Journal of Applied Physiology
Vol. 82, No. 6, pp. 1771-1775, June 1997
CONTROL OF BREATHING, CIRCULATION, AND TEMPERATURE

cAMP production in rabbit carotid body: role of adenosine

J. Chen, B. Dinger, and S. J. Fidone

Department of Physiology, University of Utah School of Medicine, Salt Lake City, Utah 84108

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Chen, J., B. Dinger, and S. J. Fidone. cAMP production in rabbit carotid body: role of adenosine. J. Appl. Physiol. 82(6): 1771-1775, 1997.In the present study, we have investigated the possible role of adenosine in the hypoxia-mediated increase in adenosine 3,5-cyclic monophosphate (cAMP) in the carotid body. cAMP levels in rabbit carotid bodies superfused in vitro for 10 min were increased in the presence of adenosine (100 µM and 1.0 mM; maximum increase = 127%, P < 0.01). These effects were reduced by the nonspecific adenosine-receptor antagonist 1,3-dipropyl-8[p-sulfophenyl]xanthine (DPSPX; 10 µM). The specific A₂-receptor agonist 2-[4(2-carboxymethyl)phenylethylamino]-5-N-ethylcarboxamido adenosine (CGS-21680; 100 nM) also elevated carotid body cAMP levels, an effect that was blocked by the specific A₂-antagonist 3,7-dimethyl-L-propargyl-xanthine (DMPX; 50 µM). Hypoxia-evoked elevations in cAMP were potentiated in the presence of the adenosine-uptake inhibitor dipyridamole (100 nM) and blocked by exposure to adenosine-receptor antagonists. Our data suggest that the rabbit carotid body contains specific adenosine receptors (A₂ subtype) that are positively coupled to adenylate cyclase and that increases in cAMP associated with hypoxia are mediated by the release of endogenous adenosine.

hypoxia; adenylate cyclase; chemoreception; chemotransduction

INTRODUCTION

THE CAROTID BODIES, small paired organs located at the bifurcation of the common carotid arteries, initiate reflex cardiopulmonary adjustments in response to hypoxia, hypercapnia, and low pH. The parenchyma of the carotid body consists of lobules of specialized type I (glomus) cells that are innervated by afferent fibers of the carotid sinus nerve (CSN), a branch of the glossopharyngeal (IXth) nerve. It is now well established that certain initial stimulus-transduction events occur within the type I chemoreceptor cells of the carotid body; however, a complete knowledge of the transduction cascade that culminates in the release of neurotransmitters and excitation of chemoafferent nerve fibers is lacking, and many steps in this process remain controversial (9).

In chemoreceptive olfactory neurons and gustatory cells, the levels of adenosine 3,5-cyclic monophosphate (cAMP) are elevated by adequate stimuli (2, 27, 33). These changes occur as a direct consequence of odorant or taste molecules, respectively, acting on G-protein-coupled receptors that activate adenylate cyclase (4, 14). Sensory transduction in olfactory neurons is mediated by intracellular cAMP, which directly gates a nonselective cation channel (33). In gustatory cells, cAMP initiates a protein phosphorylation step, which, similarly, leads to altered membrane conductance (2, 27). In these chemical senses, therefore, the generation of cAMP is an immediate step in the primary sensory transduction cascade. A possible role for cAMP in the initial stimulus transduction events in the carotid body has been suggested by a number of studies, including our own, which demonstrated elevated cAMP levels after brief hypoxic exposures in vitro (7, 22, 29, 30). Based on these data, it has been widely assumed that, as is the case in olfactory and gustatory cells, adenylate cyclase is activated as a direct consequence of the primary stimulus, i.e., hypoxia.

