously reported by us (Kou et al., 1991).
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In order to determine whether the present findings were unique, identical experiments were carried out using bovine ventrolateral medulla as previously described (Ernsberger et al., 1993). Relative to cat or rabbit carotid body, epinephrine and SK&F 86466 inhibited a larger proportion of sites (689 4 and 5993%, respectively, n=4). Conversely, imidazole-4-acetic acid and cimetidine inhibited a smaller proportion of sites (349 8 and 3496%, respectively, n = 4). Clonidine itself, or the analog moxonidine, inhibited the total population of specific [125I]p-iodoclonidine sites. Thus, the profile of [125I]p-iodoclonidine binding was similar in the ventrolateral medulla, but the ratio of I1-imidazoline to a2-adrenergic receptors was 1:3 as compared to 1:1 for cat or rabbit carotid body. The quantity of carotid body tissue available from either cat or rabbit was insufficient to conduct saturation binding studies. Using the Kd values for [125I]p-iodoclonidine determine in the bovine medulla (Ernsberger et al., 1993), we estimate the Bmax values for I1-imidazoline sites in cat and rabbit carotid bodies to be identical at 169 5 and 1692 fmol/mg protein, respectively, whereas for a2-adrenergic receptors, the estimated Bmax values are 109 2 and 23 9 2 fmol/mg protein. The estimated density of a2-adrenergic receptors is significantly greater in rabbit compared with cat carotid body.
3.2. Autoradiographic 6isualization of I1 -imidazoline and a2 -adrenergic binding sites in the cat carotid bifurcation The distribution of [125I]p-iodoclonidine binding sites in the cat carotid bifurcation is shown in Figs. 4 and 5. There was a single intensely bright area, indicating a high density of binding sites. The bright area corresponds to the carotid body as identified by hematoxylin and eosin staining, as shown in Fig. 6, which is a bright enlargement of the boxed region in Fig. 4, panel T. The carotid artery itself expresses a lower level of binding, which was most prominent in the endothelium (Figs. 4 and 5). Most of the labeling was lost in the presence of BDF-6143, a nonselective imidazoline ligand, indicating that the labeling was specific (panels labeled NS for non-specific binding). Labeling of the carotid body by [125I]p-iodoclonidine was partially inhibited with the addition of cimetidine to block I1-imidazoline sites (panels labeled A for a2-adrenergic binding), whereas the faint labeling in the carotid artery was not affected by cimetidine. Partial inhibition of carotid body labeling was also observed in the presence of epinephrine (panels labeled I for imidazoline binding), indicating that a portion of the [125I]p-iodoclonidine binding sites are labeled in the carotid body are non-adrenergic and likely to be I1-imidazoline sites. I1-imidazoline sites appear to be almost entirely lacking from the carotid artery. These data indicate that both I1-imidazoline and a2-adrenergic sites are expressed within the cat carotid body.
3.3. Effect of selecti6e stimulation of I1 -imidazoline receptors on chemosensory discharge
Fig. 3. Dose-dependent inhibition of [125I]p-iodoclonidine binding by the a2-agonist guanabenz. Inhibition of [125I]piodoclonidine (0.5 nM) binding by increasing concentrations of guanabenz. Data are the mean of two experiments, each conducted in duplicate. Details as in Fig. 1.
The role of I1-imidazoline and a2-adrenergic receptors in control of chemosensory discharge was determined in anesthetized, paralyzed and artificially respirated cats. We used guanabenz and SK&F 86466 as a selective agonist and antagonist for a2-adrenergic receptors and clonidine as a mixed I1/a2 agonist. In the absence of a2-blockade, clonidine elicited variable effects, in some cases inhibiting chemosensory discharge (Fig. 7,
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Fig. 4. Autoradiographic visualization of [125I]p-iodoclonidine binding sites in the cat carotid bifurcation. Shown are photographic prints made under identical conditions using the autoradiograms as negatives. Light areas correspond to the highest intensity of labeling. Adjacent 8 mm sections through the cat carotid bifurcation were incubated with 0.5 nM [125I]p-iodoclonidine under one of four different conditions. The section labeled T (total binding) was incubated with antioxidant vehicle alone, and shows the total binding of [125I]p-iodoclonidine. Binding sites were concentrated within the carotid body (bright region). The white rectangle corresponds to the photomicrograph in Fig. 6. The carotid body region is overexposed in order to allow visualization of the carotid arteries. The adjacent section labeled NS (non specific) was incubated with 10 mM BDF-6143 to block both I1 and a2 binding, and thus shows the distribution of non specific sites. The section labeled A (a2) was incubated with 10 mM cimetidine to selectively mask I1-imidazoline sites, and thus shows the distribution of a2-adrenergic sites. Labeling in the carotid body was partially inhibited by 10 mM cimetidine. The section on the far right, labeled I, was incubated with 10 mM epinephrine to mask a2-receptors, and thus shows the distribution of I1 binding. Note the persistence of [125I]p-iodoclonidine labeling in the carotid body, indicating the presence of non-adrenergic binding sites.
top panel), in a manner similar to that previously reported for guanabenz (Kou et al., 1991). In other cases, slightly facilitatory responses were observed (Fig. 7, bottom panel). The transient pressor response to clonidine, mediated by vascular a2-adrenergic receptors, was consistently observed (Fig. 7). Thus, indirect vascular effects on chemosensory discharge cannot account for the variable reasons to clonidine. In order to block a2-adrenergic receptors, cats were pretreated with 0.5 mg/kg SK&F 86466 i.v. The completeness of a2-blockade was tested by intracarotid infusion of the selective a2-agonist guanabenz (5.0 mg/min). Prior to treatment with SK&F 86466, guanabenz attenuated the chemosensory response to hypoxia (control D = 12 9 3; post-guanabenz D =5 9 2 impulses/sec).
After administration of SK&F 86466, guanabenz had no effect on the chemosensory response to hypoxia (D= 1193 impulses/sec). Effective a2blockade is illustrated in Fig. 8. After pretreatment with SK&F 86466, systemic administration of guanabenz (100 mg/kg i.v.) no longer inhibited chemosensory discharge or increased arterial pressure. During a2-blockade with SK&F 86466, systemic administration of clonidine stimulated chemosensory discharge under hypoxia (Fig. 9). Similar results were obtained with close arterial injection into the carotid body (20 mg/kg), or with intravenous administration (100 mg/kg). An analysis of variance for the effects of clonidine treatment (treated vs. untreated) and of oxygen tension (hyperoxia vs. normoxia vs. hypoxia) showed signifi-
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Fig. 5. Auto radiograms from sections of the carotid body from a second cat. Details as in Fig. 5.
cant effects of clonidine (F (1,30) = 10.7, P B 0.003) and of oxygen (F (2,30) =44.5, PB 0.0001) as well as a significant interaction between clonidine treatment and oxygen (F (2,30) = 3.4, P B0.05). The latter finding indicates that clonidine’s facilitatory effect was greater under hypoxia than under normoxic or hyperoxic conditions.
