Evidence for two types of nicotinic receptors in the cat carotid body chemoreceptor cells

Ž. Brain Research 754 1997 298–302 Short communication Evidence for two types of nicotinic receptors in the cat carotid body chemoreceptor cells a´aab b Ana Obeso , Maria Angeles Gomez-Nino , Laura Almaraz , Bruce Dinger , Salvatore Fidone , ´˜ Constancio Gonzalez a,) ´ aDepartamento de Bioquımica y Biologıa Molecular y Fisiologıa, Facultad de Medicina, UniÕersidad de Valladolid, 47005 Valladolid, Spain ´´ ´ b Department of Physiology, UniÕersity of Utah School of Medicine, Salt Lake City, UT, USA Accepted 4 February 1997 Abstract Current concepts on the location and functional significance of nicotinic receptors in the carotid body rest on a -bungarotoxin binding and autoradiographic studies. Using an in vitro preparation of the cat carotid body whose catecholamine deposits have been labeled by w 3 xw 3 x prior incubation with the tritiated natural precursor H tyrosine, we have found that nicotine induces release of H catecholamines in a Ž. Ž. dose-dependent manner IC s9.81 m M . We also found that mecamylamine 50 m M completely abolished the nicotine-induced 50 Ž. release, while a -bungarotoxin 100 nM; f20 times its binding Konly reduced the release by 56%. These findings indicate that d chemoreceptor cells, and perhaps other carotid body structures, contain nicotinic receptors that are not sensitive to a -bungarotoxin and force a revision of the current concepts on cholinergic mechanisms in the carotid body chemoreception. Keywords: Carotid body; Nicotinic receptor; Nicotine; a -Bungarotoxin; Catecholamine Ž. The significance of acetylcholine ACh in the carotid Ž. body CB arterial chemoreceptors has been debated throughout the history of arterial chemoreceptors. Heywx mans et al. 17,18 discovered that nicotine and exogenous ACh, acting at the level of the CB, can evoke respiratory reflexes comparable to those produced by hypoxia and wx acidosis, and Schweitzer and Wright 24 observed that Ž. prostigmine an inhibitor of ACh-esterase mimicked the action of ACh. These findings lead to the proposal that ACh would be the neurotransmitter mediating the activation of the sensory nerve endings of the carotid sinus nerve Ž. CSN . This proposal, known as the cholinergic hypothesis of CB chemoreception, was highly debated, and in the sixties Eyzaguirre’s laboratory accumulated sound experimental evidence to support a nicotinic receptor-mediated role for ACh in the genesis of the activity in the CSN, in wx response to a variety of stimuli 9 . However, the controversy continued because classical nicotinic blockers suppressed the excitatory actions of nicotinic agonists, but only reduced in a variable percentage the activation prowx duced by natural stimuli 20,21 . )Ž. Corresponding author. Fax: q34 83 423588. Biochemically, it was shown that the CB contains ACh wx 12,16 , expresses an adequate activity of choline acetyl wx transferase 16 , accumulates choline by a high affinity wx system, and exhibits a high turnover rate for ACh 10 . In spite of all these data, the role of ACh in the chemoreception process remained elusive. Radioligand binding and w125 xŽ autoradiographic studies, using I a -bungarotoxin a - . BT as ligand in normal, CSN-denervated and sympathectomized CBs, showed that the high affinity specific binding was located in chemoreceptor cells and sympathetic wx endings, but not in sensory nerve endings 4,5 . Electrophysiological studies have also shown the presence of wx nicotinic receptors in chemoreceptor cells 15,25 , and neurochemically it has been shown that chemoreceptor Ž. cells release catecholamines CA in response to nicotinic wx agonists 5,14 . These findings have been considered to indicate that ACh acts as a secondary neurotransmitter exclusively on the presynaptic side of the chemoreceptor wx cell-sensory nerve ending synapse 11,13 . There is, however, a finding that was not satisfactorily explained: while classical nicotinic blockers fully elimiwx nated the nicotine-mediated excitation of the CSN 9,20 , a -BT at a concentration near 10 times the K, inhibited by d only f50% the release of CA and CSN discharges elicited 0006-8993r97r$17.00 Copyright q1997 Elsevier Science B.V. All rights reserved. Ž. PII S0006-8993 97 00185-6 () A. Obeso et al.rBrain Research 754 1997 298–302 299 wx by nicotine 5 . In the light of recent advances in the wx biology of the nicotinic receptor 3,19,23 we have tested the possibility that chemoreceptor cells express two