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Cat carotid body chemoreceptor responses before and after nicotine receptor blockade with α-bungarotoxin
Journal of the Autonomic Nervous System, 18 (1987) 25-31
25
Elsevier JAN 00671
Cat carotid body chemoreceptor responses before and after nicotine receptor blockade with a-bungarotoxin E. Mulligan * and S. Lahiri Department of Physiology, University of Pennsyh;ania School of Medicine, Philadelphia, PA 19104 (U.S.A.) (Received 30 September 1985) (Revised version received 26 August 1986) (Accepted 28 August 1986)
Key words: Acetylcholine receptor; Hypoxia; Hypercapnia; Mecamylamine; Oxygen sensor Summary The nature of nicotine receptors in the carotid body was studied in anesthetized, paralyzed and artificially ventilated cats. Chemoreceptor discharge in single or few-fiber preparations of the carotid sinus nerve was measured during isocapnic hypoxia, hyperoxic hypercapnia and in response to nicotine injections before and after administration of a-bungarotoxin (10 cats) and after a-bungarotoxin plus rnecamylamine (7 cats) which binds to neuromuscular-type nicotine cholinergic receptors, a-Bungarotoxin caused a slight enhancement of the chemoreceptor response to hypoxia without affecting the chemoreceptor stimulation by nicotine. Mecamylamine (1-5 rag, i.v.), a ganglionic-type nicotinic receptor blocker, had no further effect on the response to hypoxia while it completely abolished the chemoreceptor stimulation by nicotine. Thus the nicotinic receptors in the cat carotid body which elicit excitation of chemosensory fibers appear to be of the ganglionic-type. Blockade of neuromuscular and ganglionic types of nicotinic receptors in the carotid body by a-bungarotoxin and mecamylamine does not attenuate the chemosensory responses to either hypoxia or hypercapnia. These nicotinic receptors therefore, do not appear to play an essential role in hypoxic or hypercapnic chemoreception in the cat carotid body.
Introduction Acetylcholine is a potent stimulus of carotid chemoreceptor activity in the cat [10]. This stimulatory effect is believed to be mediated by nicotinic cholinergic receptors because nicotine also vigorously stimulates cat carotid chemoreceptors [10]. Also, metabolic systems for acetylcholine synthesis, storage and inactivation are found in the carotid body [10]. Accordingly, acetylcholine * Present address: Department of Physiology, Temple University School of Medicine, 3420 N. Broad Street, Philadelphia, PA 19140, U.S.A. Correspondence: S. Lahiri, Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, U.S.A.
has been proposed as a neurotransmitter involved in carotid body chemoreception [10]. Research has been undertaken to localize nicotinic receptors in the tissue of the carotid body. a-Bungarotoxin (c~-BT) binding has been used as an indicator to determine the location of these receptors [4,5,7,8]. a-BT binding sites have been reported to be located on the type I or glomus cells of the carotid body and not on the nerve endings [5,8]. Since a-BT is a nicotinic receptor blocker and may bind to the receptors responsible for nicotine stimulation of carotid chemoreceptors, one would expect that it would decrease the carotid chemoreceptor response to nicotine. And, if acetylcholine is an excitatory transmitter essential for chemoreception, c~-BT would also decrease the chemoreceptor
0165-1838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
26 responses. But the hypothesis has not been adequately tested. Dinger et al. [8] reported that in the carotid body in vitro, c~-BT decreased chemoreceptor stimulation by nicotine. They also reported that hypoxic stimulation of the chemoreceptors was significantly reduced. The present study was undertaken to examine in detail the in vivo effects of a-BT administration on the carotid body chemoreceptor responses to hypoxia, hypercapnia and to nicotine in the cat. In addition, the effects on these responses of a ganglionic-type nicotinic receptor blocker, mecamylamine, were also tested. The results showed that a-BT did not decrease the chemoreceptor responses to nicotine and that after c~-BT administration, the chemoreceptor response to hypoxia was not reduced but actually was slightly enhanced. In contrast, mecamylamine completely abolished the response of chemoreceptors to nicotine and had no further effect on the hypoxic or hypercapnic responses. Materials and Methods
Ten cats (2.5-3 kg) were anesthetized with sodium pentobarbitat (Nembutal: 25 mg- k g ~, i.p. initially; 2 mg. kg -~. h~ t, i.v. subsequently), maintained at 38°C by using a blanket warmed by circulating water, paralyzed with gallamine triethiodide (Flaxedil; 3 m g - k g 1. h - l , i.v.), and ventilated with a positive pressure ventilator. Catheters were placed in the femoral arteries for continuous monitoring of blood pressure and for sampling of arterial blood for blood gas analysis and in a femoral vein for administration of drugs. To expose the carotid sinus areas, a tracheal tube was placed low in the neck and the trachea and esophagus were ligated, cut and reflected cranially. A catheter used for injection of a-BT close to the carotid body was placed in the lingual artery and advanced so that the tip was located upstream of the carotid body artery. The ipsilaterat external carotid artery was ligated to increase the percentage of the injectant reaching the carotid body. The catheter could be used to inject substances into the carotid body because bolus injections of nicotine (5 /xg) and NaCN (2 p.g) given through this catheter increased chemoreceptor activity transiently.