4. Discussion This study demonstrates that I1-imidazoline sites as well as a2-adrenergic receptors are expressed within the carotid bodies of cats and rabbits. Direct labeling of binding sites in situ demonstrates both receptor types are present in high density throughout the carotid body and are expressed at much higher levels than in surrounding arterial or connective tissues. Functional studies of chemosensory discharge suggest that I1-imidazoline receptors may facilitate the response to hypoxia, whereas a2-adrenergic receptors inhibit chemosensory function. The latter finding confirms previous studies of the function of a2-adrenergic receptors in the cat carotid body (Kou et al., 1991). The precise nature of the
mechanism coupling I1-imidazoline receptor stimulation to facilitation of chemosensory responses cannot be ascertained from the present data. However, activation of I1-imidazoline receptors in pheochromocytoma cells elicits the release of prostaglandin E2 (Ernsberger et al., 1995b), and this mediator is also released by the carotid body in response to hypoxia (Go´mez-Nin˜o et al., 1994). Unknown at present are the roles played by cellular mediators in the response to stimulation of either I1-imidazoline or a2-adrenergic receptors within the carotid body. There are relatively few prior reports of radioligand binding studies of receptor sites in the carotid body. Muscarinic acetylcholine receptors have been identified by membrane binding and autoradiography (Dinger et al., 1991), endothelin receptors have been localized within the regions of the carotid body by autoradiography (Nichols and Ruffolo, 1991), dopamine D2 receptors have been detected by in situ hybridization to complementary RNA (Czyzyk-Krzeska et al., 1992), and we previously identified a2-adrenergic receptors (Kou et al., 1991). The primary obstacle to the study of neurotransmitter receptors in the carotid body is the small mass of the tissue (0.8 and 0.3
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mg in the cat and rabbit, respectively) relative to the requirements of standard radioligand binding assays (60 mg per triplicate data point). In the present assay, we reduced the assay volume (250 ml), and used an iodinated probe with high specific activity (2200 Ci/mmol) and high affinity for I1-imidazoline and a2-adrenergic binding sites. A limitation of the present study was that insufficient tissue was available to conduct saturation experiments in order to determine radioligand affinity and the absolute number of binding sites. However, if the assumption is made that the affinity of [125I]p-iodoclonidine does not vary significantly between tissues, which has already been documented (Ernsberger et al., 1995b),
Fig. 6. Photomicrograph of a carotid body section stained for histological reference. The section used to generate the autoradiogram of total binding for Fig. 4 was stained with hematoxylin and eosin. The area shown corresponds to the boxed area in Fig. 4.
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then the density of a2-adrenergic binding sites can be estimated using affinity constants obtained in the bovine brain (Section 3). The estimated density (Bmax) for a2-adrenergic receptors is 18 fmol per mg protein in cat carotid body and 76 fmol per mg protein in the rabbit. These predicted receptor densities fall within the range of values reported for tissues known to respond to a2adrenergic receptors agonists, including spinal cord, platelets, ileum, and the kidney (Bylund and U’Prichard, 1983), suggesting that the level of expression of a2-adrenergic receptors in the carotid body is compatible with physiological responses to receptor activation. Previous studies testing the function of putative I1-imidazoline receptors have focused on cardiovascular responses in the RVLM of the brainstem (Bousquet et al., 1984; Tibiric¸a et al., 1989; Ernsberger et al., 1990b; Haxhiu et al., 1992). Microinjection of I1-antagonists into the RVLM blocks the vasodepressor action of systemic imidazolines, whereas blockade of a2-adrenergic receptors with the specific antagonist SK&F 86466 has no effect (Gomez et al., 1991). However, because both I1imidazoline and a2-adrenergic receptors lower blood pressure when activated within the brainstem, precisely parceling out the effects mediated by the two receptors is difficult. In the present study, a2-adrenergic stimulation inhibited chemosensory discharge, but I1-imidazoline stimulation after blockade of a2-receptors facilitated chemosensory discharge. Thus, the separation of a2-adrenergic and I1-imidazoline receptor mediated effects is more distinct than in the brainstem, since the two receptors appear to mediate opposite effects. This interpretation is supported by the contrasts between guanabenz and clonidine, two a2-adrenergic agonists expressing nearly identical affinity and efficacy at a2-receptors (Bylund and U’Prichard, 1983). Without a2-blockade, guanabenz inhibited nearly two-thirds of the chemosensory response to hypoxia, confirming our previous results (Kou et al., 1991). Clonidine, in contrast, had inconsistent effects when given alone. After blockade of a2-adrenergic receptors with the specific antagonist SK&F 86466, guanabenz had no effect. In contrast, a2-blockade unmasked a stimulatory effect of clonidine. These
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Fig. 7. Inconsistent effects of clonidine on chemosensory discharge prior to a2-adrenergic blockade. Representative tracings show strongly inhibitory (top) and weakly excitatory (bottom) effects of clonidine (100 mg/kg i.v.) delivered at the arrow. CA, integrated chemoreceptor activity; AP, action potentials; ABP, arterial blood pressure; CO2, end-tidal PCO2. Pressor response to clonidine is an acute effect mediated by vascular a2-adrenergic receptors.
data demonstrate that clonidine acts on a receptor other than the a2-adrenergic receptor to facilitate chemosensory discharge. The present findings concerning the action of clonidine on chemoreceptor activity are paralleled by studies of clonidine’s effects on insulin secretion from pancreatic islets (Schulz and Hasselblatt, 1989).
Clonidine suppresses insulin release when administered alone, but after blockade of a2-adrenergic receptors, a stimulatory action is unmasked. These data support the hypothesis that I1-imidazoline binding sites are functional receptors and are functionally distinct from a2-adrenergic receptors.
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Fig. 8. Lack of inhibitory or pressor effects of guanabenz following a2-adrenergic blockade. Representative tracing showing the absence of any effect of guanabenz (100 mg/kg i.v.) on chemoreceptor activity (CA and AP) or on arterial pressure (AP) following blockade of a2-adrenergic receptors with SK&F 86466 (0.5 mg/kg i.v.). These data are consistent with effective blockade of a2-adrenergic responses.
