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The carotid body (CB) is a polymodal chemosensor of arterial blood located next to the internal carotid artery. The basic chemosensing unit is composed of the neurotransmitter (NT)-containing glomus cells (GCs) and the sensory afferent fibers synapsing onto the GCs. Nicotinic and muscarinic receptors have been found on both the sensory afferent fibers and on the GCs. Neural output from the CB (CBNO) increases when arterial blood perfusing it is hypoxic, hypoglycemic, hypercapnic, or acidic. The increased CBNO due to GC release of excitatory NTs must be preceded by an entrance of calcium into the GCs. With repeated release of ACh from the GCs, cholinergic receptors could become desensitized, particularly nicotinic receptors which function as calcium channels. The purpose of the present study was to see if adenosine (ADO), known to alter receptor sensitivities, could attenuate or eliminate any desensitization of the nicotinic receptors occurring during the repeated application of ACh. Cat CBs were harvested with techniques approved by the University's Animal Care/Use Committee. The GCs were cultured and prepared for detecting [Ca++]i with standard techniques. Repeated application of ACh produced a progressively decreasing increase in [Ca++]i. With the use of ADO or an A2a ADO receptor agonist the decrease was avoided. Though ADO also increased GC [Ca++]i, the sum of ADO increase and ACh increase, when superfused separately, was less than the increase when they were both included in the same superfusion. This suggested the possible involvement of a new path in the action. Potential mechanisms to explain the phenomena are discussed.
Obstructive Sleep Apnea produces repeated bouts of hypoxia intermittently. Hypoxia provokes the release of acetylcholine (ACh) within the carotid body (CB). ACh generates increased neural activity in the abutting sensory afferent nerves in several species, but also acts back on the GC from which it is released. In a previous conference we reported that ACh increased the intracellular calcium in the glomus cells (GCs) of the cat CB. In the present study we wished to see if GCs exposed to intermittent ACh challenges gave a consistent increase in the intracellular calcium ([Ca++]i). But there was concern that the cholinergic receptors on the GCs might become desensitized (Giniatullin, et al., 2005). It has been reported that adenosine (ADO) increased CB neural output (Monteiro, Ribeiro, 1987). That would indicate that ADO might act directly on the sensory afferent nerves, or facilitate the release of other excitatory neurotransmitters, or decrease the release of attenuating/inhibiting neurotransmitters (Fitzgerald, et al., 2004). We had previously reported that ADO did augment the hypoxia-induced increased release of ACh and reduced the hypoxia-induced increased release of catecholamines (dopamine [DA] and norepinephrine [NE]; Fitzgerald, et al., 2004). The former transmitter is generally accepted as excitatory in the cat and rat, while the latter transmitters are generally reported to be attenuating or inhibitory in cat and rat. Further, it has been recently reported that ADO increases intracellular calcium in the GCs of the rat (Xu, et al., 2006.).
The present study focuses on the interacting of ACh and ADO in the control of [Ca++]i in the GCs of the cat CB. Several questions were addressed: (1) Did repeated ACh challenges consistently increase GC intracellular calcium ([Ca++]i)? (2) What is the impact of ADO on the response to repeated ACh challenges? (3) What ADO receptors are involved? (4)What effect does ADO alone have on the [Ca++]i of the cat GCs? (5) Since both ACh and ADO will likely elevate [Ca++]i in the cat GCs, is their effect additive or more than additive? (6) Does ADO's effect have a cholinergic component?
The “A” component of the following figures is intended to provide the reader with a clearer picture of precisely when and how the agents were applied to the preparation. Component “B” presents the total [Ca++]i signal to the challenge maneuver while component “C” presents the increase above a common baseline.
In summary and focusing on the questions presented in the Introduction: (1) Intermittent brief superfusions of GCs with 100 μM ACh produced an increase in [Ca++]i which deteriorated on each subsequent superfusion. The increase was not consistent in spite of an intervening 13.5 min of superfusion with Krebs solution. (2) Including 100 μM ADO with the 100μM ACh in the second superfusion abolished the decrease in the [Ca++]i signal. The response was indistinguishable from the first perfusion. If the ADO was included in the third ACh superfusion, the [Ca++]i signal was again returned to the size of the first ACh superfusion. (3) Because the A2A ADO receptor agonist, CGS 21680, had the same effect as ADO and blocking any A1 ADO receptor effect with DPCPX also produced the same effect as ADO, it seems reasonable to conclude that it is the A2A ADO receptors on the GCs which are mediating the effect of ADO in restoring the [Ca++]i signal strength to that of the original ACh superfusion. (4) The effect of three brief superfusions of 100 μM ADO on the [Ca++]i signal was quite the opposite of that generated by the three brief superfusions of 100 μM ACh; the second superfusion showed a small decrease in the signal (borderline significant; P=0.046) while the third superfusion significantly elevated the [Ca++]i signal. (5) The Sum of the increase in the [Ca++]i signal generated by ACh plus that generated by ADO was significantly less than the signal when the superfusion fluid contained both ACh and ADO. (6) This suggested the possibility that ADO might have an influence beyond that which was the result of ADO acting on the A2A receptor.