functional subtypes of nicotinic receptors, one sensitive and another insensitive to a -BT. Using an in vitro preparation Ž. of cat CB whose catecholamine CA deposits have been w3x labeled by incubation with the natural precursor H tyrosine, we have found that a -BT, at concentrations 20 times above its binding K, inhibits the nicotine evoked release d w3x of H CA by only 56%, while the classical nicotine blocker mecamylamine at 50 m M inhibited the nicotineevoked release by 100%. These findings imply that chemoreceptor cells express nicotinic receptors containing wx a - a subunits and thereby sensitive to a -BT 19,23 , and 78 other subtypes of nicotinic receptors with different a subunits insensitive to the toxin. Experiments were performed with CBs of adult cats Ž. 2.5–3.5 kg . The animals were anesthetized with sodium Ž. pentobarbital 40 mgrkg; i.p. , and after tracheotomy, the carotid bifurcations were identified, removed and placed in Ž a lucite chamber containing ice-cold modified Tyrode in . mM : NaCl, 112; KCl, 4.7; CaCl , 2.2; MgCl , 1.1; 22 .wx sodium glutamate, 42; HEPES, 5; glucose, 5.5 2 at pH 7.40 and equilibrated with 100% O . 2 Under a dissecting microscope the CBs were cleaned of surrounding tissue and thereafter incubated in glass vials containing 2 ml of Tyrode placed in a metabolic shaker at a constant temperature of 378C. The incubating solution Ž contained 100 m M 6-methyl-tetrahydropterine a tyrosine .Ž hydroxylase cofactor , 1 mM ascorbic acid as a cofactor .w3xŽ of dopamineb -hydroxylase and 40 m M H tyrosine 20 . CirmM; Amersham , the natural precursor of catecholŽ. amines CA . After 2 h of incubation CA deposits were labeled so that each CB has synthesized f12 pmol of w3xŽ. 5 H dopamine DA , equivalent to f3=10 d.p.m., and w3xŽ. f1 pmol of H norepinephrine NE , equivalent to f3 =104d.p.m. After the labeling period the organs were transferred to new vials containing 4 ml of precursor-free Tyrode continuously bubbled with 100% O saturated with 2 water vapor. The solution was renewed every 30 min during 2 h and discarded; in this washing period most of the precursor as well as the labile pool of labeled CA were w3x lost, and afterwards the basal release of H CA was stable wx for several hours 1 . Thereafter the incubating solutions were renewed every 10 min and collected for the ulterior w3x analysis in their H CA content. The collection of incubating solutions was grouped in stimulation cycles, each cycle consisting in a control sample to determine the basal release, a stimulus sample corresponding to the incubating period in the presence of Ž. the agent s tested, and several post-stimulus samples corresponding to periods of incubation with control solutions w3xŽ until the release of H CA returned to the basal level see .Ž Fig. 1A . Nicotinic blockers i.e., mecamylamine and a - .Ž. BT were included in the control period s prior to nicotine application to assure an adequate diffusion of the drug and block of nicotinic receptors. A given preparation could be subjected to one or more stimulation cycles depending on the reversibility of the effect of the agent under study. The w3x analysis of the released H CA included: acidification of the collected incubating solutions to pH 3.2 with a mixture of glacial acetic and ascorbic acid to avoid degradation of CA, bulk adsorption of all catechols released into alumina at a pH of 8.6, intense washing of alumina columns with distilled water and bulk elution of all catechols with 1 N HCl. Part of the eluate was used to determine the total w3x amount of H CA released in each period by liquid scintillation spectrometry, and the rest was pooled with correspondent samples of a total of four experiments, dried under vacuum, resuspended in the mobile phase containing unlabeled catechols as carriers of the labeled substances, and high performance liquid chromatographied to identify w3x Fig. 1. A: a stimulation cycle to show the time course of the release of H catecholamines induced by 100 m M nicotine in the cat carotid body. The line Ž. Ž. crossing the histogram separates graphically the basal release below from the evoked release above . B: log dose-response curve for nicotine on the w3x release of H catecholamine by the cat carotid body. The release response is expressed as times basal release. Data were fitted to the following function: wŽ. p x ysAyAr1qxrx , where A is the maximum effect, x is the concentration of nicotine, x is the IC and pis the Hill coefficient. The IC 22 0 2 050 50 was 9.81 m M. Data