The resolution of the receptor autoradiographic technique is not sufficient to determine which cell type(s) within the carotid body express I1-imidazoline and a2-adrenergic receptors. In the brain, I1-imidazoline sites are probably neuronal, because they are absent in primary cultures of glial astrocytes (Ernsberger et al., 1995b), whereas I2sites are primarily localized on glia (Regunathan et al., 1993). Because type II carotid body cells resemble glia, they most likely express I2-sites (Youngson et al., 1995), while the neuron-like type I cells might possibly express the I1-subtype. The binding characteristics of the a2-adrenergic receptors identified in cat and rabbit carotid bodies correlate closely with the results of physiological studies. The a2-adrenergic component of binding was potently blocked by the antagonist SK&F 86466, and this antagonist potently blocks the action of the a2-agonist guanabenz and elevates basal chemosensory discharge by blocking tonic catecholaminergic activity (Kou et al., 1991). The a2-agonist guanabenz inhibited [125I]piodoclonidine binding in a dose-dependent man-
ner with and high affinity (2 nM), and was also a potent inhibitor of chemosensory discharge in the cat (Kou et al., 1991). Although clonidine can interact with a1-adrenergic receptors at high doses (Bylund and U’Prichard 1983), it is unlikely that a1-receptors are responsible for this effect at the doses used. Furthermore, the a1-agonist phenylephrine increases chemosensory discharge only slightly (B 2 impulses/sec) and, unlike clonidine, does not selectively enhance the response to hypoxia (Kou et al., 1991). We conclude that I1-imidazoline sites are associated with peripheral chemoreceptors, and I1-imidazoline receptors may stimulate chemosensory discharge, while a2-receptors may elicit opposite effects.
Acknowledgements Supported by the National Institutes of Health (HL45780 and HL44514). We gratefully acknowledge the expert technical assistance of Laura A.
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Fig. 9. Effects of clonidine on chemosensory discharge following a2-adrenergic blockade. Chemoreceptor activity was recorded from a fine filament of the cut carotid sinus nerve in seven cats which had been pretreated with SK&F 86466 (0.5 mg/kg i.v.). This treatment completely blocked the effect of a2-adrenergic receptor stimulation with guanabenz administered by close intracarotid injection. Activity was recorded for 5 min during each of three conditions: hyperoxia (100% O2), normoxia (room air), and hypoxia (12% O2 in 88% N2). Clonidine was then administered i.v. (100 mg/kg) or by close intracarotid injection (20 mg/kg) and chemoreceptor activity recordings were repeated 5 min later under hyperoxia, normoxia and hypoxia. Data represent mean 9SE (n= 7) and were obtained as mean impulse frequency during the last minute of each condition. Because intravenous and intracarotid injections produced indistinguishable effects, the data were combined. Two-way analysis of variance demonstrated significant main effects of oxygen level and clonidine treatment and a significant interaction between oxygen level and clonidine treatment. Post-hoc Tukey tests showed that clonidine significantly enhanced chemosensory discharge during hypoxia (P B 0.05).
Collins for studies.
autoradiographic
and
histologic
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P. Ernsberger et al. / Respiration Physiology 112 (1998) 239–251 Haxhiu, M.A., Dreshaj, I.A., Erokwu, B., Collins, L.A., Ernsberger, P., 1995. Effect of I1-imidazoline receptor activation on responses of hypoglossal and phrenic nerve to chemical stimulation. Ann. New York Acad. Sci. 763, 445 – 462. Haxhiu, M.A., McFadden, C.B., Ernsberger, P., Kaan, E.C., Erokwu, B., Dreshaj, I.A., 1996. Activation of I1-imidazoline receptors inhibits reflexly induced tracheal smooth muscle contraction. Atemweg. Lungenkrank. 22, 370–373. Heath, D., Khan, Q., Smith, P., 1990. Histopathology of the carotid bodies in neonates and infants. Histopathology 17, 511 – 520. Kou, Y.R., Ernsberger, P., Cragg, P.A., Cherniack, N.S., Prabhakar, N.R., 1991. Role of a2-adrenergic receptors in the carotid body response to isocapnic hypoxia. Respir. Physiol. 83, 353 – 364. Liedtke, C.M., Furin, J., Ernsberger, P., 1993. a2-Adrenergic, but not imidazoline, agonists activate NaCl co-transport in rabbit tracheal epithelial cells. Am. J. Physiol. Cell Physiol. 264, C568 – C576. Nichols, A.J., Ruffolo, R.R. Jr., 1991. Structure-activity relationships for a-adrenoceptor agonists and antagonists. Prog. Basic Clin. Pharmacol. 8, 75–114. Prabhakar, N.R., Landis, S.C., Kumar, G.K., Mullikin-Kilpatrick, D., Cherniack, N.S., Leeman, S., 1989. Substance P and neurokinin A in the cat carotid body: Localization, exogenous effects and changes in content in response to arterial PO2. Brain Res. 481, 205–214. Prabhakar, N.R., 1994. Neurotransmitters in the carotid body. Adv. Exp. Med. Biol. 360, 57–69. Prabhakar, N.R., Shenoy, B.C., Simonson, M.S., Cherniack, N.S., 1995. Cell selective induction and transcriptional activation of immediate early genes by hypoxia. Brain Res. 697, 266 – 270.
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Regunathan, S., Feinstein, D.L., Reis, D.J., 1993. Expression of non-adrenergic imidazoline sites in rat cerebral cortical astrocytes. J. Neurosci. Res. 34, 681 – 688. Schulz, A., Hasselblatt, A., 1989. Dual action of clonidine on insulin release: Suppression, but stimulation when a2adrenoceptors are blocked. N-S Arch. Pharmacol. 340, 712 – 714. Separovic, D., Kester, M., Ernsberger, P., 1996. Coupling of I1-imidazoline receptors to diacylglyceride accumulation in PC12 rat pheochromocytoma cells. Mol. Pharmacol. 49, 668 – 675. Separovic, D., Kester, M., Haxhiu, M.A., Ernsberger, P., 1997. Activation of phosphatidylcholine-selective phospholipase C by I1-imidazoline receptors in PC12 cells and rostral ventrolateral medulla. Brain Res. 749, 335 – 339. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76 – 85. Tesson, F., Limon-Boulez, I., Urban, P., Puype, M., Vandekerckhove, J., Coupry, I., Pompon, D., Parini, A., 1995. Localization of I2-imidazoline binding sites on monoamine oxidases. J. Biol. Chem. 270, 9856 – 9861. Tibiric¸a, E., Mermet, C., Feldman, J., Gonon, F., Bousquet, P., 1989. Correlation between the inhibitory effect on catecholaminergic ventrolateral medullary neurons and the hypotension evoked by clonidine: A voltammetric approach. J. Pharmacol. Exp. Ther. 250, 642 – 647. Youngson, C., Regunathan, S., Wang, H., Li, G., Reis, D.J., 1995. Co-expression of imidazoline receptors and agmatine in rat carotid body. Ann. New York Acad. Sci. 763, 440 – 444.