Ascribing with certainty a detailed, step-by-step mechanism responsible for the phenomena presented above is difficult since ADO receptors interact with so many other neurotransmitter systems potentially capable of elevating [Ca2+] i. For example, A2A ADO receptor interacts with other receptors (Ribeiro, 1999; Sebastiao, Ribeiro, 2000), including cholinergic nicotinic (Correia-de-Sa, Ribeiro, 1994) and muscarinic receptors (Oliveira, et al., 2002).
Curiously, ADO given with ACh in the second or third superfusion elevated the GCs' [Ca++]i signal back to the same level as the first ACh superfusion. The signal is statistically indistinguishable from the signal to the first ACh superfusion, neither more nor less. Since Giniatullin and his colleagues (Giniatullin, et al., 2005) had described α4β2 subunit- and α3β4 subunit-containing nicotinic receptors as being moderately or very prone to desensitization, and the cat CB GCs contains α3, α4, β2, and β4 subunits (Hirasawa, et al., 2002), we initially hypothesized that the effect of ADO was to attenuate or eliminate desensitization of the nicotinic receptors. The proposed mechanism for counteracting desensitization is based on a rise in [Ca++]i, followed by an activation of PKC and/or PKA which promotes recovery of the receptor (Giniatullin, et al., 2005). Some of our data was consistent with the possibility that the ADO was preventing any desensitization of the nicotinic receptors; ADO was simply restoring the GC to the initial status
On the other hand, hexamethonium would have taken the nAChRs out of the picture entirely. And ADO had the same impact on the [Ca++]i signal in the presence of hexamethonium as in the absence of hexamethonium, the drop in the signal post ADO. It would seem unlikely, then, that nAChR desensitization is involved since in this case they were blocked from the start. So ADO must be working on another system/pathway. However, ADO raised the GCs' [Ca++]i, and atropine significantly reduced the impact of ADO acting alone on the [Ca++]i signal. This suggested the possibility that ADO may exercise its cholinergic effect via a muscarinic pathway.
Kobayashi et al. (2000) using isolated GCs from the rat CB in whole cell voltage-clamp studies found that ADO inhibited the voltage-dependent Ca++ currents; the phenomenon was abolished by a selective A2A receptor antagonist. They also.showed that exogenously applied ADO significantly attenuated the hypoxia-induced increase in [Ca++]i.
However, Xu et al. (2006) found in the GCs of the rat that ADO, acting via the A2A ADO receptors, coupled to adenylate cyclase and the PKA pathway, reduced the background TASK-like K+ current to trigger depolarization and [Ca++]i rise. Perhaps this same system was functioning in the cat GCs.
But one could then ask why does atropine have the effect that it does on the ADO-generated [Ca++]i signal? If what happens in the rat GC also happens in the cat, then one could say that ADO acting on the A2A receptor depolarizes the GC, extracellular Ca++ enters, provoking the release of ACh which acts back on the GCs' M1 autoreceptors to release more ACh which acts on nicotinic autoreceptors. This enhances/prolongs the depolarization. More extracellular Ca++ enters. In the presence of atropine this second step is blocked.
Further, a process like this might also explain why ADO with ACh generates a larger [Ca++]i signal than the ADO signal summed with the ACh signal. The ADO-generated increase in [Ca++]i provokes the release of GC-contained ACh which, acting on GC M1 autoreceptors initiates a second influx of extracellular Ca++. Such a presynaptic influence by ADO on ACh release is consistent with the modulation by ADO of both muscarinic M1-facilitated and M2-inhibition of ACh release from rat phrenic nerve terminals (Oliveira et al., 2002).
Vandier and colleagues (Vandier, et al.,1999) suggest the possibility of a different mechanistic cascade altogether. They report that ADO inhibits 4-AP-sensitive K+ current in isolated type I cells of the rat CB. They observed that depolarization of Type I cells produced an outward current that was significantly decreased by 10 μM ADO. Zero calcium also decreased outward current, and zero calcium plus ADO reduced it even further. 4-AP abolished the effect of ADO. They thought it was the A2A ADO receptor which was involved. This receptor is widely accepted as being coupled to GS protein which stimulates adenylyl cyclase activity. Elevations in cAMP levels have been shown to decrease 4-AP-sensitive outward current. Hence ADO in the present study, acting on the A2A ADO receptor, may be acting to decrease outward flow of K+ through the channels, and promoting depolarization and an increase in [Ca 2+] i
A definitive mechanistic schema to explain the above results seems to require further experimentation, perhaps with different concentrations of ADO. To our knowledge, however, this report presents for the first time the negative impact of a muscarinic antagonist on the impact of ADO on the [Ca++]i signal in the rat or cat GCs.