are means"S.E.M.; ns6. () A. Obeso et al.rBrain Research 754 1997 298–302300 w3xŽ the actual H catechol released for chromatographic dewx. tails see ref. 14 . The effect of a given agent on the w3x release of H CA was calculated in two different ways: Ž. first, the evoked release above the dashed line in Fig. 1A was referred to the basal release prior the application of the stimulus and expressed as times basal release, and second, the evoked release by a given stimulus was referred to the tissue content and expressed as percent of the tissue content. The significance of the effects observed was assessed with a two-tailed Student t-test for paired or unpaired data according to experimental design. [3] Effects of nicotine on the release of H CA. Fig. 1A shows a stimulation cycle with 100 m M nicotine. Nicow3x tine-induced release of H CA is represented by the d.p.m. above the dashed line crossing the histogram. Note that nearly 80% of the evoked release is collected during the 10 min period corresponding to the nicotine application, and that during the post-stimulus periods there is a slow decline of the release to reach basal pre-stimulation levels. This slow phase of the evoked release is represented by the w3xw 3 x washing-out of H CA catabolites; a part the H CA released during the stimulus period is taken out by the w3x tissues and degraded, and the H catabolites are slowly disposed by the cells. Fig. 1B shows a dose-effect curve w3x for nicotine on the release of H CA. The evoked release is expressed as times basal release. At the lowest concenŽ. tration tested 0.1 m M , nicotine increased the release of w3x H CA by a factor of 0.65 above basal release; maximal response, corresponding to a release of 14.4 times above basal was obtained with 50 m M nicotine, and the nicotine Ž. concentration producing half maximal effect IC was 50 9.81 m M. The same IC was obtained if the evoked 50 release was expressed as percentage of tissue content, the w3x Fig. 2. Analytical profile of the H catechol released. In basal conditions w3xŽ. H dihydroxyphenyl acetic acid DOPAC , the main catabolite of w3xw 3 x H dopamine, represented 60% of the total H catechols released. Durw3x ing hypoxic stimulation H DOPAC amounted to 20% of the released material and during nicotinic stimulation it represented 17%. The catabow3x lites of H norepinephrine were below detection. Fig. 3. Effects of mecamylamine and a -bungarotoxin on the release of w3x H catecholamines elicited by nicotine. The left group of columns w3x represents the release of H CA elicited by 50 m M nicotine during the Ž. Ž . stimulus S and the post-stimulus PS periods. The middle group of Ž. columns shows the complete inhibition of the nicotine 50 m M release response produced by 50 m M mecamylamine, and the right group of w3xŽ columns corresponds to the release of H CA produced by nicotine 50 . m M in the presence of 100 nM a -bungarotoxin. Data are means"S.E.M.; ns6 for nicotine and nicotine plus mecamylamine and ns8 for nicotine plus a -bungarotoxin. )P-0.001 in all the cases. basal release in a 10 min period representing 0.5% of the tissue content. w3x Fig. 2 shows the analytical profile of the H catechols found in basal samples, in incubating samples correspondŽ. ing to the stimulation with nicotine 50 m M , and for comparative purposes in incubating samples corresponding Ž to stimulation with hypoxia incubation with 10% O -equi2 .w3xw 3 x librated solutions . The ratio for H DArH NE found in basal samples was 10.5, indicating that 91% of the total w3xw 3 x H CA released was H DA and the remaining 9% was w3x H NE. These percentages are comparable to those found wx in the tissues 22 , indicating that in basal conditions both w3x H CA are released at a rate proportional to their tissue levels. During hypoxic stimulation the release of both w3xw 3 xw 3 x H CA increased, and the H DArH NE ratio in the collected samples increased to 23, indicating a preferential w3x release of H DA. On the contrary, during nicotinic stimuw3xw 3 x lation the ratio of H DArH NE in the collected samples dropped to a value of 4, indicating a preferential w3x release of H NE. Even a more marked preferential rew3x lease of H NE during nicotinic stimulation has been wx previously reported in the rabbit CB 14 . Fig. 3 shows the effect of a -BT and mecamylamine on w3x the release of H CA elicited by nicotine. Since in previous experiments it was found that maximal a -BT binding Žwx. was attained in 30 min see ref. 5 , the toxin was included