Effects of dopamine superfusion on the activity of rabbit carotid chemoreceptors in vitro

NeuroscienceVol. 16, No. 2, pp. 431438, Printed in Great Britain

0306-4522/85$3.00+ 0.00 Pergamon Press Ltd 0 1985IBRO

1985

EFFECTS OF DOPAMINE SUPERFUSION ON THE ACTIVITY OF RABBIT CAROTID CHEMORECEPTORS IN VITRO L.-M. LEITNER* and M. ROUMY Laboratoire de Physiologie, ERA CNRS No. 070846, 133, route de Narbonne, 31062 Toulouse Cedex, France Abstract-The effect of different concentrations (0.001, 0.01, 0.1 and 1mM) of dopamine on chemoafferent activity was studied in the rabbit carotid body superfused in vitro. Excitation was the sole effect observed: it was always present for dopamine tests at 0.1 and 1mM but was found in only 4 out of 9 tests at 0.01 mM and in 1 out of 5 tests at 0.001 mM. By comparison with a natural stimulus like hypoxia, dopamine excitation was delayed and had a much slower time course. Dopamine antagonists, ( + )-butaclamol and haloperidol did not affect the responses to dopamine and to hypoxia. The results were not significantly altered when CO, was added to the superfusing medium. It is concluded that dopamine is not a likely excitatory transmitter for chemoreception in the rabbit carotid body.

Recent biochemical investigations into the synthesis and release of dopamine (DA) by the carotid body studies of the type I cells”’ as well as physiological effects of catecholamine depletion on the responses to hypoxia lend support to the idea that DA plays a major role in the genesis of the chemoafferent

response to hypoxia. The possibility that DA is the excitatory transmitter at the synapse between type I cells and afferent nerve endings should be carefully examined.* Indeed, the increased release of DA during hypoxia” together with the increase in chemoafferent activity induced by exogenously applied DA in the rabbit carotid body in vitro24 support this possibility. However, the lack of effect of DA antagonists on the response to hypoxia,5,6.'9.28 as well as the disappearance of the excitatory effect of exogenous DA in catecholaminedepleted carotid bodies in vitro I8 contradict this proposal. In addition among DA receptors studied in the central nervous system, those having the lowest affinity for DA have a KM of about 1 PM and are thought by some authors to represent postsynaptic DA receptors. 26 Such a KM value did not compare with the concentrations of DA injected into the stream of fluid superfusing the rabbit carotid body in vitro24 (2-20 mM). Although dilution undoubtedly occurred, a dilution factor of 100 would still yield concentrations from 0.02 to 0.2mM. It is therefore worthwhile investigating the responses of chemoafferent units to superfusions with DA solutions of known concentrations, to test whether or not the sensitivity to DA of chemoafferent units is consistent with its action on a postsynaptic DA receptor. Our interest was also focused on the time course of the *To whom correspondence

should be addressed.

Abbreviation: DA, dopamine.