Pursuing the interelationships among the transmitters and modulators in response to brief intermittent exposures to hypoxia or hypoxia plus hypercapnia might reveal some therapeutic strategies helpful in managing Obstructive Sleep Apnea, a condition in which apneic episodes can occur as frequently as 10-15 times per hour. And insofar as the CB is the sentinel arousing the OSA subject to capture a breath due to the hypoxic/hypercapnic-induced release of ACh (and ATP), with subsequent sensory nerve excitation, it is important to know that the presence of ADO from ATP breakdown or otherwise prevents the deterioration of ACh's effect on the GCs' [Ca++]i signal, an effect that may have a muscarinic component. The autocrine/paracrine action of the slower excitatory activity of serotonin release on the GCs would also be affected, as would be the modulating actions of GABA on the GCs.
The purpose-bred cats were purchased from Liberty Laboratory, and used for tissue harvesting as described below. All animal protocols were approved by the Animal Care and Use Committee of The Johns Hopkins University.
CBs were harvested from 3-4 month-old cats of either sex and GCs cultured. The method has been described previously (Shirahata, et al., 1994). In brief, each cat was anesthetized with ketamine (50mg/kg), and an intravenous injection of heparin (40mg) followed. The cat was then sacrificed with pentobarbital sodium (~100mg/kg); the head was rapidly removed and the carotid arteries flushed with ice-cold L-15 media (Sigma). The CBs were isolated, cleaned of connective tissue, and washed with ice-cold L-15 media. A 45min incubation of the CBs in 0.2% collagenase (Sigma-Aldrich; a-Aldrich, P.O. Box 932594, Atlanta, GA 31193-2594; sigma-aldrich.com/order)) followed. This was followed by, and then replaced with, cell culture medium containing 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with bovine serum albumin, bovine transferring, bovine insulin, sodium selenite, 7s-nerve growth factor, and pyruvate (Sigma-Aldrich). The CBs were incubated for 1hr and gently triturated using 20-30G needles. The cells were divided into 7 wells which consisted of plastic cylinders (5mm in diameter) set on round cover slips (25mm in diameter). The cells were cultured in medium and kept in incubators (21% O2/5% CO2) at 37°C for more than 48hrs or until used in experiments.
Cells cultured on a glass coverslip were incubated with Fura-w/AM (Molecular Probes; 2μM) for 90 min. The coverslip with cells was then moved to the recording chamber on an inverted microscope and washed for 5-10 min by superfusing Krebs solution at a flow of 1.5ml/minute. The composition of Krebs solution was (in mM): NaC1, 118; KC1, 4.7; CaCl2, 2.2; MgSO4-7H2O, 1.2; KH2PO4,1.2; NaHCO3, 25; EDTA, 0.0016; and glucose, 11.1; pH was 7.4 equilibrated with 5% CO2/air at 37°C. Every 2.5 sec images were collected through a 510 nm interference filter with a cooled CCD camera (Photometrics Tucson, AZ) during alternate excitations at 340 nm and 380 nm. A PC based computer and ImageMaster software (Photon Technology International Inc. Birmingham, NJ) were used for the acquisition of the data. Several cells or clusters in one frame were selected. After subtracting background (taken from the area without cells) data for each cell or cluster of cells was analyzed by averaging the fluorescent ratio values in all pixels within the selected field. Relative changes from the control value (defined as “1”) were used for describing [Ca2+]i changes.