in the incubating solution for the three 10 min control periods prior to and during nicotine application; () A. Obeso et al.rBrain Research 754 1997 298–302 301 mecamylamine was included in the incubating solution for a single 10 min control period prior to and during nicotine Ž. application. Note that mecamylamine 50 m M completely blocked the release induced by an identical concentration of nicotine. However, a -BT at a concentration of 100 nM, wx which is nearly 20 times its Kin binding experiments 5 , d only inhibited the nicotine-induced release by 56%. This level of inhibition is identical to that obtained with 50 nM wx a -BT 5 , and therefore it was not necessary to test higher concentrations. Neither nicotinic blocker affected the conw3x trol unstimulated or basal release of H CA. The main finding of this work is that mecamylamine w3x completely inhibits the release of H CA while a -BT only inhibits 56%, indicating that there is a set of nicotinic receptors in the CB that is insensitive to a -BT. These results also imply that the current concepts concerning the identification and location of nicotinic receptors in the CB, which are based on a -BT binding and autoradiographic experiments, need to be revised. w3x The dose-response for nicotine on the release of H CA obtained in the present study is displaced to the right when wx compared to that obtained by Eyzaguirre and Zapata 7 for ACh on the cat CSN discharges. They reported a threshold Žy8. for the response of 68 nM 10 grml , an IC of 50 f0.68 m M and a maximal response at f6.8 m M; these values indicate a displacement to the right by more than an order of magnitude of the dose-response curve reported here. A ready explanation for this difference could be that ACh increases CSN discharges acting on sensory nerve endings and not on chemoreceptor cells; alternatively, the CA released from chemoreceptor cells could represent the drivers of the CSN discharges, in such a way that small increases in the release above basal produce significant increases in the CSN discharges, and submaximal release Ž produces maximal CSN action potential frequency see . below . It is impossible to compare our data with those obtained in other studies, because in most of them ACh or nicotine were administered intra-arterially as bolus injections or in the inflowing tubing to the recording chamber. w3x The preferential release of H NE observed during nicotinic stimulation correlates with the observation that nearly 40% of the a -BT binding sites disappear after chronic CB wx sympathectomy 5 and indicate that a significant part of w3x the H NE released comes from intraglomic sympathetic endings. In fact, and contrary to the situation in the rabbit wx w 3x 14 , the preferential release of H NE observed under nicotinic stimulation in the cat CB disappears after sympaŽ. thectomy Gomez-Nino, unpublished . ´˜ Our findings with mecamylamine vs. a -BT clearly demonstrate that part of the nicotinic receptors in the cat CB are not sensitive to the snake toxin. Although the present pharmacological study does not allow to identify the subtypes of nicotinic receptors, it is clear that chemoreceptor cells should express nicotinic receptors with a or 7 a subunits, because only these subunits bind a -BT; it is 8 also clear that they must express nicotinic receptors with other a subunits, probably of the subtypes a or a 35 which are the most commonly found in sympathetic neuwx rons, and PC12 cells 19 which are embryologically related to the CB. Our findings in turn indicate that previous identification of nicotinic receptors on the basis of a -BT binding and autoradiographic studies have let out part of the nicotinic receptors in the CB, and the possibility exists that a part of these missed receptors are located in the sensory nerve endings. The unambiguous answer to this possibility would require the demonstration of the receptors in the sensory nerve endings andror the demonstration of the message for them in the chemoreceptor neurons of the petrosal ganglion by immunohistochemical andror in situ hybridization techniques. However, the fact that nicotinic effects on CSN discharges are greatly diminished 2q2qwx or abolished in Ca -free or Mg -rich solutions 8,9 suggests that nicotinic receptors are predominantly or exclusively located in chemoreceptor cells, and thereby that nicotinic effects on CSN discharges are mediated via the Ca2q-dependent release of other neurotransmitters. 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