Carotid body I1-imidazoline receptors: Binding, visualization and modulatory function P. Ernsberger a,*, Y.R. Kou b, N.R. Prabhakar b,c a b
Department of Nutrition, Case Western Reser6e School of Medicine, 10900 Euclid A6enue, Cle6eland, OH 44106 -4906, USA Department of Medicine, Case Western Reser6e School of Medicine, 10900 Euclid A6enue, Cle6eland, OH 44106 -4906, USA c Department of Physiology and Biophysics, Case Western Reser6e School of Medicine, 10900 Euclid A6enue, Cle6eland, OH 44106 -4970, USA Accepted 23 February 1998
Abstract The carotid body is influenced by many neurotransmitter receptors. A novel receptor specific for imidazolines has been implicated in cardiorespiratory regulation in the brain. To test for both I1-imidazoline and a2-adrenergic receptors, which also recognize imidazolines, specific [125I]p-iodoclonidine binding to carotid body membranes was characterized. The specific a2-agents epinephrine (100 mM) or SK&F 86466 (10 mM) inhibited only a portion of specific [125I]p-iodoclonidine binding in both cat and rabbit carotid bodies, indicating the presence of I1-imidazoline as well as a2-adrenergic sites. The distribution of [125I]p-iodoclonidine binding sites was visualized autoradiographically. The cat carotid body was intensely labeled by [125I]p-iodoclonidine, with both I1-imidazoline and a2-adrenergic sites expressed. The relevance of I1-imidazoline receptors in modulation of chemosensory discharge was determined in seven cats after a2-adrenergic blockade. Clonidine (100 mg/kg) facilitated chemosensory activity particularly under hypoxia. We conclude that I1-imidazoline receptors are expressed within the carotid body and may potentiate chemosensory discharge, in contrast to the inhibitory action of a2-adrenergic receptors. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Carotid body, imidazoline receptors; Control of breathing, carotid body, transmitter substances; Mammals, cat, rabbit; Receptors, a2-adrenergic, imidazoline I2
1. Introduction
* Corresponding author. Tel.: + 1 216 3684738; fax: +1 216 3684752; e-mail: [email protected]
In its function as a peripheral sensory organ detecting changes in the partial pressure of arterial oxygen, the carotid body is influenced by
0034-5687/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0034-5687(98)00021-8
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many neurotransmitters and neuromodulators (Prabhakar, 1994). Glomus cells of the carotid body resemble chromaffin cells and are of similar neural crest original (Heath et al., 1990). Chromaffin cells within the adrenal medulla have been found to express a high density of I1-imidazoline sites (Ernsberger et al., 1993, 1995b). These sites may be functional receptors, because imidazolines induce expression of catecholamine synthesizing genes in direct proportion to their binding affinity at I1-imidazoline sites (Evinger et al., 1995). Tumor cells derived from adrenal medulla, pheochromocytoma PC12 cells, also express I1-imidazoline receptors (Separovic et al., 1996). PC12 cells resemble carotid body type I cells in their response to hypoxia (Prabhakar et al., 1995; Bright et al., 1996). We hypothesized that the chromaffin-like cells of the carotid body, like adrenal chromaffin cells and the PC12 line, might express I1-imidazoline sites. In contrast to the I1-subtype, which is localized to the plasma membrane (Ernsberger and Shen, 1997), I2-imidazoline sites are localized to mitochondria and may regulate monoamine oxidase (Tesson et al., 1995). Mitochondrial I2-imidazoline sites have been localized to the carotid body (Youngson et al., 1995), but the possible association of I1-imidazoline receptors with peripheral chemoreceptors has not been investigated. In the ventral medulla oblongata, putative I1imidazoline receptors may mediate the cardiovascular actions of imidazoline drugs (Ernsberger et al., 1990b; Haxhiu et al., 1994). A specific cellular signaling pathway involving the generation of diglycerides from phosphatidylcholine appears to be involved (Separovic et al., 1996, 1997). Furthermore, in the ventral medulla these receptors may modulate the central control of breathing and airway tone (Haxhiu et al., 1995, 1996). Therefore, in the present study, we sought to determine whether I1-imidazoline sites as well as a2-adrenergic receptors are localized to the carotid body and to assess a possible modulatory role of I1imidazoline receptors in the chemosensory response to hypoxia.
2. Methods
2.1. Tissue preparation In a group of nine cats anesthetized with pentobarbital (45 mg/kg) and ventilated with room air, 18 carotid bodies were dissected out and frozen in liquid nitrogen. Intact carotid bifurcations (four) were removed from another group of cats and frozen on dry ice. Carotid bodies were also removed from 20 rabbits. No adrenergic drugs were administered to the animals at any time. For purposes of comparison, in some experiments tissue from the bovine ventrolateral medulla oblongata was processed as previously described (Ernsberger et al., 1993), and assayed in parallel to the carotid body samples.
2.2. Membrane preparation Carotid bodies were thawed on ice, pooled, and incubated in 25 ml of 20 mM HEPES-Tris buffer (pH 7.4) containing 0.5 mg/ml collagenase (Sigma, type I) and the protease inhibitors EDTA (5.0 mM), 1,10-phenanthroline (0.1 mM) and phenylmethylsulfonyl fluoride (50 mM) in order to inhibit degradation of receptor protein. The digested and lysed tissue was cooled on ice and homogenized with a ground glass homogenizer. The homogenate was centrifuged at 1000× g for 5 min at 4°C to remove nuclei and debris. The pellets were resuspended in 20 ml of homogenization buffer using a polytron (Tecmar Tissuemizer, setting 60), and centrifuged again at 1000 × g for 5 min. The supernatants were combined and centrifuged at 40000× g for 20 min at 4°C, and the resulting P2 pellet was resuspended in 25 ml of 50 mM Tris–HCl buffer (pH 7.7) containing protease inhibitors. After centrifugation again at 40000× g for 20 min, the resulting membrane pellet was resuspended a final time in Tris–HCl alone, centrifuged, flash-frozen, and stored at − 70°C for up to 2 weeks.