response to DA and whether or not it compares with the time course of the response to hypoxia. These experiments were also aimed at determining why the inhibitory effect of DA observed in vivo6 was not seen in vitro.24 We investigated whether the lack of CO2 in the superfusing medium in the in vitro experiments could be the source of this discrepancy. EXPERIMENTAL PROCEDURES The right carotid body with its Hering’s nerve were excised from 43 rabbits, under sodium pentobarbital (Clin-Midy, 40mg/kg iv.) anesthesia. A detailed description of the dissection of the carotid body and of the methods for recording chemoafferent activity have already been published.” The preparations were superfused, at a flow rate of 1.5 ml/min, 4th a medium heated to 37°C and containing (in mMj: NaCI. 1IO: KCI. 5: M&I,. 0.5: CaCl,. 2.2: elucose. 5.5; s&rose, jq; HEPEs Buier -~N-i-hydroxyetj;ylpiper: a&e-N’-2-ethanesulfonic acid), 5 @H was adjusted to 7.40 with N-NaOH at room temperature). Normoxia, hypoxia and hyperoxia were obtained by equilibrating this solution with air (PO,, 18.9-19.3 kPa), -lOvO0, in I$ (PO,, 10% 11.4kPa1. 100% 0, (85.8-96.8 kPaj. resnectivelv. In all cases the mea&red fCd,‘was less than 6.4 k’Pa. Whkn the superfusing medium was changed, the time lag due to the dead space of the experimental set-up was approximately 1min. Two media, equilibrated at 37°C with, respectively: 5% CO,, 20% O,, 75% N, and 8% CO,, 20% 0, and 72% N, were prepared. They contained HEPES Buffer (5 mM) and NaHCO, and had a pH of 7.40. The amount of added NaHCO, was calculated using the CO, solubility coefficient and H&O, dissociation constantsI and the NaCl concentration was reduced accordingly. These media were permanently recirculated to prevent CO, desaturation. Dual measurements for the 5% CO2 medium gave the following values: 18.4-18.5 kPa for PO_,, 4.44.5 kPa for PCO, and 7,39-7.39 for pH. In the 8% CO, medium PO, was: 21.121.3 kPa, PCO, was 8.0-8.1 kPa and pH was 7.37-7.38. Dopamine concentrations used were 1mM, 0.1 mM, 0.01 mM and 0.001 mM for each experiment. Two ml of a 50 mM DA (Dopamine HCI, Sigma) solution were prepared with glucose (0.3 M) as an antioxidant and the solution was

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protected from light with aluminum foil. This solution was diluted with the superfusing medium to give DA concentrations of 0.001-0.1 mM. The glucose concentration of these test media was increased by at most 0.6 mM and their osmolaiity was not significantly altered compared to that of the normal superfusing medium. When DA concentratjons of 1mM or more were to be assayed. glucose was omitted from the stock solution which was kept at 4 ‘C to minimize oxidation. Prior to each superfusion the necessary amount of stock solution was diluted with the superfusing medium, placed in a dark amber flask and kept at room temperature. The level of light was reduced as much as possible during superfusions with the DA-containing medium. No reducing agent was added to the test DA solutions since: (1) they stimulate chemoafferent activities” (also. Leitner and Roumy, unpublished observations) and (2) ascorbic acid inhibits binding of DA agonists and antagonists to DA receptors.” Furthermore, in the present experiments DA solutions were used in most cases for less than 15 min. thus it is unlikely that oxidation of DA significantly lowered its concentration in the test media. ~laloperidol (Janssenf, ( + )-butaclamal hydrochloride (Ayers& chlorpromazine hydrochloride (Sigma) and ( + )-propranolol hydrochloride (Sigma) were used. Results are presented as mean k the standard error of the mean @EM). RESULTS Effects of dopamine superfusions Normoxic and CO,-free medium. Because of the very slow time course of the responses to DA superfusion it was not possible to measure significant dose-response relationshjps. To test 3 different DA concentrations with the same nerve filament would have taken about 3 h and it would have been particularly difficult to maintain constant recording conditions for such a period of time. Consequently we will describe the responses to different DA concentrations used in several preparations. The chemoafferent responses to 1 m&I DA superfusions, lasting 2-5 min, were tested in 11 multiunit filaments and in all cases an increase in discharge frequency was recorded. The latency for the increase