Several studies investigated whether elevated cAMP production in type I cells might also be triggered by neurotransmitter action on type I cell autoreceptors. It was found that in the absence of Ca²⁺, the hypoxia-induced increase in cAMP persisted, although it was altered in magnitude (7, 22, 29), which suggested that the activity of adenylate cyclase within the stimulus transduction cascade could be modulated through feedback control by released neurotransmitters. Among the neurotransmitters liberated from type I cells during hypoxia are the catecholamines, dopamine and norepinephrine, and there is evidence that these agents activate adenylate cyclase and increase cAMP levels in the carotid body (22).

Adenosine, another agent known to increase the concentration of cAMP in a variety of tissues (24, 28), has been shown to elevate both CSN activity and ventilation (15, 16, 18, 23, 31). Moreover, it has been found that ventilation is facilitated in rats receiving intracarotid doses of inhibitors of adenosine degradation and uptake (20) and that these effects are abolished by CSN section or after administration of adenosine-receptor antagonists (20). Because adenosine is released in the heart, brain, and other tissues during ischemia/hypoxia (3, 8), the present study investigated the possibility that adenosine, perhaps also released in the carotid body, is in part responsible for increased carotid body cAMP levels associated with hypoxia. Interestingly, the data suggest that the action of adenosine may fully account for this hypoxia-induced increase in cAMP, a result that calls into question the notion that cAMP generation is an immediate step in the primary chemotransduction cascade in type I cells. A preliminary report of some of these findings has been published in abstract form (5).

MATERIALS AND METHODS

Under pentobarbital sodium (35 mg/kg ip) anesthesia, the carotid bifurcations along with the carotid bodies were rapidly removed from adult New Zealand White rabbits. With the aid of a dissecting microscope, the carotid bodies were cleaned of surrounding connective tissue in a lucite chamber containing 100% O₂-equilibrated modified Tyrode solution at 0-4°C (in mM: NaCl 112, KCl 4.7, CaCl₂ 2.2, MgCl₂ 1.1, Na-glutamate 42, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer 5, glucose 5.6, pH = 7.4). The tissues were weighed on a Cahn electrobalance equipped with a humidified weighing chamber (mean weight of rabbit carotid body = 388 µg) and then transferred to glass scintillation vials in a water bath shaker for a 30-min preincubation in 1 ml of 100% O₂ Tyrode solution at 37°C. Experimental procedures involved incubation in 5% or 100% O₂-equilibrated solutions with or without selected drugs. After incubation, carotid bodies were immersed in 600 µl of cold (4°C) 6% trichloroacetic acid. The tissues were homogenized in a glass-glass homogenizer, and the homogenates were centrifuged at 13,000 g for 10 min (4°C). The supernatant was extracted three times in 3 ml of water-saturated ethyl ether, the remaining aqueous phase was dried under vacuum (Savant), and the sealed samples were stored at 4°C for up to 1 mo before radioimmunoassay (RIA). The assays were performed by using commercially available cAMP RIA kits (DuPont NEN), and the results are expressed as picomoles cAMP per milligram tissue. Data were evaluated with analysis of variance with post hoc analysis of Bonferroni's P values (Instat: GraphPad, San Diego, CA). Statistical comparisons were limited to data obtained from a single assay because basal cAMP values varied somewhat between assays due to slight differences in RIA standard curves. Drugs were obtained from Research Biochemical International, Natick, MA.

RESULTS

Our experiments showed that 10-min exposures to 10 µM adenosine did not alter basal cAMP levels but that 100 µM and 1 mM adenosine each evoked significant elevations in cyclic nucleotide content (Fig. 1). Activation of adenylate cyclase by high micromolar concentrations of adenosine is consistent with the presence of an A₂-receptor subtype, in accord with the interpretation of van Calker et al. (28) from studies of A₁- and A₂-receptor subtypes in cultured glial cells. The data in Fig. 2 show that the cAMP response to 1 mM adenosine is reduced in the presence of the nonspecific adenosine-receptor antagonist 1,3-dipropyl-8[p-sulfophenyl]xanthine (DPSPX; 10 µM), suggesting the existence of adenylate cyclase-coupled adenosine receptors in the rabbit carotid body. In other experiments shown in Fig. 3, incubation (10 min) in 100 nM of the selective high-affinity A₂-receptor agonist 2-[4-(2-carboxymethyl)phenethylamino]-5N-ethylcarboxamido adenosine (CGS-21680; Ref. 13), resulted in elevated carotid body cAMP content, and this response was blocked by the selective A₂-receptor antagonist 3,7-dimethyl-L-propargyl-xanthine (DMPX; 50 µM).