Single or few-fiber carotid chemoreceptor affer, ent activity was recorded from a fine strand of the cut carotid sinus nerve as described previously [16,17]. To insure that only the responses of the carotid body were measured, the sympathetic supply to the carotid body ioe. the ipsilateral ganglioglomerular nerve, was c u t Thus, the carotid body was neurally isolated Airway tidal O, and CO 2 partial pressures were also recorded (Beckman OM-11 for 0 2 and LB-2 for CO2), The experimental protocols were as follows: (1) to measure the isocapnic carotid chemoreceptor 0 2 response, the inspired 0 2 level was changed in steps from about 10% 02 up to 100% 02• Arterial blood samples were withdrawn during the steady, state, 3 min after each step in inspired O, concentration and were analyzed for pO 2, pCO 2 and pHa (Radiometer). (2) The hyperoxic (pat, > 300 Tort) CO 2 response of the chemoreceptor fiber was measured by raising the inspired CO 2 concentration in steps from 0% CO 2 up to about 7% CO 2. Blood samples were again withdrawn and analyzed at each steady-state end-tidal pCO 2 level (5 rain after each step in pCO2). (3) The chemoreceptor dose-response curve to nicotine (Nicotine bitartrate; 20, 40, 60, 80/~g, i.v.; doses taken from a stock solution of 200 /~g-ml 1 in saline) was measured while the Cat was ventilated with air ( p a t 2 about 85 Torr). Saline blank injections were made which had no effect on chemoreceptor activity. Then. ~x,BT (1 rag; Sigma; 1 m g . m t 1 dissolved in salinc~ was injected intra-arterially via the lingual artery catheter over about 1 min and protocols 1- 3 were repeated In some experiments from 1 to 3 mg more a-BT was injected, 1 mg at a time, and protocols 1-3 were repeated after each 1 mg dose o f a-BT. Subsequent to this, in 7 of the I0 cats, mecamylamine (0.8-1.6 m g . k g - 1 ) was administered and protocols l - 3 were repeated once again•
Results
Intra-carotid injections of c~-BT (200-1000 l~g) during normoxia (paO2 about 85 Torr) were followed immediately by increases in carotid chemo, receptor activity which declined to a value above
27
the control level within about a minute. Injections of similar volumes of saline, the vehicle, did not cause similar changes in carotid chemoreceptor activity. Also, intra-carotid bolus injections of nicotine (5 #g) or sodium cyanide (2 #g) through the same catheter excited the chemoreceptors transiently. These results suggest that the injected a-BT did reach the carotid chemoreceptors. Further supporting evidence came from one experiment in which the cat breathed spontaneously. Intra-carotid injections (in 200/~g installments) of a-BT were followed by decreases in ventilation due to neuro-muscular blockade and consequent increases in the blood gas stimuli which were associated with increases in carotid chemoreceptor activity. After a cumulative dose of 1 mg a-BT, spontaneous breathing stopped and the cat had to be ventilated artificially. Thus it is reasonable to assume that enough a-BT was present in the extracellular fluid in the carotid body and was readily accessible to the corresponding binding sites. Since the affinity of a-BT for its binding sites is high [1,15,18], it is also reasonable to assume that the sites were saturated with a-BT. After the total dose of a-BT, there was the normal rapid change in chemoreceptor activity at the onset and withdrawal of hypoxia or hypercapnia and after nicotine injections. Thus there is no reason to suppose that a-BT had altered the blood circulation to the carotid body. An example of the carotid chemoreceptor afferent responses to pO 2 before and after a-BT ad-
-> 5
>-
b
ministration is shown in Fig. 1. PaCO2 and pHa remained constant at about 24 Torr and 7.45, respectively, throughout the measurement of O 2 responses. Both response curves show the typical hyperbolic shape of a chemoreceptor O 2 response curve. However, after a-BT the curve is steeper, i.e. the discharge rate is greater at lower paO2 values below about 90 Torr. During hyperoxia, the activities before and after were identical, showing that a-BT itself was not excitatory and its excitatory effect was dependent on hypoxic stimulation. In order to compare the results such as those in Fig. 1 of different experiments, the differences in chemoreceptor activity at various paO 2 levels were compared using a one-way analysis of variance with repeated measures. These results are shown in Fig. 2. In this figure, the difference in steadystate chemoreceptor activity after a-BT is plotted against the PaO2 level. Thus, after ct-BT administration, the response to hypoxia is augmented. The activities of the same carotid chemoreceptor afferents at several levels of paCO2 (20-70 Torr) during hyperoxia were also compared before and after a-BT administration. The paCO 2 response curves were linear. In some cats the p~CO 2 response curve after a-BT was steeper than before ct-BT. However, when the data from all experiments were taken together, there was no statistically significant change in either the magnitude or the slope of the PaCO2 response after a-BT. The responses to various doses of nicotine were
I0
~ngarotoxin 5
a~ 7
'~ 3
EE o
\
5 Contr,
\
o
~5
~
;775 25
50
75
I00 " >300
PoOz (Torr)
Fig. 1. Effect of a-bungarotoxin (1 rag, i.a.) on the steady-state isocapnic paO2 response of a carotid chemoreceptor afferent fiber in one cat (paCO 2 about 24 Torr).
0 ¢/
4L5
6~5
8'5
Po02(Torr) Fig. 2. Change in carotid chemoreceptor steady-state paO2 response after a-bungarotoxin (1-4 mg, i.a.). Chemoreceptor activity is significantly greater ( P < 0.05) after a-bungarotoxin. (mean_+ SE; , = 9 cats).
28
8°I
A Control !:
CAROTID 30I CHEMORECEPTOR ACTIVITY (imp-so(')
0
z 6o 0
!:!!!
L
• Control j / • After a-bungarotoxm • After bunQorotoxmandmecarnylOm;r~ a -
/ a/ / - //
o_~, 40
Impulses
g~ • _. v.) B. After a-bungarotoxin
11; c) c2
20
S
r
I1:
~802
50 I
i/ ic
(imp sec-')
40 DOSE OF NICOTINE
o
Impulses C. After a-bunoorotoxin and mecamylamine
(imp.sec-') Impulses
I
I
IOsec
~ NICOTINE C780 180#.g, i.v)
Fig. 3. Records of responses of the same carotid chemoreceptor afferents to nicotine (80 ~tg, i.v.) before (Panel A), and after a-bungarotoxin (1 rag, i.a.; Panel B), and subsequently after a-bungarotoxin plus mecamylamine (5 mg, i.v.; Panel C). Mecamylamine inhibits the nicotine response whereas ,-~bungarotoxin does not.