2.3. [ 125I]p-iodoclonidine radioligand binding microassays The small mass of the carotid body demanded development of a sensitive binding assay. One
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method of increasing assay sensitivity is by using a radioligand with high specific activity, such as one bearing an 125I atom. We have recently characterized the binding properties of [125I]p-iodoclonidine, a high-affinity ligand for I1-imidazoline as well as a2-adrenergic binding sites (Kou et al., 1991; Ernsberger et al., 1993; Liedtke et al., 1993; Ernsberger et al., 1995c). Radioligand binding microassays with [125I]p-iodoclonidine for determination of specific binding to I1-imidazoline sites and a2-adrenergic receptors were performed by a modification of these methods. Membranes were slowly thawed and resuspended in Tris–HCl buffer at a concentration of 10 mg protein/ml. Assays were conducted in a total volume of 250 ml consisting of 125 ml membrane suspension, 25 ml radioligand, and 100 ml drug or vehicle. Incubations were initiated by the addition of membrane and were carried out for 40 min at 25°C. Non specific binding was defined in the presence of 10 mM phentolamine, an imidazoline adrenergic agent. Specific a2-adrenergic binding was defined by inhibition with (− )epinephrine (0.1 mM). In experiments using catecholamines, all samples contained ascorbic acid in a final concentration of 0.001%. Incubations were terminated by vacuum filtration using a cell harvester (Brandel), equipped with Teflon tubing to reduce absorption of the radioligand, over glass fiber filters (Schleicher and Schuell c 34) which were preincubated for 4 h at 4°C in 0.03% polylethylimine to reduce non specific binding to the filter. The filters were washed four times with 5 ml ice-cold Tris– HCl, placed in scintillation vials, covered with 4 ml scintillation cocktail (BioSafe II, Research Products International), and counted at 50% efficiency (Beckman LS5801). Protein was assayed by the bicinchoninic acid method (Smith et al., 1985).
2.4. Receptor autoradiography The carotid bifurcation was mounted on a cryostat chuck with mounting medium (M-1, Lipshaw) and sectioned on a Hacker cryostat ( − 18°C) at 8 mm thickness. Sections were collected by thaw-mounting on acid-cleaned, gelatin-subbed glass slides, dried in a vacuum
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desiccator for 1 h and stored at − 20°C for 24–48 h to insure adherence of the tissue to the slide before transfer to a − 70°C freezer for longer storage (up to 6 months). Adjacent sections were fixed in formalin without drying and stained with hematoxylin and eosin for histological reference. Autoradiography of I1-imidazoline and a2adrenergic receptor sites were conducted using [125I]p-iodoclonidine as the radioligand according to methods described previously (Ernsberger et al., 1995b; Haxhiu et al., 1995). Iso-osmotic buffers containing sucrose were used throughout in order to preserve the integrity of the tissue. Immediately prior to assay, slide-mounted sections were gradually warmed to room temperature in a vacuum desiccator and pre-incubated in Coplin jars in 20 mM Tris–HCl buffer (pH 7.4) containing 280 mM sucrose and 5.0 mM EDTA for 1 h and then washed in the same buffer without EDTA for 15 min to allow dissociation of any endogenous ligand bound to receptors within the tissue. Non-specific binding was determined in parallel incubations containing the imidazoline BDF-6143 (10 mM). a2-Adrenergic binding was defined in the presence of 10 mM epinephrine. To prevent uptake and degradation of epinephrine, the uptake blocker desipramine (10 mM), the monoamine oxidase inhibitor pargyline (10 mM), and the antioxidants ascorbic acid (10 mM) and dimethylsulfoxide (1% v/v) were included in the assay medium. I1-Imidazoline binding was defined in the presence of the selective inhibitor cimetidine (10 mM). After the incubation and wash periods, the slides were dipped in ice-cold distilled water to remove buffer salts which can cause chemography. Finally, sections were rapidly dried in a closed container by a stream of air passed through a column containing CaSO4 pellets and 5 ˚ molecular sieves and through a trap immersed A in a mixture of dry ice and ethanol, and then stored in a vacuum desiccator for 24–48 h at 4°C. The dried slides were arranged in X-ray cassettes and exposed to tritium-sensitive film (Hyperfilm, Amersham) for 96 h. The films were developed (D-19) for 4 min at 17°C, rinsed briefly, and fixed (Rapid Kodak Fixer) for 10 min. The sections
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were then fixed in formalin and stained with hematoxylin and eosin for histological reference.
given for 5 min and terminated by switching back to 100% O2.
2.5. In 6i6o studies
2.6. Data analysis
Seven adult cats (2 – 3 kg) were anesthetized with pentobarbital (45 mg/kg, i.p.) and femoral artery and vein cannulated for measuring arterial pressure and for intravenous administration of drugs. Additional doses of anesthetic (2 – 3 mg/kg i.v.) were given to maintain an absence of corneal and pain reflexes. Animals were tethered in a supine position and intubated caudal to the larynx. The carotid bifurcation was exposed by ligating and retracting the trachea and esophagus. Intracarotid infusion of drugs was performed in two cats through a catheter placed in the external carotid artery with its tip close to the carotid body. To maintain arterial blood gases constant, the anesthetized animals were paralyzed with gallamine (4 mg/kg, i.v.) and ventilated with a Harvard respirator. End-tidal CO2 was continuously monitored with an infrared CO2 analyzer (Beckman). Arterial blood samples were collected for measurements of PO2, PCO2 and pH in a blood gas analyzer (IL model 1303). An infusion of 8.4% NaHCO3 was given i.v. as necessary to maintain blood gases and pH. The body temperature of the animals was kept at 389 1°C by means of a servo-controlled heating blanket. Chemoreceptor activity was recorded as described in detail elsewhere (Prabhakar et al., 1989). Briefly, either the left or the right carotid sinus nerve was sectioned, desheathed, and successively split until activity from one to three fibers was obtained. Action potentials were amplified and displayed on an oscilloscope. The impulses were then converted to standard pulses (Winston Rad II), which were passed on to a rate meter (Frederick Haer). Chemoreceptor fibers were identified by their: (1) spontaneous sporadic discharge; (2) increase in activity when switched from room air to hypoxia (10% O2 in N2); and (3) decrease in activity when switched form room air to hyperoxia (100% O2). Isocapnic hypoxia was induced by switching the inspiratory port of the respirator from 100% O2 to a reservoir bag containing 10% O2 in N2. The hypoxic challenge was
Radioligand binding data were obtained as dpm, transformed to percent of total specific binding, and compared by analysis of variance. For in vivo experiments, arterial blood pressure (mmHg), chemosensory activity (impulses/sec) and arterial blood gasses were measured. Chemoreceptor activity was measured 1 min prior to the hypoxic challenge, and during the final min of each challenge. All data were presented as mean9 SEM. Statistical comparisons were made by analysis of variance (ANOVA) followed by comparison of group means with the Newman– Keuls test.
2.7. Materials [125I]p-iodoclonidine (2200 Ci/mmol) was obtained from New England Nuclear (Boston, MA), stored at − 70°C in ethanol and diluted in water prior to assay. Imidazole-4-acetic acid, epinephrine, phentolamine, clonidine and guanabenz were all purchased from Sigma (St. Louis, MO).