The possibility that release and reuptake of endogenous adenosine are involved in the production of cAMP in the carotid body was explored with the use of the adenosine-uptake blocker dipyridamole (100 nM). We found that this drug elevated the cAMP content of carotid bodies superfused (10 min) in media equilibrated with 100% O₂ and that this increased level of cAMP was similar to that obtained after 10 min of incubation in media equilibrated with 5% O₂ without the drug (Fig. 4). Furthermore, the addition of dipyridamole to the low-O₂ media further potentiated the cAMP response to hypoxia.

The role of endogenous adenosine was further explored by examining the effects of adenosine-receptor blockers on the low-O₂-induced increase in cAMP. These experiments utilized DPSPX (nonspecific, but slightly more selective for A₁ receptors; Ref. 6), DMPX (A₂ selective; Ref. 26), and aminophylline, a water-soluble theophylline derivative that is also nonselective for A₁ vs. A₂ adenosine receptors. Figure 5 shows the effects of these antagonists on the cAMP content of carotid bodies incubated in either 100% O₂ or 5% O₂-equilibrated solutions. In each experiment, carotid bodies were first preincubated with the selected blocker for 10 min in 100% O₂ media before final incubation (10 min) in either 100% O₂ or 5% O₂ media. The data show that, in contrast with the effects of dipyridamole (Fig. 4), exposure to the receptor antagonists did not significantly alter basal levels of cAMP (100% O₂ media). In each experiment (Fig. 5, A-C), hypoxia evoked a two- to threefold increase in cAMP content, and this effect was blocked in the presence of the receptor antagonist (i.e., no significant difference between cAMP content in control vs. hypoxic carotid bodies incubated with the antagonists). Increases in carotid body cAMP content evoked by hypoxia are thus fully blocked in the presence of adenosine-receptor antagonists.

DISCUSSION

Previous studies in numerous other tissues have revealed two types of adenosine receptors that are either positively (A₂) or negatively (A₁) coupled, respectively, to adenylate cyclase (28, 32). These receptors can be distinguished based on the relative potencies of various adenosine-receptor agonists (28, 32). Pharmacological studies of the effects of adenosine analogs on CSN activity and ventilation have suggested the presence of A₂ receptors in the carotid body (16, 18). However, other investigators have reported that adenosine had little effect on the cAMP content in this tissue (17). Our results indicate that the application of adenosine to the carotid body activates adenylate cyclase and increases cAMP content in this chemosensory tissue. Similarly, low concentrations of the high-affinity A₂-receptor agonist CGS-21680 elevate carotid body cAMP levels. These effects are blocked by low concentrations of adenosine-receptor antagonists, including the highly specific A₂ antagonist DMPX. These findings are consistent with previous pharmacological studies of adenosine-mediated increases in ventilation and CSN discharge, which implicated the involvement of A₂ receptors in the carotid body (16, 18). More recently, it has been reported that adenosine agonists, including CGS-21680, elevate cAMP levels in the rat carotid body (21). A role for endogenous adenosine in chemoexcitation has also been suggested by experiments that demonstrated that inhibitors of adenosine transport and degradation increase ventilation and potentiate the effects of hypoxia (20). Changes in ventilation evoked by these agents are absent after bilateral transection of the CSN, clearly establishing the involvement of the peripheral chemoreceptors of the carotid body (20).