also tested before and after the administration of a-BT. Fig. 3 shows an example of the chemosensory response to one dose level of nicotine at the 3 stages of the experiment. Nicotine (80 ftg) was injected i.v. as a bolus where indicated. In the control (Panel A), nicotine caused a vigorous, transient stimulation of carotid chemoreceptor activity. After a-BT (Panel B), nicotine still caused vigorous chemoreceptor activity. Administration of additional doses of a-BT caused no reduction in the response to nicotine. After m e c a m y l ~ n e administration, however, the chemoreceptors no longer showed a response to nicotine (Panel C),
Y
80
160
~9.~v
Fig. 4. Dose-response curves for carotid chemoreceptor re sponses to nicotine under control condiiions (O), after abungarotoxin (2 mg, i.a.: In), and after a-btmgarotoxin and mecamylamine (2 mg, i.v.: A) during normoxia (paO2 about 89 Torr, paCO2 about 30 Torr] m one experiment. Responses are the number of impulses generated over the baseline rates of activity during the 30 s following the injection of nicotine. Mecanlylamine inhibits the response to "all doses of nicotine whereas a-bungaratoxin has virtually no effect
but they still responded to both O 2 and C O 2. Fig. 4 shows a graph of the chemoreceptor afferent responses in one cat to different doses of nicotine at the 3 stages of the experiment. The chemoreceptor responses (in A impulses) are expressed as the number of impulses generated over the baseline rate of activity during the 30 s following the injection of the nicotine (see ref. 161. In the control curve, before a-BT. the chemoreceptor response increases as the dose of nicotine increases. After a-BT administration the chemoreceptor responses resemble that of the control state. There is definitely no diminuUon in the response to nicotine. After mecamytamine however, there is essentially no response to any dose of nicotine. Even a dose of nicotine of 160 I~g caused virtually no stimulation of chemoreceptor activity. The results on the effect of nicotine on carotid chemoreceptor activity before a-BT. after a-BT alone, and after a-BT followed by mecamylamine for all experiments are summarized in Table I. The responses to nicotine after a-BT and after
29 TABLE I
The effect of et-bungarotoxin and e~-bungarotoxin plus meeamvlamine on the carotid chemoreceptor response to nicotine Response to nicotine
% of control response
Control response 100 After a-bungarotoxin (n = 10) 133.6 + 12.0 After c~-BT plus mecamylamine (n = 7) 4.0_+ 2.4 (a-BT, 1 - 4 mg, i.a.; mecamylamine, 0.8-1.6 m g . k g I i.v.; nicotine, 80-150/*g, i.v. Nicotine dose was constant within one experiment. Data from highest dose of nicotine administered in each experiment was used for tabulation. Values are m e a n s ± S.E.M.).
a-BT and mecamylamine together are expressed as a percentage of the control responses. After a-BT alone, there was no reduction in the carotid chemoreceptor stimulation by the same dose of nicotine. The response may have even increased. Subsequently, after mecamylamine was administered~ there was a 96% reduction in the response to nicotine i.e. there was essentially no stimulation of the chemoreceptors by nicotine. Thus mecamylamine blocks the chemoreceptor response to nicotine whereas c~-BT does not. After mecamylamine, the chemoreceptor responses to PaO2 and PaCO2 did not diminish when compared with the responses before administration of mecamylamine (see also ref. 19).
Discussion These results show that, in vivo, administration of a-BT, a toxin which binds irreversibly to neuromuscular nicotinic receptors, does not diminish or block the cat carotid chemoreceptor responses to nicotine or to hypoxia or hypercapnia. In contrast, mecamylamine, a ganglionic-type nicotinic receptor blocker, when given after a-BT, completely blocks the cat carotid chemoreceptor stimulation by nicotine. This effect of mecamylamine is in agreement with data presented by McQueen [12] and Sampson [19]. But mecamylamine, like a-BT, did not block chemoreceptor response to hypoxia or hypercapnia. a-Bungarotoxin has been shown to have specific
binding sites in the carotid body of the rat [4,5] and the cat [7,8]. However, since a-BT does not block the chemoreceptor response to nicotine, these a-BT binding sites are most likely not the nicotinic receptors which function in the stimulation of the carotid body chemoreceptor by nicotine. After a-BT, administration of mecamylamine is effective in blocking the chemoreceptor response to nicotine. This indicates that the receptors in the carotid body that are responsible for nicotine stimulation are of the ganglionic (C6) type nicotinic receptor rather than the neuromuscular (C10) type [1,15], as was also suggested by McQueen [12,13]; he noted little or no decrease in acetylcholine stimulation after a-BT. It is improbable that a-BT binds to these C 6 type ganglionic receptors and thus most likely binds to receptors other than those which cause chemoreceptor stimulation by nicotine and which are blocked by mecamylamine. Similar results have been shown for other tissues such as the sympathetic ganglion and spinal Renshaw cells [see e.g. refs. 1-3,6,18] where a-BT binds specifically to the tissue but has no physiological effect on the neural activity. This has led to a controversy in the literature as to whether a-BT can bind to some types of nicotinic receptors and not affect them physiologically or whether the a-BT binding sites are distinct from the nicotinic receptors in these tissues that are the physiologically active receptor [1,15,181. The c~-BT administered in this study (1-4 mg) was injected into the carotid sinus, close to the carotid body. Thus the greatest concentration of ~-BT circulated through the carotid body. In one experiment, the cat was allowed to breathe spontaneously as the a-BT (1 mg, i.a.) was administered. Following a-BT administration, paralysis of the respiratory muscles developed gradually. The smallest dose of c~-BT used was therefore enough to block the neuromuscular nicotinic sites in a 2.5 kg cat. We estimate that after a 1 mg dose of c~-BT, the concentration of a-BT in the extracellular fluid of a 2.5 kg cat is 150 nM. This exceeds the concentrations of ~-BT used by those who have localized c~-BT binding sites in the carotid body [4,5,7,8]. In this study, it was also found that a-BT