3. Results
3.1. Radioligand binding microassays in isolated membranes Specific [125I]p-iodoclonidine binding was determined in membranes from the cat carotid body. As shown in Fig. 1, [125I]p-iodoclonidine binding was only partially inhibited by a high concentration (100 mM) of the endogenous a2-agonist ( − )epinephrine (379 3% inhibition). A similar fraction of total specific [125I]p-iodoclonidine binding was blocked by the specific a2-antagonist SK&F 86466 (449 3% inhibition at 10 mM). In contrast, clonidine, which binds to both I1-imidazoline and a2-adrenergic sites, inhibited the total population of specific sites. These findings imply that only 40% of [125I]p-iodoclonidine binding
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sites in the cat carotid body are a2-adrenergic receptor sites. Imidazole-4-acetic acid, which binds to I1-imidazoline sites but not a2-adrenergic receptors (Ernsberger et al., 1987, 1990a,b), inhibited 7296% of the specific [125I]p-iodoclonidine binding sites at a concentration of 100 mM, consistent with the presence of specific I1-imidazoline binding. Significant inhibition of binding was also observed for cimetidine, a histamine H2 blocker with moderate affinity for I1-imidazoline sites (Ki :100 nM) but which does not bind to a2-receptors (Ernsberger et al., 1987, 1990a,b). A total of 1.5 fmol of specific binding sites per carotid body were labeled by 0.6 nM [125I]p-iodoclonidine, of which 0.9 fmol were insensitive to epinephrine and thus non-adrenergic. The remaining 0.6 fmol per carotid body had the characteristics of a2-adrenergic binding sites. Specific binding sites for [125I]p-iodoclonidine were also present in rabbit carotid body membranes (Fig. 2). In this species, specific [125I]piodoclonidine binding was 0.309 0.01 fmol per carotid body or 399 2 fmol per mg protein. About one-half of specific [125I]p-iodoclonidine binding could be inhibited by agents specific for a2-adrenergic receptors such as SK&F 86466 and
Fig. 1. Effects of a2-selective and I1-selective agents on [125I]piodoclonidine binding to cat carotid body membranes in a radioligand binding microassay. Inhibition of [125I]p-iodoclonidine (0.5 nM) binding by a maximal concentration of several competing drugs. Data are expressed as percent of total specific [125I]p-iodoclonidine binding as defined in the presence of 10 mM phentolamine. IAA, imidazole-4-acetic acid.
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Fig. 2. Inhibition of [125I]p-iodoclonidine binding to rabbit carotid body membranes by imidazolines and non-imidazolines. Inhibition of [125I]p-iodoclonidine (0.5 nM) binding by a maximal concentration of several competing drugs. Data are expressed as percent of total specific [125I]p-iodoclonidine binding as defined in the presence of 0.1 mM piperoxan. The non-imidazole a2-adrenergic antagonist SK&F 86466 (10 nM and 10 mM) dose-dependently inhibited slightly more than half of total specific [125I]p-iodoclonidine binding. The binding values are within the range of data obtained with full inhibition curves in brainstem membranes (Ernsberger et al., 1993). Guanabenz (0.1 mM), a non-imidazole a2-adrenergic agonist, also inhibited only slightly more than half of the specific [125I]p-iodoclonidine binding sites. Imidazole-4-acetic acid (IAA, 10 mM) inhibited a portion of specific binding. These results indicate that, as for cat carotid body, about half of the specific sites labeled by 0.5 nM [125I]p-iodoclonidine are a2adrenergic receptors while the other half are I1-imidazoline binding sites.
guanabenz (Fig. 2). Conversely, about one-half the binding sites recognized imidazole-4-acetic acid (0.1 mM). The affinity of guanabenz for carotid body a2-receptors was determined using three concentrations of guanabenz ranging from 1 to 100 nM in two separate experiments (Fig. 3). Guanabenz potently inhibited [125I]p-iodoclonidine binding in both experiments (IC50 values of 2.1 and 2.4 nM; pseudo-Hill slopes (nH) of 0.73 and 0.88). These affinity values for guanabenz at a2-adrenergic binding sites are in agreement with previous results obtained in the bovine ventrolateral medulla oblongata (79 1 nM; Ernsberger et al., 1990b), and are consistent with the potent inhibitory action of guanabenz on chemoreceptor activity previously reported by us (Kou et al., 1991).
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In order to determine whether the present findings were unique, identical experiments were carried out using bovine ventrolateral medulla as previously described (Ernsberger et al., 1993). Relative to cat or rabbit carotid body, epinephrine and SK&F 86466 inhibited a larger proportion of sites (689 4 and 5993%, respectively, n=4). Conversely, imidazole-4-acetic acid and cimetidine inhibited a smaller proportion of sites (349 8 and 3496%, respectively, n = 4). Clonidine itself, or the analog moxonidine, inhibited the total population of specific [125I]p-iodoclonidine sites. Thus, the profile of [125I]p-iodoclonidine binding was similar in the ventrolateral medulla, but the ratio of I1-imidazoline to a2-adrenergic receptors was 1:3 as compared to 1:1 for cat or rabbit carotid body. The quantity of carotid body tissue available from either cat or rabbit was insufficient to conduct saturation binding studies. Using the Kd values for [125I]p-iodoclonidine determine in the bovine medulla (Ernsberger et al., 1993), we estimate the Bmax values for I1-imidazoline sites in cat and rabbit carotid bodies to be identical at 169 5 and 1692 fmol/mg protein, respectively, whereas for a2-adrenergic receptors, the estimated Bmax values are 109 2 and 23 9 2 fmol/mg protein. The estimated density of a2-adrenergic receptors is significantly greater in rabbit compared with cat carotid body.
3.2. Autoradiographic 6isualization of I1 -imidazoline and a2 -adrenergic binding sites in the cat carotid bifurcation The distribution of [125I]p-iodoclonidine binding sites in the cat carotid bifurcation is shown in Figs. 4 and 5. There was a single intensely bright area, indicating a high density of binding sites. The bright area corresponds to the carotid body as identified by hematoxylin and eosin staining, as shown in Fig. 6, which is a bright enlargement of the boxed region in Fig. 4, panel T. The carotid artery itself expresses a lower level of binding, which was most prominent in the endothelium (Figs. 4 and 5). Most of the labeling was lost in the presence of BDF-6143, a nonselective imidazoline ligand, indicating that the labeling was specific (panels labeled NS for non-specific binding). Labeling of the carotid body by [125I]p-iodoclonidine was partially inhibited with the addition of cimetidine to block I1-imidazoline sites (panels labeled A for a2-adrenergic binding), whereas the faint labeling in the carotid artery was not affected by cimetidine. Partial inhibition of carotid body labeling was also observed in the presence of epinephrine (panels labeled I for imidazoline binding), indicating that a portion of the [125I]p-iodoclonidine binding sites are labeled in the carotid body are non-adrenergic and likely to be I1-imidazoline sites. I1-imidazoline sites appear to be almost entirely lacking from the carotid artery. These data indicate that both I1-imidazoline and a2-adrenergic sites are expressed within the cat carotid body.