In the present experiments, the adenosine-uptake inhibitor dipyridamole elevated basal cAMP levels in the carotid body and potentiated the effects of hypoxia-induced increases in cyclic nucleotide content. Although these data are consistent with the involvement of endogenous adenosine in the increased cAMP response, it should be noted that the absolute magnitude of the dipyridamole effect was nearly the same in both 100% and 5% O₂-equilibrated solutions, and, thus, these results do not reveal whether low O₂ elevates the release of endogenous adenosine in the carotid body. However, the observation that adenosine-receptor blockers, including the selective A₂ antagonist DMPX, fully inhibit the low-O₂-induced cAMP response supports a possible role for endogenous adenosine in the organ. These findings suggest that adenosine (and/or its precursor ATP) is released in the hypoxic carotid body and that adenosine action on receptors coupled to adenylate cyclase contributes significantly to increased generation of cAMP in this chemosensory tissue. Adenosine-receptor blockers did not, however, alter basal levels of cAMP, suggesting that, in the presence of ample O₂, extracellular adenosine levels are too low to activate adenylate cyclase, perhaps because of the presence of a highly efficient uptake mechanism.

Although our data strongly favor a role for adenosine, they cannot unequivocally rule out the possible interaction of other endogenous neurotransmitter agents such as dopamine and norepinephrine, which have been shown in pharmacological experiments to elevate cAMP levels in chemosensory tissue (22). The cAMP response to noradrenergic drugs is likely mediated by -adrenergic receptors, and the relative roles of type I cells vs. other tissue elements (e.g., vascular smooth muscle) in this response is not known (see Ref. 9). Other evidence suggests that dopaminergic D₁ receptors coupled to adenylate cyclase are located apart from lobules of type I cells, possibly in the extensive carotid body vasculature (1). Putative D₂-receptor subtypes have been localized to type I cells (see Ref. 9), where they may modulate cAMP responses. In other tissues, D₂ receptors are negatively coupled to adenylate cyclase and, although they apparently inhibit catecholamine release from type I cells (10), their function with respect to cAMP generation has not been rigorously studied in the carotid body. Finally, it is possible that the agents used in the present study may have effects in addition to those known to occur via adenosine receptors, although the utilization of low drug concentrations and multiple drugs with analogous results suggests minimal involvement of nonadenosine receptor sites.

It is necessary to consider these results and our interpretation in respect to previously published data regarding the role of cAMP in the chemoresponse to hypoxia. Three different laboratories, including our own, have reported that in zero Ca²⁺ superfusion solutions, the cAMP response to hypoxia remains intact (7, 22, 29). This finding suggests that the hypoxia-induced activation of adenylate cyclase occurs independently of increased neurotransmitter secretion from type I cells during low-O₂ exposure. A role for adenosine in the absence of extracellular Ca²⁺ is nonetheless possible, because studies in brain tissue have revealed a pool of adenosine whose release is Ca²⁺ independent (11, 12, 25). Furthermore, the absence of Ca²⁺ may augment the release of adenosine in brain slices (25), and we have shown that in the absence of extracellular Ca²⁺ cAMP levels in the carotid body are elevated even under normoxic conditions (29).

In conclusion, our present data indicate that exogenous adenosine and related agonists elevate cAMP in the carotid body via an A₂ adenosine receptor that is positively coupled to adenylate cyclase. Moreover, it appears that hypoxia increases the release of endogenous adenosine in the carotid body, which can then lead to elevation of cAMP levels, as has been shown to occur in type I cells after exposure to low O₂ (30). Our findings are in accord with other studies that also implicate endogenous adenosine in this response (19, 20) and they provide further support for the notion that cAMP enhances hypoxia-evoked chemoexcitation. Finally, our findings do not support the notion that cAMP generation induced by hypoxia is independent of the A₂ receptor-mediated effects of endogenous adenosine.

ACKNOWLEDGEMENTS

This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-07938 and NS-12636.

FOOTNOTES

Address for reprint requests: S. J. Fidone, Dept. of Physiology, Univ. of Utah School of Medicine, 410 Chipeta Way, Research Park, Salt Lake City, UT 84108.

Received 30 October 1996; accepted in final form 7 February 1997.

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