30
administration slightly but consistently enhanced the chemoreceptor hypoxic response but had no significant effect on the hypercapnic response. By contrast Dinger et al. [8] reported that in the carotid body in vitro, a-BT significantly reduced the rate of chemoreceptor activity as well as the amount of dopamine released from the carotid body during hypoxia. If dopamine release is decreased by a-BT in vivo as well, then since dopamine is inhibitory to chemoreceptor activity this could result in an enhanced chemoreceptor O 2 response as seen in this study. This enchancement is similar to that seen when dopamine receptors are blocked with haloperidol [9,11]. Similarly, Dinger et al. [8] reported that a-BT reduced the amount of dopamine released by the carotid body during exposure to nicotine. If this also occurs in vivo, it would offer an explanation as to why the nicotine response may be enhanced after a-BT (Table I). Dinger et al. [8] also reported that after a-BT in the carotid body in vitro, there was a reduction in the chemoreceptor response to nicotine; this was not seen in the present study in vivo. The reduction in the response in vitro (about 30%) is small when compared to the virtually complete (about 96%, Table I) inhibition of the response that can be obtained with mecamylamine in vivo (see Figs. 3, 4 and Table I). In our study, after o~-BT, mecamylamine administration blocked the nicotine response but had no further effect on the chemoreceptor 02 and CO 2 response [see also ref. 19]. This suggests that the nicotinic receptor responsible for the chemoreceptor nicotine stimulation is not an integral part of the process of natural O 2 and CO 2 chemoreception. In summary, it has been found that, in vivo, administration of a-BT does not block the stimulatory response of carotid chemoreceptors to nicotine whereas mecamylamine does block this response. Thus it is highly unlikely that a-BT binds to the receptors responsible for chemoreceptor stimulation by nicotine. It is possible that there is more than one type of nicotinic receptor in the carotid body. Those receptors responsible for cholinergic stimulation of carotid body chemoreceptors have yet to be localized with certainty. Blockade of the nicotinic cholinergic receptors by
a-BT and mecamylamine does not attenuate the carotid chemosensory responses to hypoxia and hypercapnia, the natural physiological stimuli.
Acknowledgements The authors are grateful to Mrs. E. Hopkin and Mr. A. Mokashi for their assistance Supported in part by the NIH Grants HL-I9737-10, NS-2106802, 5-T32-HL-07027 and the Puritan-Bennett Foundation.
References 1 Brown. D.A.. Neurotoxins and the ganglionic (C6) type of nicotinic receptor. Adv. Cytopharmacol.. 3 (1979) 2 2 5 - 2 3 0 2 Brown. D . A and Fumagalli. L.. Dissociation of abungarotoxin binding and receptor block in the rat superior cervical ganglion. Brain Res., 129 11977~ 165-168. 3 Carbonetto. S.T.. Fambrough, D.M. and Muller, K.J_ Non-equivalence of a-bungarotoxin receptors and acetylcholine receptors in chick sympathetic neurons, Proc. ,VatL Acad Sci. U.S.A.. 75 0978) 1016--1020. 4 Chen, 1., Mascorro. J.A. and Yates, R.D., Autoradiographic localization of a-bungarotoxin bindi~ng sites in the carotid body of the rat. Cell Tissue Res.. 219 (1981) 6 0 9 - 6 1 8 5 Chen. I. and Yates. R.D.. Two types of glomus cells in the rat carotid body as revealed by a-bungarotoxin binding. J. Neurot vtol., 13 (1984) 281-302, 6 Chou. T.C. and Lee. C.Y., Effect of whole and fractionated cobra venom on sympathetic ganglion transmission, Eur..L Pharmacol.. 8 ~1969~ 326-330. - Dinger, B., Gonzalez. C.. Yoshizaki, K and Fidone, S., Alpha-bungarotoxin binding in the rat carotid body, Bruin Res.. 205 (1981) 187-193. 8 Dinger, B.. Gonzalez. C., Yoshizaki. K, and Fidone. S.. Localization and function of cat carotid body nicotine receptors. Brain Res.. 339 (1985) 295-304, 9 Donnetly, D.F.. Smith, E.J. and Dutton, R.E.. Neural response of carotid chemoreceptors following dopamine blockade. J. Appl. PhysioL, 50 (1981) 172-177. 10 Ey~aguirre, C.. Fitzgerald. R.S. L'ahiri. S. and Zapata. P.. Arterial Chemoreceptors. In Handbook of Physiology:. l'he Cardiovascular System. Sect. 3, Vol. III, Am. Physiol. Sot.. Bethesda. M.D. 1983, pp. 557-621 11 LahirL S., Nishino. T., Mokashi, A. and Mulligan, E.. Interaction of dopamine and haloperidol with O2 and CO2 chemoreception in carotid body, J. Appl. Physiol., 4 9 (1980) 45-51. 12 MeQueen. D.S.. A quantitative study of the effects of cholinergic drugs on carotid chemoreceptors in the cat, d. Phvsiol. tLondon), 273 (1977) 515-532.
31 13 McQueen, D.S., Effects of dihydro-fl-erythroidine on the cat carotid chemoreceptors, Q. J, Exp. Physiol., 65 (1980) 229 237. 14 Morley, B.J., The properties of brain nicotine receptors, Pharrnacol. Ther., 15 (1981) 111-122. 15 Morely, B.J. and Kemp, G.E., Characterization of a putative nicotinic acetylcholine receptor in mammalian brain, Brain Res. Rev., 3 (1981) 81-104. 16 Mulligan, E. and Lahiri, S., Dependence of carotid chemoreceptor stimulation by metabolic agents on paO2 and paC02, .l. Appl. Physiol., 50 (1981) 884-891.
17 Mulligan, E. and Lahiri, S., Separation of carotid body chemoreceptor responses to 02 and CO2 by oligomycin and by antimycin A, Am. J. Pltvsiol., 242 (Cell Physiol. 11) (1982) C200-C206. 18 Oswald, R.E. and Freeman, J.A., Alpha-bungarotoxin binding and central nervous system nicotinic acetylcholine receptors, Neuroscience, 6 (1981) 1-14. 19 Sampson, S.R., Effects of mecamylamine on responses of carotid body chemoreceptors in vivo to physiological and pharmacological stimuli, J. Physiol. (London), 212 (1971) 655-666.