3.3. Effect of selecti6e stimulation of I1 -imidazoline receptors on chemosensory discharge
Fig. 3. Dose-dependent inhibition of [125I]p-iodoclonidine binding by the a2-agonist guanabenz. Inhibition of [125I]piodoclonidine (0.5 nM) binding by increasing concentrations of guanabenz. Data are the mean of two experiments, each conducted in duplicate. Details as in Fig. 1.
The role of I1-imidazoline and a2-adrenergic receptors in control of chemosensory discharge was determined in anesthetized, paralyzed and artificially respirated cats. We used guanabenz and SK&F 86466 as a selective agonist and antagonist for a2-adrenergic receptors and clonidine as a mixed I1/a2 agonist. In the absence of a2-blockade, clonidine elicited variable effects, in some cases inhibiting chemosensory discharge (Fig. 7,
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Fig. 4. Autoradiographic visualization of [125I]p-iodoclonidine binding sites in the cat carotid bifurcation. Shown are photographic prints made under identical conditions using the autoradiograms as negatives. Light areas correspond to the highest intensity of labeling. Adjacent 8 mm sections through the cat carotid bifurcation were incubated with 0.5 nM [125I]p-iodoclonidine under one of four different conditions. The section labeled T (total binding) was incubated with antioxidant vehicle alone, and shows the total binding of [125I]p-iodoclonidine. Binding sites were concentrated within the carotid body (bright region). The white rectangle corresponds to the photomicrograph in Fig. 6. The carotid body region is overexposed in order to allow visualization of the carotid arteries. The adjacent section labeled NS (non specific) was incubated with 10 mM BDF-6143 to block both I1 and a2 binding, and thus shows the distribution of non specific sites. The section labeled A (a2) was incubated with 10 mM cimetidine to selectively mask I1-imidazoline sites, and thus shows the distribution of a2-adrenergic sites. Labeling in the carotid body was partially inhibited by 10 mM cimetidine. The section on the far right, labeled I, was incubated with 10 mM epinephrine to mask a2-receptors, and thus shows the distribution of I1 binding. Note the persistence of [125I]p-iodoclonidine labeling in the carotid body, indicating the presence of non-adrenergic binding sites.
top panel), in a manner similar to that previously reported for guanabenz (Kou et al., 1991). In other cases, slightly facilitatory responses were observed (Fig. 7, bottom panel). The transient pressor response to clonidine, mediated by vascular a2-adrenergic receptors, was consistently observed (Fig. 7). Thus, indirect vascular effects on chemosensory discharge cannot account for the variable reasons to clonidine. In order to block a2-adrenergic receptors, cats were pretreated with 0.5 mg/kg SK&F 86466 i.v. The completeness of a2-blockade was tested by intracarotid infusion of the selective a2-agonist guanabenz (5.0 mg/min). Prior to treatment with SK&F 86466, guanabenz attenuated the chemosensory response to hypoxia (control D = 12 9 3; post-guanabenz D =5 9 2 impulses/sec).
After administration of SK&F 86466, guanabenz had no effect on the chemosensory response to hypoxia (D= 1193 impulses/sec). Effective a2blockade is illustrated in Fig. 8. After pretreatment with SK&F 86466, systemic administration of guanabenz (100 mg/kg i.v.) no longer inhibited chemosensory discharge or increased arterial pressure. During a2-blockade with SK&F 86466, systemic administration of clonidine stimulated chemosensory discharge under hypoxia (Fig. 9). Similar results were obtained with close arterial injection into the carotid body (20 mg/kg), or with intravenous administration (100 mg/kg). An analysis of variance for the effects of clonidine treatment (treated vs. untreated) and of oxygen tension (hyperoxia vs. normoxia vs. hypoxia) showed signifi-
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Fig. 5. Auto radiograms from sections of the carotid body from a second cat. Details as in Fig. 5.
cant effects of clonidine (F (1,30) = 10.7, P B 0.003) and of oxygen (F (2,30) =44.5, PB 0.0001) as well as a significant interaction between clonidine treatment and oxygen (F (2,30) = 3.4, P B0.05). The latter finding indicates that clonidine’s facilitatory effect was greater under hypoxia than under normoxic or hyperoxic conditions.
4. Discussion This study demonstrates that I1-imidazoline sites as well as a2-adrenergic receptors are expressed within the carotid bodies of cats and rabbits. Direct labeling of binding sites in situ demonstrates both receptor types are present in high density throughout the carotid body and are expressed at much higher levels than in surrounding arterial or connective tissues. Functional studies of chemosensory discharge suggest that I1-imidazoline receptors may facilitate the response to hypoxia, whereas a2-adrenergic receptors inhibit chemosensory function. The latter finding confirms previous studies of the function of a2-adrenergic receptors in the cat carotid body (Kou et al., 1991). The precise nature of the
mechanism coupling I1-imidazoline receptor stimulation to facilitation of chemosensory responses cannot be ascertained from the present data. However, activation of I1-imidazoline receptors in pheochromocytoma cells elicits the release of prostaglandin E2 (Ernsberger et al., 1995b), and this mediator is also released by the carotid body in response to hypoxia (Go´mez-Nin˜o et al., 1994). Unknown at present are the roles played by cellular mediators in the response to stimulation of either I1-imidazoline or a2-adrenergic receptors within the carotid body. There are relatively few prior reports of radioligand binding studies of receptor sites in the carotid body. Muscarinic acetylcholine receptors have been identified by membrane binding and autoradiography (Dinger et al., 1991), endothelin receptors have been localized within the regions of the carotid body by autoradiography (Nichols and Ruffolo, 1991), dopamine D2 receptors have been detected by in situ hybridization to complementary RNA (Czyzyk-Krzeska et al., 1992), and we previously identified a2-adrenergic receptors (Kou et al., 1991). The primary obstacle to the study of neurotransmitter receptors in the carotid body is the small mass of the tissue (0.8 and 0.3
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mg in the cat and rabbit, respectively) relative to the requirements of standard radioligand binding assays (60 mg per triplicate data point). In the present assay, we reduced the assay volume (250 ml), and used an iodinated probe with high specific activity (2200 Ci/mmol) and high affinity for I1-imidazoline and a2-adrenergic binding sites. A limitation of the present study was that insufficient tissue was available to conduct saturation experiments in order to determine radioligand affinity and the absolute number of binding sites. However, if the assumption is made that the affinity of [125I]p-iodoclonidine does not vary significantly between tissues, which has already been documented (Ernsberger et al., 1995b),
Fig. 6. Photomicrograph of a carotid body section stained for histological reference. The section used to generate the autoradiogram of total binding for Fig. 4 was stained with hematoxylin and eosin. The area shown corresponds to the boxed area in Fig. 4.