t

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M. ROUMY

in discharge was 108 + 19 s (n = I I). The responses were variable in time course and magnitude: in 7 multiunits filaments the increase in discharge after 120-150 s DA superfusion was 12.2 f 2.5 imp/s, while in the 4 other filaments excitation was only recorded after longer DA superfusions. A response to 1 mM DA superfusion is displayed in Fig. 1 together with the response to hypoxia recorded in the same multiunit filament. The discharge increased 114 s after the start of DA superfusion (against a latency of 78 s for the hypoxic response) and the rate of increase of discharge frequency was as large as during hypoxia. However, in 4 recordings in which the increases in discharge during hypoxic and DA superfusions were sufficiently linear to be compared, the rate of increase of discharge during DA test was 58 f 20% of that recorded during hypoxic superfusion (range 1693%). In IO multiunit filaments, there was no evidence that a new steady level of discharge had been reached by the end of DA superfusions lasting 2-5 min (Fig. I). Only in one instance did the frequency of discharge reach a plateau before the end of a 5-min DA superfusion. It is clear in Fig. 1 that when hypoxic superfusion was terminated, the discharge quickly returned to its control value, whereas after terminating DA superfusion, the frequency decreased very slowly and it took 22 min to attain its control values. The range of recovery times was 15-30 min. In all 16 multiunit filaments, the responses to 0.1 mM DA superfusions, lasting 5-30 min, varied in amplitude and time course in different recordings. The increase in discharge occurred with a mean latency of 190 rfi 28 s (n = 16). The increase in discharge after 5 min DA superfusions was 7.7 ? 3 imp/s (n = 16), i.e. it was extremely variable. In 6 of these multiunit filaments, the increase in discharge was 1 + 0.4impjs after 5 min DA and it increased to 8.9 f 5 imp/s after 10 min superfusion. A response to DA is displayed in Fig. 2 together with the

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Time (min) Fig. 1. Comparison between chemoreceptor responses to 90-s hypoxia (left) and 150-s superfusion with 1 mM DA (right) recorded in the same multiunit filament.

Effects of dopamine on carotid chemoreceptor activity

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DOPAMINE, 0.1 mM

1

10 Time(min)

Fig. 2. Chemoreceptor responses to 90-s hypoxic stimulation and to IO-min DA 0.1 mM superfusion recorded in the same multiunit filament. Note transient increase in discharge when DA superfusion was ended.

response to hypoxia recorded in the same multiunit filament. The increase in discharge occurred much later than during hypoxia and the rate of increase in discharge was smaller. In 5 multiunit filaments where they could be compared, the rate of increase of discharge during DA superfusions was 11 + 3.5% (n = 5) of that observed during hypoxic stimulation. In 15 filaments DA superfusions lasted 5-l 1 min and in all cases but one there was no evidence that the discharge had reached a steady level by the end of DA superfusion. In one case a plateau was attained after 5.5 min superfusion. In one filament, a 30-min DA superfusion was performed and a plateau was reached after 18 min. The discharge remained stable at this new level until DA was removed from the medium when the discharge frequency returned very slowly towards its control value. Recovery times varied from 16 to 35 min. This again contrasted with the very fast decrease in discharge recorded after the end of hypoxic stimulation (Fig. 2). In 8 filaments a transient increase in discharge was recorded when DA superfusion was ended (see Fig. 2). Responses to 0.01 mM DA superfusions lasting 8-18 min were tested in 9 multiunit filaments and 4 of them exhibited an increase in discharge. The responses were similar to those obtained with 0.1 mM DA. In 5 other nerve filaments no significant changes in discharge frequency were seen although they responded to 0.1 mM DA superfusions. Further decreasing DA concentration to 0.001 mM resulted in a slowly developing excitation in only one out of the 5 nerve filaments tested. No responses were ever observed with lower DA concentrations. Hyperoxic and CO,-free medium. When DA (0.01 mM, 0.1 mM) was applied in an hyperoxic medium, the discharge frequency did not increase during DA superfusion (Fig. 3). However, upon return to the normoxic medium, without DA, an excitation developed that decayed slowly (Fig. 3). This excitation did not exist when DA superfusion