25
Elsevier JAN 00671
Cat carotid body chemoreceptor responses before and after nicotine receptor blockade with a-bungarotoxin E. Mulligan * and S. Lahiri Department of Physiology, University of Pennsyh;ania School of Medicine, Philadelphia, PA 19104 (U.S.A.) (Received 30 September 1985) (Revised version received 26 August 1986) (Accepted 28 August 1986)
Key words: Acetylcholine receptor; Hypoxia; Hypercapnia; Mecamylamine; Oxygen sensor Summary The nature of nicotine receptors in the carotid body was studied in anesthetized, paralyzed and artificially ventilated cats. Chemoreceptor discharge in single or few-fiber preparations of the carotid sinus nerve was measured during isocapnic hypoxia, hyperoxic hypercapnia and in response to nicotine injections before and after administration of a-bungarotoxin (10 cats) and after a-bungarotoxin plus rnecamylamine (7 cats) which binds to neuromuscular-type nicotine cholinergic receptors, a-Bungarotoxin caused a slight enhancement of the chemoreceptor response to hypoxia without affecting the chemoreceptor stimulation by nicotine. Mecamylamine (1-5 rag, i.v.), a ganglionic-type nicotinic receptor blocker, had no further effect on the response to hypoxia while it completely abolished the chemoreceptor stimulation by nicotine. Thus the nicotinic receptors in the cat carotid body which elicit excitation of chemosensory fibers appear to be of the ganglionic-type. Blockade of neuromuscular and ganglionic types of nicotinic receptors in the carotid body by a-bungarotoxin and mecamylamine does not attenuate the chemosensory responses to either hypoxia or hypercapnia. These nicotinic receptors therefore, do not appear to play an essential role in hypoxic or hypercapnic chemoreception in the cat carotid body.
Introduction Acetylcholine is a potent stimulus of carotid chemoreceptor activity in the cat [10]. This stimulatory effect is believed to be mediated by nicotinic cholinergic receptors because nicotine also vigorously stimulates cat carotid chemoreceptors [10]. Also, metabolic systems for acetylcholine synthesis, storage and inactivation are found in the carotid body [10]. Accordingly, acetylcholine * Present address: Department of Physiology, Temple University School of Medicine, 3420 N. Broad Street, Philadelphia, PA 19140, U.S.A. Correspondence: S. Lahiri, Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, U.S.A.
has been proposed as a neurotransmitter involved in carotid body chemoreception [10]. Research has been undertaken to localize nicotinic receptors in the tissue of the carotid body. a-Bungarotoxin (c~-BT) binding has been used as an indicator to determine the location of these receptors [4,5,7,8]. a-BT binding sites have been reported to be located on the type I or glomus cells of the carotid body and not on the nerve endings [5,8]. Since a-BT is a nicotinic receptor blocker and may bind to the receptors responsible for nicotine stimulation of carotid chemoreceptors, one would expect that it would decrease the carotid chemoreceptor response to nicotine. And, if acetylcholine is an excitatory transmitter essential for chemoreception, c~-BT would also decrease the chemoreceptor
0165-1838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
26 responses. But the hypothesis has not been adequately tested. Dinger et al. [8] reported that in the carotid body in vitro, c~-BT decreased chemoreceptor stimulation by nicotine. They also reported that hypoxic stimulation of the chemoreceptors was significantly reduced. The present study was undertaken to examine in detail the in vivo effects of a-BT administration on the carotid body chemoreceptor responses to hypoxia, hypercapnia and to nicotine in the cat. In addition, the effects on these responses of a ganglionic-type nicotinic receptor blocker, mecamylamine, were also tested. The results showed that a-BT did not decrease the chemoreceptor responses to nicotine and that after c~-BT administration, the chemoreceptor response to hypoxia was not reduced but actually was slightly enhanced. In contrast, mecamylamine completely abolished the response of chemoreceptors to nicotine and had no further effect on the hypoxic or hypercapnic responses. Materials and Methods
Ten cats (2.5-3 kg) were anesthetized with sodium pentobarbitat (Nembutal: 25 mg- k g ~, i.p. initially; 2 mg. kg -~. h~ t, i.v. subsequently), maintained at 38°C by using a blanket warmed by circulating water, paralyzed with gallamine triethiodide (Flaxedil; 3 m g - k g 1. h - l , i.v.), and ventilated with a positive pressure ventilator. Catheters were placed in the femoral arteries for continuous monitoring of blood pressure and for sampling of arterial blood for blood gas analysis and in a femoral vein for administration of drugs. To expose the carotid sinus areas, a tracheal tube was placed low in the neck and the trachea and esophagus were ligated, cut and reflected cranially. A catheter used for injection of a-BT close to the carotid body was placed in the lingual artery and advanced so that the tip was located upstream of the carotid body artery. The ipsilaterat external carotid artery was ligated to increase the percentage of the injectant reaching the carotid body. The catheter could be used to inject substances into the carotid body because bolus injections of nicotine (5 /xg) and NaCN (2 p.g) given through this catheter increased chemoreceptor activity transiently.