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then the density of a2-adrenergic binding sites can be estimated using affinity constants obtained in the bovine brain (Section 3). The estimated density (Bmax) for a2-adrenergic receptors is 18 fmol per mg protein in cat carotid body and 76 fmol per mg protein in the rabbit. These predicted receptor densities fall within the range of values reported for tissues known to respond to a2adrenergic receptors agonists, including spinal cord, platelets, ileum, and the kidney (Bylund and U’Prichard, 1983), suggesting that the level of expression of a2-adrenergic receptors in the carotid body is compatible with physiological responses to receptor activation. Previous studies testing the function of putative I1-imidazoline receptors have focused on cardiovascular responses in the RVLM of the brainstem (Bousquet et al., 1984; Tibiric¸a et al., 1989; Ernsberger et al., 1990b; Haxhiu et al., 1992). Microinjection of I1-antagonists into the RVLM blocks the vasodepressor action of systemic imidazolines, whereas blockade of a2-adrenergic receptors with the specific antagonist SK&F 86466 has no effect (Gomez et al., 1991). However, because both I1imidazoline and a2-adrenergic receptors lower blood pressure when activated within the brainstem, precisely parceling out the effects mediated by the two receptors is difficult. In the present study, a2-adrenergic stimulation inhibited chemosensory discharge, but I1-imidazoline stimulation after blockade of a2-receptors facilitated chemosensory discharge. Thus, the separation of a2-adrenergic and I1-imidazoline receptor mediated effects is more distinct than in the brainstem, since the two receptors appear to mediate opposite effects. This interpretation is supported by the contrasts between guanabenz and clonidine, two a2-adrenergic agonists expressing nearly identical affinity and efficacy at a2-receptors (Bylund and U’Prichard, 1983). Without a2-blockade, guanabenz inhibited nearly two-thirds of the chemosensory response to hypoxia, confirming our previous results (Kou et al., 1991). Clonidine, in contrast, had inconsistent effects when given alone. After blockade of a2-adrenergic receptors with the specific antagonist SK&F 86466, guanabenz had no effect. In contrast, a2-blockade unmasked a stimulatory effect of clonidine. These
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Fig. 7. Inconsistent effects of clonidine on chemosensory discharge prior to a2-adrenergic blockade. Representative tracings show strongly inhibitory (top) and weakly excitatory (bottom) effects of clonidine (100 mg/kg i.v.) delivered at the arrow. CA, integrated chemoreceptor activity; AP, action potentials; ABP, arterial blood pressure; CO2, end-tidal PCO2. Pressor response to clonidine is an acute effect mediated by vascular a2-adrenergic receptors.
data demonstrate that clonidine acts on a receptor other than the a2-adrenergic receptor to facilitate chemosensory discharge. The present findings concerning the action of clonidine on chemoreceptor activity are paralleled by studies of clonidine’s effects on insulin secretion from pancreatic islets (Schulz and Hasselblatt, 1989).
Clonidine suppresses insulin release when administered alone, but after blockade of a2-adrenergic receptors, a stimulatory action is unmasked. These data support the hypothesis that I1-imidazoline binding sites are functional receptors and are functionally distinct from a2-adrenergic receptors.
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Fig. 8. Lack of inhibitory or pressor effects of guanabenz following a2-adrenergic blockade. Representative tracing showing the absence of any effect of guanabenz (100 mg/kg i.v.) on chemoreceptor activity (CA and AP) or on arterial pressure (AP) following blockade of a2-adrenergic receptors with SK&F 86466 (0.5 mg/kg i.v.). These data are consistent with effective blockade of a2-adrenergic responses.
The resolution of the receptor autoradiographic technique is not sufficient to determine which cell type(s) within the carotid body express I1-imidazoline and a2-adrenergic receptors. In the brain, I1-imidazoline sites are probably neuronal, because they are absent in primary cultures of glial astrocytes (Ernsberger et al., 1995b), whereas I2sites are primarily localized on glia (Regunathan et al., 1993). Because type II carotid body cells resemble glia, they most likely express I2-sites (Youngson et al., 1995), while the neuron-like type I cells might possibly express the I1-subtype. The binding characteristics of the a2-adrenergic receptors identified in cat and rabbit carotid bodies correlate closely with the results of physiological studies. The a2-adrenergic component of binding was potently blocked by the antagonist SK&F 86466, and this antagonist potently blocks the action of the a2-agonist guanabenz and elevates basal chemosensory discharge by blocking tonic catecholaminergic activity (Kou et al., 1991). The a2-agonist guanabenz inhibited [125I]piodoclonidine binding in a dose-dependent man-
ner with and high affinity (2 nM), and was also a potent inhibitor of chemosensory discharge in the cat (Kou et al., 1991). Although clonidine can interact with a1-adrenergic receptors at high doses (Bylund and U’Prichard 1983), it is unlikely that a1-receptors are responsible for this effect at the doses used. Furthermore, the a1-agonist phenylephrine increases chemosensory discharge only slightly (B 2 impulses/sec) and, unlike clonidine, does not selectively enhance the response to hypoxia (Kou et al., 1991). We conclude that I1-imidazoline sites are associated with peripheral chemoreceptors, and I1-imidazoline receptors may stimulate chemosensory discharge, while a2-receptors may elicit opposite effects.
Acknowledgements Supported by the National Institutes of Health (HL45780 and HL44514). We gratefully acknowledge the expert technical assistance of Laura A.
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Fig. 9. Effects of clonidine on chemosensory discharge following a2-adrenergic blockade. Chemoreceptor activity was recorded from a fine filament of the cut carotid sinus nerve in seven cats which had been pretreated with SK&F 86466 (0.5 mg/kg i.v.). This treatment completely blocked the effect of a2-adrenergic receptor stimulation with guanabenz administered by close intracarotid injection. Activity was recorded for 5 min during each of three conditions: hyperoxia (100% O2), normoxia (room air), and hypoxia (12% O2 in 88% N2). Clonidine was then administered i.v. (100 mg/kg) or by close intracarotid injection (20 mg/kg) and chemoreceptor activity recordings were repeated 5 min later under hyperoxia, normoxia and hypoxia. Data represent mean 9SE (n= 7) and were obtained as mean impulse frequency during the last minute of each condition. Because intravenous and intracarotid injections produced indistinguishable effects, the data were combined. Two-way analysis of variance demonstrated significant main effects of oxygen level and clonidine treatment and a significant interaction between oxygen level and clonidine treatment. Post-hoc Tukey tests showed that clonidine significantly enhanced chemosensory discharge during hypoxia (P B 0.05).
Collins for studies.
autoradiographic
and
histologic
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