was omitted. Since oxidation of DA could have been accelerated by the hyperoxic medium, this result was checked in two other ways. First DA (0.01, 0.1 mM) was dissolved in the normoxic medium and the usual excitation recorded. Then the DA-containing medium was replaced by an hyperoxic one (without DA): it resulted in a very fast decrease in discharge frequency to the very low values normally found during hyperoxia. Second, the level of oxygenation of the carotid body was altered by varying the flow speed of the superfusing medium: increasing the flow from 0.85 to 2.1 ml/min resulted in increased latency, decreased magnitude and faster recovery phase of the response to DA. Thus increasing the oxygen partial pressure decreased the chemoreceptor response to DA. Normoxic hypercapnic medium. Superfusions with a medium equilibrated with 5 or 8% CO2 at pH 7.40 led to large increases in discharge. Although partial adaptation was present, total adaptation was not observed and a steady level of discharge was maintained for more than one hour in the longest recordings (as previously observed)‘. Dopamine superfusions (0.1, 1 mM) never caused inhibition of discharges. The excitation reported in the absence of CO* was still present and even enhanced in a few instances (Fig. 4). However, particularly at 0.1 mM concentrations, the increase in discharge was small and frequently difficult to see.

Effects of dopamine antagonists Normoxic and C02-free medium.

Butaclamol and haloperidol Superfusions with either ( + )- or ( - )-butaclamol (0.2 pM-O.5 PM) or haloperidol(0.5 PM) for as long as 30 min had no effect on the normoxic frequency of discharge. The carotid body was made hypoxic until a steady level of discharge was reached and the active enantiomer ( + )-butaclamol (0.5 @M) or haloperidol

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ROUMY

-1

NORMOXIA

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DOPAMINE,O.O 1 mM f-

h 7 u! E” i5 5; IO 5 2 t

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20 Timetmin)

Fig. 3. Effects of hyperoxia on the chemoreceptor response to DA. At the first arrow the preparation was superfused with the hyperoxic medium. After 150-s hyperoxia, DA (0.01 mM) was added to the hyperoxic medium and superfused for IOmin. At the end of the period. the carotid body was superfused with a normoxic medium without DA. The inset (time base, 40 s) shows the action potentials recorded during the period indicated on the graph by the upward arrowheads.

(0.5 PM) was added to the hypoxic medium and then superfused for 20 min. However, no changes in the

0

10 TFme (mid

Fig. 4. Chemoreceptor response to a 1 mM DA superfusion (5 min) in CO,-free medium (upper trace) and with an 8% CO,-enriched solution at pH 7.4 (lower trace). Note enhancement of the excitatory response to DA by the CO,-enriched medium and absence of inhibitory effect of DA.

frequency of discharge were recorded. After the response to 0.1 mM DA superfusion had been recorded as a control, a 20-min ( -t- )-butaclamol or haloperidol superfusion was started when the frequency of discharge had returned to its control value. Afterwards, DA and the antagonist were superfused, but there was no significant change in the response to DA (Fig. 5). The concentration of ( + )-butaciamo1 was once increased to 5 ph4 in one experiment without producing any effect. Other antagonists During suFer~~s~5~ with chZorpro~uzj~e (2-10 ~1M) the frequency of discharge increased. However, at concentrations from 20 to 100 PM it was followed by a decrease in discharge and complete cessation of activity was obtained in about 10 min with IOOpM and in about 30min with 20pM chlorpromazine. This block of activity lasted approximately 90min after a 10 min superfusion with 100 PM, after which the activity slowly recovered. During these 90min, the chemoafferent units did not respond to hypoxia, DA (I mM) superfusion or injection of KC1 (110 mM) close to the carotid body. Perphenazine dose had the same effects and propranolol (100 PM) a P-blocker produced the same changes in discharge, including cessation of activity, as chlorpromazine did. However, the block of activity by chlorpromazine was accompanied by a decrease in spike amplitude. Finally, we had the opportunity to record activity from a few mechanoreceptor units located inside the carotid body. After 6min superfusion, chlorpromazine (100 PM) produced a reversible block of the mechanosensitive discharge.

Effects of dopamine on carotid chemoreceptor

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Time (min) Fig.5.Chemoreceptor Normoxic-hypercupnic

responses to DA 0.1 mM, before (left) and during (right) superfusion with 0.5 PM ( + )-butaclamol for 20 min. medium.