Single or few-fiber carotid chemoreceptor affer, ent activity was recorded from a fine strand of the cut carotid sinus nerve as described previously [16,17]. To insure that only the responses of the carotid body were measured, the sympathetic supply to the carotid body ioe. the ipsilateral ganglioglomerular nerve, was c u t Thus, the carotid body was neurally isolated Airway tidal O, and CO 2 partial pressures were also recorded (Beckman OM-11 for 0 2 and LB-2 for CO2), The experimental protocols were as follows: (1) to measure the isocapnic carotid chemoreceptor 0 2 response, the inspired 0 2 level was changed in steps from about 10% 02 up to 100% 02• Arterial blood samples were withdrawn during the steady, state, 3 min after each step in inspired O, concentration and were analyzed for pO 2, pCO 2 and pHa (Radiometer). (2) The hyperoxic (pat, > 300 Tort) CO 2 response of the chemoreceptor fiber was measured by raising the inspired CO 2 concentration in steps from 0% CO 2 up to about 7% CO 2. Blood samples were again withdrawn and analyzed at each steady-state end-tidal pCO 2 level (5 rain after each step in pCO2). (3) The chemoreceptor dose-response curve to nicotine (Nicotine bitartrate; 20, 40, 60, 80/~g, i.v.; doses taken from a stock solution of 200 /~g-ml 1 in saline) was measured while the Cat was ventilated with air ( p a t 2 about 85 Torr). Saline blank injections were made which had no effect on chemoreceptor activity. Then. ~x,BT (1 rag; Sigma; 1 m g . m t 1 dissolved in salinc~ was injected intra-arterially via the lingual artery catheter over about 1 min and protocols 1- 3 were repeated In some experiments from 1 to 3 mg more a-BT was injected, 1 mg at a time, and protocols 1-3 were repeated after each 1 mg dose o f a-BT. Subsequent to this, in 7 of the I0 cats, mecamylamine (0.8-1.6 m g . k g - 1 ) was administered and protocols l - 3 were repeated once again•
Results
Intra-carotid injections of c~-BT (200-1000 l~g) during normoxia (paO2 about 85 Torr) were followed immediately by increases in carotid chemo, receptor activity which declined to a value above
27
the control level within about a minute. Injections of similar volumes of saline, the vehicle, did not cause similar changes in carotid chemoreceptor activity. Also, intra-carotid bolus injections of nicotine (5 #g) or sodium cyanide (2 #g) through the same catheter excited the chemoreceptors transiently. These results suggest that the injected a-BT did reach the carotid chemoreceptors. Further supporting evidence came from one experiment in which the cat breathed spontaneously. Intra-carotid injections (in 200/~g installments) of a-BT were followed by decreases in ventilation due to neuro-muscular blockade and consequent increases in the blood gas stimuli which were associated with increases in carotid chemoreceptor activity. After a cumulative dose of 1 mg a-BT, spontaneous breathing stopped and the cat had to be ventilated artificially. Thus it is reasonable to assume that enough a-BT was present in the extracellular fluid in the carotid body and was readily accessible to the corresponding binding sites. Since the affinity of a-BT for its binding sites is high [1,15,18], it is also reasonable to assume that the sites were saturated with a-BT. After the total dose of a-BT, there was the normal rapid change in chemoreceptor activity at the onset and withdrawal of hypoxia or hypercapnia and after nicotine injections. Thus there is no reason to suppose that a-BT had altered the blood circulation to the carotid body. An example of the carotid chemoreceptor afferent responses to pO 2 before and after a-BT ad-
-> 5
>-
b
ministration is shown in Fig. 1. PaCO2 and pHa remained constant at about 24 Torr and 7.45, respectively, throughout the measurement of O 2 responses. Both response curves show the typical hyperbolic shape of a chemoreceptor O 2 response curve. However, after a-BT the curve is steeper, i.e. the discharge rate is greater at lower paO2 values below about 90 Torr. During hyperoxia, the activities before and after were identical, showing that a-BT itself was not excitatory and its excitatory effect was dependent on hypoxic stimulation. In order to compare the results such as those in Fig. 1 of different experiments, the differences in chemoreceptor activity at various paO 2 levels were compared using a one-way analysis of variance with repeated measures. These results are shown in Fig. 2. In this figure, the difference in steadystate chemoreceptor activity after a-BT is plotted against the PaO2 level. Thus, after ct-BT administration, the response to hypoxia is augmented. The activities of the same carotid chemoreceptor afferents at several levels of paCO2 (20-70 Torr) during hyperoxia were also compared before and after a-BT administration. The paCO 2 response curves were linear. In some cats the p~CO 2 response curve after a-BT was steeper than before ct-BT. However, when the data from all experiments were taken together, there was no statistically significant change in either the magnitude or the slope of the PaCO2 response after a-BT. The responses to various doses of nicotine were
I0
~ngarotoxin 5
a~ 7
'~ 3
EE o
\
5 Contr,
\
o
~5
~
;775 25
50
75
I00 " >300
PoOz (Torr)
Fig. 1. Effect of a-bungarotoxin (1 rag, i.a.) on the steady-state isocapnic paO2 response of a carotid chemoreceptor afferent fiber in one cat (paCO 2 about 24 Torr).
0 ¢/
4L5
6~5
8'5
Po02(Torr) Fig. 2. Change in carotid chemoreceptor steady-state paO2 response after a-bungarotoxin (1-4 mg, i.a.). Chemoreceptor activity is significantly greater ( P < 0.05) after a-bungarotoxin. (mean_+ SE; , = 9 cats).
28
8°I
A Control !:
CAROTID 30I CHEMORECEPTOR ACTIVITY (imp-so(')
0
z 6o 0
!:!!!
L
• Control j / • After a-bungarotoxm • After bunQorotoxmandmecarnylOm;r~ a -
/ a/ / - //
o_~, 40
Impulses
g~ • _. v.) B. After a-bungarotoxin
11; c) c2
20
S
r
I1:
~802
50 I
i/ ic
(imp sec-')
40 DOSE OF NICOTINE
o
Impulses C. After a-bunoorotoxin and mecamylamine
(imp.sec-') Impulses
I
I
IOsec
~ NICOTINE C780 180#.g, i.v)
Fig. 3. Records of responses of the same carotid chemoreceptor afferents to nicotine (80 ~tg, i.v.) before (Panel A), and after a-bungarotoxin (1 rag, i.a.; Panel B), and subsequently after a-bungarotoxin plus mecamylamine (5 mg, i.v.; Panel C). Mecamylamine inhibits the nicotine response whereas ,-~bungarotoxin does not.