During the 30 second control period preceding each challenge there were 12 such measurements; all were virtually identical with the initial ratio, 1, except when hexamethonium was used(cf. Fig. 1A). However, when the KRB containing 100 μM ACh was started at the 30 sec mark, there was, after the initial wash-out, a rapid rise in the ratio. The peak ratio was attained in approximately 2 min (the ACh-containing KRB perfusion was terminated at the 90 sec mark). Perfusion of ACh-free KRB started again and lasted for 3.5 min during which period the ratio values decreased toward the control level. The same ACh-free KRB perfusion was provided for the subsequent 10 min rest period and the ratio values hovered around the “1” value. Recording RLU values was sometimes extended. But provision of fluids followed a constant format. Immediately at the 15 min mark the process of control, ACh challenge, recovery was repeated. This was followed by a third ACh challenge. The experiments in which there were three successive 100 μM ACh challenges constituted the first group (n = 27). In a second group (n = 35) the initial 100 μM ACh challenge was followed by a second superfusion in which 100 μM ADO was included with the 100 μM ACh. In a third group (n = 20) the ADO was included in the third ACh challenge rather than the second ACh challenge. In a fourth group (n = 33) the standard 100 μM ACh challenge was followed by a second challenge in which the superfusion contained, in addition to the 100 μM ACh and 100 μM ADO, 10 μM of 1, 3 dipropyl-8-cyclopebntylxanthine (DPCPX), the ADO A1 receptor blocker. In a fifth group (n = 21) the second 100 μM ACh challenge contained, instead of ADO, 1 nM of 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamido-adenosine hydrochloride (CGS 21680; Sigma-Aldrich), the ADO A2A receptor agonist. In a sixth group (n = 17) there were three successive 100 μM ADO challenges. In a seventh group (n = 22) an ADO challenge was followed by an ACh challenge and lastly by a challenge superfusion fluid containing both ADO and ACh. In an eighth group (n = 31) the impact of ADO on the GCs being perfused with ACh + hexamethonium. And lastly in a ninth group (n = 12) the effect of atropine on three superfusions of ADO was studied.
Data was recorded for 5 or more min. In each of the three ACh-challenge sequences in the five groups of experiments data was recorded for 5 min. Figures 1A, ,2A,2A, ,3A,3A, ,4A,4A, and and5A5A are recordings illustrating the emission intensity ratios over the course of the five minutes before, during, and after the ACh-containing superfusions. Peak ratios (maximum intensity in relative light units [RLU]) were selected from each of the three superfusions in each of the five groups. The mean values for the several experiments in each group were calculated, and are displayed as bar graphs in Figures 1B, ,2B,2B, ,3B,3B, ,4B,4B, and and5B5B (Mean [±SEM]). Since three or more measurements were made from the same cell or cluster of cells, the first test used to detect any differences was a One-Way Repeated Measures Analysis of Variance. This was followed by a Tukey Test. Subsequently a paired t-Test instrument was used when comparing the 1st superfusion with the 2nd or 3rd, or the 2nd with 3rd, etc.
Figures 1C, ,2C,2C, ,3C,3C, ,4C,4C, ,5C5C show the increases in GC[Ca2+]i from the baseline of 1. They were included to visualize the differences more easily. Increases among the three ACh challenges were again determined with a One-Way Repeated Measures Analysis of Variance. If a significant difference was found, and All Pairwise Multiple Comparison Procedure (Tukey Test) was used to locate the difference(s). The paired t-Test procedure followed. The tools were provided by the Sigma Stat Package (Jandel Scientific Software, P.O. Box 7005, San Rafael, CA 94912-7005).
This same formula was followed for the data presented in Figures 7 (three superfusions with 100 μM ADO), 8 (for ACh, ADO, and ACh+ADO), 9 (the effect of hexamethonium), and 10 (the effect of atropine on the response to ADO). In Figures 9B (9C) the maximum signal (increase) was first normalized to the preceding pre-stimulus control when it was determined that these four control values were significantly different from each other, and not 1 as in the previous superfusions. The metanalysis presented in Figure 6 is explained in the text Results.
The carotid bodies were harvested as described above and immersed in zinc fixative, embedded in paraffin, sectioned at 4-5 μm thickness, and mounted on poly-L-lysine-coated slides. After deparaffinization, the sections were treated in boiling 10 mM citric acid buffer (pH 8.0) for 5 min to retrieve antigens (Shi, et al., 1997). The endogeneous peroxidase was quenched with 1% H2O2 in phosphate buffered saline (PBS). Endogenous biotin was blocked (avidin biotin-blocking kit: Vector Laboratories, CA.), and other nonspecific binding was blocked with normal goat serum (1:75) and casein (CAS block, Zymed Laboratories, CA). A polyclonal antibody against the A2A ADO receptor (Chemicon International, Temecula, CA 92590; dilution 1:100; made in the rabbit) was prepared in PBS containing 8% cat serum and 2% goat serum. These solutions were kept at 4° C for 24 hours before applying to the tissue sections. The tissues were incubated with either the treated anti-A2A ADO receptor antibody or the negative control solution overnight at room temperature followed by an application of biotinylated anti-rabbit IgG made in the goat (1:2,000) for 1 hr at room temperature. Subsequently, standard avidin-biotin peroxidase techniques were applied. As a chromogen, Vector SG (Vector) was used. Between each step, the slides were washed in 0.1M PBS.
The authors gratefully acknowledge support for this study from the National Institutes of Health, National Heart Lung Blood Institute, specifically awards HL 50712 and HL 61596.
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