( + )-Butaclamol

(up to 1 PM) and haloperidol (up to 1 ,uM) had either no effect upon the frequency of discharge, or they caused a transient decrease of activity (Fig. 6, lower

trace). In no case there was a suppression of the response Lo COz. The inactive stereoisomer (- )butaclamol produced only small variable changes in discharge frequency (Fig. 6, upper trace).

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Time (min) Fig. 6. Effects of I PM ( - )- and ( + )-butaclamol on chemoafferent activity recorded from same multiunit nerve filament from carotid body superfused with 8% CO1 in air. Dotted lines represent mean frequency of discharge during 100 s preceding the test.

L.-M. LEITNERand M. ROUMY

436 DISCUSSION

The present work agrees with a previous study,24 to the extent that DA has only excitatory effects in the rabbit carotid body in vitro. The DA-induced inhibition of chemoafferent activity found in vivo could not be demonstrated in the present experiments, whether or not superfusing medium contained CO*. The only indication that endogenous DA could exert an inhibitory influence on chemoafferent activity was the slight excitation observed during superfusion with chlorpromazine. However, its physiological significance is rather dubious since other DA antagonists (haloperidol, ( +)-butaclamol) had no effects. An excitatory influence of endogenous DA has been suggested on the basis that benztropine, a DA-uptake inhibitor, and pargyline, a monoamine oxidase inhibitor, potentiat~ the chemor~eptor response to cyanide in the cat carotid body in V~VO.~ This could be in keeping with the decrease in chemoafferent discharge induced by ( + )-butaclamol in the presence of CO2 (Fig. 6, lower trace). However, this effect was inconstant and transient and cannot be taken as an argument favoring an excitatory effect of endogenous DA. Furthermore, DA uptake in the carotid body is of the low-affinity type” and it is not established that if benztropine inhibits it, it would significantly increase the DA concentration in the extracellular space. It is therefore difficult to draw any firm conclusion concerning the effects of endogenous DA on the chemoafferent activity in the rabbit carotid body. There is now convincing evidence in the literature that no diffusion barrier prevents DA or DA-related drugs from entering or escaping quickly from the extracellular spaces of the rabbit carotid body in vitro. First, binding of labelled antagonists revealed a single type of DA receptors with an equal affinity for the labelled ligands, whether the experiments were performed with the whole organ incubated in vitro3 or with particulate membrane fractions isolated from the carotid bodyt3 demonstrating the lack of steadystate concentration gradient for the labelled antagonist in the incubated organ. Second, the half time for the washout of [‘Hlinulin is about 1 min.9 Third, when a carotid body which has been synthesizing [3H]DA is superfused for Smin with an N2-equilibrated medium, the large amount of released [‘HIDA is immediately recovered in the No-equilibrated medium demonstrating that DA released from type I cells by natural stimuli quickly gains access to the medium superfusing the carotid body.’ Therefore a possible diffusion barrier cannot explain the slow time course of the DA responses and the low sensitivity of chemoafferent units to this catecholamine. Does DA induced chemoafferent excitation require the binding of DA to a specific dopaminergic receptor? Direct study of the binding of [‘Hlspiroperidol to the rabbit carotid body in vitro has shown the existence of only one type of DA receptor with high