also tested before and after the administration of a-BT. Fig. 3 shows an example of the chemosensory response to one dose level of nicotine at the 3 stages of the experiment. Nicotine (80 ftg) was injected i.v. as a bolus where indicated. In the control (Panel A), nicotine caused a vigorous, transient stimulation of carotid chemoreceptor activity. After a-BT (Panel B), nicotine still caused vigorous chemoreceptor activity. Administration of additional doses of a-BT caused no reduction in the response to nicotine. After m e c a m y l ~ n e administration, however, the chemoreceptors no longer showed a response to nicotine (Panel C),
Y
80
160
~9.~v
Fig. 4. Dose-response curves for carotid chemoreceptor re sponses to nicotine under control condiiions (O), after abungarotoxin (2 mg, i.a.: In), and after a-btmgarotoxin and mecamylamine (2 mg, i.v.: A) during normoxia (paO2 about 89 Torr, paCO2 about 30 Torr] m one experiment. Responses are the number of impulses generated over the baseline rates of activity during the 30 s following the injection of nicotine. Mecanlylamine inhibits the response to "all doses of nicotine whereas a-bungaratoxin has virtually no effect
but they still responded to both O 2 and C O 2. Fig. 4 shows a graph of the chemoreceptor afferent responses in one cat to different doses of nicotine at the 3 stages of the experiment. The chemoreceptor responses (in A impulses) are expressed as the number of impulses generated over the baseline rate of activity during the 30 s following the injection of the nicotine (see ref. 161. In the control curve, before a-BT. the chemoreceptor response increases as the dose of nicotine increases. After a-BT administration the chemoreceptor responses resemble that of the control state. There is definitely no diminuUon in the response to nicotine. After mecamytamine however, there is essentially no response to any dose of nicotine. Even a dose of nicotine of 160 I~g caused virtually no stimulation of chemoreceptor activity. The results on the effect of nicotine on carotid chemoreceptor activity before a-BT. after a-BT alone, and after a-BT followed by mecamylamine for all experiments are summarized in Table I. The responses to nicotine after a-BT and after
29 TABLE I
The effect of et-bungarotoxin and e~-bungarotoxin plus meeamvlamine on the carotid chemoreceptor response to nicotine Response to nicotine
% of control response
Control response 100 After a-bungarotoxin (n = 10) 133.6 + 12.0 After c~-BT plus mecamylamine (n = 7) 4.0_+ 2.4 (a-BT, 1 - 4 mg, i.a.; mecamylamine, 0.8-1.6 m g . k g I i.v.; nicotine, 80-150/*g, i.v. Nicotine dose was constant within one experiment. Data from highest dose of nicotine administered in each experiment was used for tabulation. Values are m e a n s ± S.E.M.).
a-BT and mecamylamine together are expressed as a percentage of the control responses. After a-BT alone, there was no reduction in the carotid chemoreceptor stimulation by the same dose of nicotine. The response may have even increased. Subsequently, after mecamylamine was administered~ there was a 96% reduction in the response to nicotine i.e. there was essentially no stimulation of the chemoreceptors by nicotine. Thus mecamylamine blocks the chemoreceptor response to nicotine whereas c~-BT does not. After mecamylamine, the chemoreceptor responses to PaO2 and PaCO2 did not diminish when compared with the responses before administration of mecamylamine (see also ref. 19).
Discussion These results show that, in vivo, administration of a-BT, a toxin which binds irreversibly to neuromuscular nicotinic receptors, does not diminish or block the cat carotid chemoreceptor responses to nicotine or to hypoxia or hypercapnia. In contrast, mecamylamine, a ganglionic-type nicotinic receptor blocker, when given after a-BT, completely blocks the cat carotid chemoreceptor stimulation by nicotine. This effect of mecamylamine is in agreement with data presented by McQueen [12] and Sampson [19]. But mecamylamine, like a-BT, did not block chemoreceptor response to hypoxia or hypercapnia. a-Bungarotoxin has been shown to have specific
binding sites in the carotid body of the rat [4,5] and the cat [7,8]. However, since a-BT does not block the chemoreceptor response to nicotine, these a-BT binding sites are most likely not the nicotinic receptors which function in the stimulation of the carotid body chemoreceptor by nicotine. After a-BT, administration of mecamylamine is effective in blocking the chemoreceptor response to nicotine. This indicates that the receptors in the carotid body that are responsible for nicotine stimulation are of the ganglionic (C6) type nicotinic receptor rather than the neuromuscular (C10) type [1,15], as was also suggested by McQueen [12,13]; he noted little or no decrease in acetylcholine stimulation after a-BT. It is improbable that a-BT binds to these C 6 type ganglionic receptors and thus most likely binds to receptors other than those which cause chemoreceptor stimulation by nicotine and which are blocked by mecamylamine. Similar results have been shown for other tissues such as the sympathetic ganglion and spinal Renshaw cells [see e.g. refs. 1-3,6,18] where a-BT binds specifically to the tissue but has no physiological effect on the neural activity. This has led to a controversy in the literature as to whether a-BT can bind to some types of nicotinic receptors and not affect them physiologically or whether the a-BT binding sites are distinct from the nicotinic receptors in these tissues that are the physiologically active receptor [1,15,181. The c~-BT administered in this study (1-4 mg) was injected into the carotid sinus, close to the carotid body. Thus the greatest concentration of ~-BT circulated through the carotid body. In one experiment, the cat was allowed to breathe spontaneously as the a-BT (1 mg, i.a.) was administered. Following a-BT administration, paralysis of the respiratory muscles developed gradually. The smallest dose of c~-BT used was therefore enough to block the neuromuscular nicotinic sites in a 2.5 kg cat. We estimate that after a 1 mg dose of c~-BT, the concentration of a-BT in the extracellular fluid of a 2.5 kg cat is 150 nM. This exceeds the concentrations of ~-BT used by those who have localized c~-BT binding sites in the carotid body [4,5,7,8]. In this study, it was also found that a-BT
30
administration slightly but consistently enhanced the chemoreceptor hypoxic response but had no significant effect on the hypercapnic response. By contrast Dinger et al. [8] reported that in the carotid body in vitro, a-BT significantly reduced the rate of chemoreceptor activity as well as the amount of dopamine released from the carotid body during hypoxia. If dopamine release is decreased by a-BT in vivo as well, then since dopamine is inhibitory to chemoreceptor activity this could result in an enhanced chemoreceptor O 2 response as seen in this study. This enchancement is similar to that seen when dopamine receptors are blocked with haloperidol [9,11]. Similarly, Dinger et al. [8] reported that a-BT reduced the amount of dopamine released by the carotid body during exposure to nicotine. If this also occurs in vivo, it would offer an explanation as to why the nicotine response may be enhanced after a-BT (Table I). Dinger et al. [8] also reported that after a-BT in the carotid body in vitro, there was a reduction in the chemoreceptor response to nicotine; this was not seen in the present study in vivo. The reduction in the response in vitro (about 30%) is small when compared to the virtually complete (about 96%, Table I) inhibition of the response that can be obtained with mecamylamine in vivo (see Figs. 3, 4 and Table I). In our study, after o~-BT, mecamylamine administration blocked the nicotine response but had no further effect on the chemoreceptor 02 and CO 2 response [see also ref. 19]. This suggests that the nicotinic receptor responsible for the chemoreceptor nicotine stimulation is not an integral part of the process of natural O 2 and CO 2 chemoreception. In summary, it has been found that, in vivo, administration of a-BT does not block the stimulatory response of carotid chemoreceptors to nicotine whereas mecamylamine does block this response. Thus it is highly unlikely that a-BT binds to the receptors responsible for chemoreceptor stimulation by nicotine. It is possible that there is more than one type of nicotinic receptor in the carotid body. Those receptors responsible for cholinergic stimulation of carotid body chemoreceptors have yet to be localized with certainty. Blockade of the nicotinic cholinergic receptors by
a-BT and mecamylamine does not attenuate the carotid chemosensory responses to hypoxia and hypercapnia, the natural physiological stimuli.
Acknowledgements The authors are grateful to Mrs. E. Hopkin and Mr. A. Mokashi for their assistance Supported in part by the NIH Grants HL-I9737-10, NS-2106802, 5-T32-HL-07027 and the Puritan-Bennett Foundation.
References 1 Brown. D.A.. Neurotoxins and the ganglionic (C6) type of nicotinic receptor. Adv. Cytopharmacol.. 3 (1979) 2 2 5 - 2 3 0 2 Brown. D . A and Fumagalli. L.. Dissociation of abungarotoxin binding and receptor block in the rat superior cervical ganglion. Brain Res., 129 11977~ 165-168. 3 Carbonetto. S.T.. Fambrough, D.M. and Muller, K.J_ Non-equivalence of a-bungarotoxin receptors and acetylcholine receptors in chick sympathetic neurons, Proc. ,VatL Acad Sci. U.S.A.. 75 0978) 1016--1020. 4 Chen, 1., Mascorro. J.A. and Yates, R.D., Autoradiographic localization of a-bungarotoxin bindi~ng sites in the carotid body of the rat. Cell Tissue Res.. 219 (1981) 6 0 9 - 6 1 8 5 Chen. I. and Yates. R.D.. Two types of glomus cells in the rat carotid body as revealed by a-bungarotoxin binding. J. Neurot vtol., 13 (1984) 281-302, 6 Chou. T.C. and Lee. C.Y., Effect of whole and fractionated cobra venom on sympathetic ganglion transmission, Eur..L Pharmacol.. 8 ~1969~ 326-330. - Dinger, B., Gonzalez. C.. Yoshizaki, K and Fidone, S., Alpha-bungarotoxin binding in the rat carotid body, Bruin Res.. 205 (1981) 187-193. 8 Dinger, B.. Gonzalez. C., Yoshizaki. K, and Fidone. S.. Localization and function of cat carotid body nicotine receptors. Brain Res.. 339 (1985) 295-304, 9 Donnetly, D.F.. Smith, E.J. and Dutton, R.E.. Neural response of carotid chemoreceptors following dopamine blockade. J. Appl. PhysioL, 50 (1981) 172-177. 10 Ey~aguirre, C.. Fitzgerald. R.S. L'ahiri. S. and Zapata. P.. Arterial Chemoreceptors. In Handbook of Physiology:. l'he Cardiovascular System. Sect. 3, Vol. III, Am. Physiol. Sot.. Bethesda. M.D. 1983, pp. 557-621 11 LahirL S., Nishino. T., Mokashi, A. and Mulligan, E.. Interaction of dopamine and haloperidol with O2 and CO2 chemoreception in carotid body, J. Appl. Physiol., 4 9 (1980) 45-51. 12 MeQueen. D.S.. A quantitative study of the effects of cholinergic drugs on carotid chemoreceptors in the cat, d. Phvsiol. tLondon), 273 (1977) 515-532.
31 13 McQueen, D.S., Effects of dihydro-fl-erythroidine on the cat carotid chemoreceptors, Q. J, Exp. Physiol., 65 (1980) 229 237. 14 Morley, B.J., The properties of brain nicotine receptors, Pharrnacol. Ther., 15 (1981) 111-122. 15 Morely, B.J. and Kemp, G.E., Characterization of a putative nicotinic acetylcholine receptor in mammalian brain, Brain Res. Rev., 3 (1981) 81-104. 16 Mulligan, E. and Lahiri, S., Dependence of carotid chemoreceptor stimulation by metabolic agents on paO2 and paC02, .l. Appl. Physiol., 50 (1981) 884-891.
17 Mulligan, E. and Lahiri, S., Separation of carotid body chemoreceptor responses to 02 and CO2 by oligomycin and by antimycin A, Am. J. Pltvsiol., 242 (Cell Physiol. 11) (1982) C200-C206. 18 Oswald, R.E. and Freeman, J.A., Alpha-bungarotoxin binding and central nervous system nicotinic acetylcholine receptors, Neuroscience, 6 (1981) 1-14. 19 Sampson, S.R., Effects of mecamylamine on responses of carotid body chemoreceptors in vivo to physiological and pharmacological stimuli, J. Physiol. (London), 212 (1971) 655-666.