affinity (EC,= 0.16 nM) for the antagonist3 and it has been demonstrated that this receptor was of the D, type. 22 This justified our use of haloperidol (more potent antagonist of D, than of D, receptors)16 and of ( + )-butaclamol (equipotent for both types).” Binding of [3H]spiroperidol reaches an equifibrium after 20 min exposure to spiroperidol which, even at near saturation concentration, was totally displaced from the receptor by 0.2 p M ( + )-butaclamoL3 In the present experiments, the antagonists were superfused for at least 20min before testing DA or hypoxic superfusions so that the equilibrium between the DA receptor and the antagonist could be achieved. Minimal butaclamol and haloperidol concentrations were 0.2pM, i.e. sufficient to interfere strongly with the binding of DA to the DA receptor. However they did not modify DA-induced chemoafferent excitations which cannot be due to the interaction of DA with the receptor identified in biochemical studies. The low sensitivity of the chemoafferent units to DA (half of the units were unaffected by 0.01 mM DA superfusions) contrasts with the high affinity of the d,-receptors for DA (iu, in the nM range) and further supports this conclusion. When the inhibitory action of DA upon cat and rabbit chemoafferent activity in viva has been antagonized by haloperidol, DA induced an excitation suppressed by B-adrenergic antagonists, propranolol and metroprolol which also blocked or strongly reduced the response to hypoxia.” Other authors’7,“0 reported a much Iesser reduction of the response to hypoxia by these two P-antagonists in both cat and rabbit carotid bodies in oivo. However, it must be noted that most P-antagonists have membranestabilizing properties and although metroprolol is much less potent than propranolol in this respect, it is not entirely devoid of local anesthetic properties.’ We presented clear evidence that both propranolol and chlorpromazine blocked chemoafferent activity by a local anesthetic action as already shown for dichloroisoproterenol in the cat carotid body in citm2’ The /i’-adrenoreceptor is linked to adenylate cyclase in virtually all tissues.” Isoprenaline stimulates adenosine 3’,5’-phosphate production in the rabbit23 and rat*’ carotid bodies, an effect suppressed by @,-antagonists in the rat carotid body.” However, neither DA nor hypoxia altered the adenosine 3’S’-phosphate production,*’ suggesting that DA is not acting on /%-receptors which do not seem to be implicated in the response to hypoxia. It is expected that further direct characterization of the @-receptors in the carotid body will help to interpret these conflicting results. Binding of DA to a membrane receptor is certainly not the only phenomenon that should be considered in trying to explain the excitatory effect of DA. It has been demonstrated that DA is taken up by the rabbit carotid body in vitro, most likely by the type I cells.” This uptake has low affinity for DA (KM = 0.564 mM), a y,,,,, of 1804 pmol/mg protein/min and DA

Effects of dopamine on carotid chemoreceptor accumulation is linear up to 15 min at 0.01 mM DA and up to 4min at 0.9mM DA.13 Thus under our

experimental conditions, DA uptake undoubtedly occurred and the results obtained in the rabbit carotid body incubated in vitro are directly applicable to our experimental situation. If DA had to be taken up to excite chemoafferent units, then a KM of 0.564 mM would explain the rather low sensitivity of chemoafferent units to exogenous DA. It would also account for the faster increase in discharge when DA concentration was raised from 0.1 to 1 mM. If we assume that the carotid body contains about 200 pmol DA*’ and 50-6Opg proteins, we can estimate the amount of DA taken up under our experimental conditions.

For

10min

superfusions

or 0.01 mM it would amount control 60pmol

with DA 0.001

to 1.8 pmol (1% of

content) and 18 pmol (9%), respectively; (30%) for 4 min 0.1 mM DA and 135 pmol

(67%) for 2 min, 1 mM DA superfusions.

It is clear

activity

43-l

then that at DA concentrations which consistently excited chemoafferent units (0.1-l mM), a large amount of DA would be expected to be taken up. That DA excited chemoafferent units through an action on type I cells has already been suggested” on the grounds that excitation induced by exogenous DA was abolished in catecholamine depleted carotid bodies. It might be also in keeping with the suppression of DA excitatory effect by hyperoxia. How DA taken up by type I cells would excite chemoafferent units has yet to be discovered. Acknowledgements-We

thank Ayerst Research Laborato-

ries for the gift of butaclamol and Janssen Pharmaceutics for the gift of haloperidol. We also express our thanks to Drs C. Eyzaguirre, J. Lamarche and A. Verna for their helpful comments and criticisms of the manuscript and Mrs M. Racaud for typing the manuscript. This research was supported by the Centre National de la Recherche gcien t’fi 1 que and the Conseil Scientifique, Facultt de

Mkdecine Toulouse-Purpan.

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