Tetrodotoxin-resistant response to nicotine in rabbit bronchial preparation

European Journal of Pharmacology, 104 (1984) 351-356

351

Elsevier

T E T R O D O T O X I N - R E S I S T A N T R E S P O N S E T O N I C O T I N E IN R A B B I T B R O N C H I A L PREPARATION ISSEI TAKAYANAGI *, YASUO KIZAWA and TSUYOSHI HIRUTA Department of Chemical Pharmacologic', Toho University School of Pharmaceutical Sciences, Miyama, Funabashi, Chiba 274, Japan

Received 21 February 1984, revised MS received 8 May 1984, accepted 26 June 1984

I. TAKAYANAGI, Y. KIZAWA and T. HIRUTA, Tetrodotoxin-resistant response to nicotine in rabbit bronchial preparation, European J. Pharmacol. 104 (1984) 351-356. The mode of action of nicotine was studied in a rabbit bronchial preparation. Nicotine (3 X 10 5-10 3 M) produced a phasic contraction. No inhibitory response to nicotine was observed. The contractile response to nicotine was inhibited by hexamethonium, pentolinium and atropine but not by tetrodotoxin. Nicotine increased the efflux of tritium from preparations which had been labelled with [3H]choline. Tetrodotoxin did not inhibit the nicotine-evoked tritium release from the bronchial preparation. The results indicate that the release of acetylcholine evoked by nicotine was not influenced by tetrodotoxin in this preparation, and that the nicotine-induced response may be produced mainly through a sodium action potential-independent process. We could not rule out a contribution by acetylcholine released from the acetylcholine store in the smooth muscle in the contractile mechanisms for nicotine. Sodium potential-independent process

Nicotine

1. Introduction N i c o t i n e has been shown to p r o d u c e a biphasic response in the guinea-pig trachea consisting of an initial c o n t r a c t i o n followed by a large relaxation, this biphasic response to nicotine being blocked by tetrodotoxin a n d h e x a m e t h o n i u m (Jones et al., 1980). However, it was recently f o u n d that the contractile responses of the m a i n bronchial strip from guinea-pig to nicotine a n d a nicotinic stimulant, 1 , 1 - d i m e t h y l - 4 - p h e n y l p i p e r a z i n i u m iodide ( D M P P ) were inhibited by the ganglionic blockers h e x a m e t h o n i u m a n d p e n t o l i n i u m b u t not by atropine a n d tetrodotoxin, suggesting that the contractile response to nicotine a n d D M P P did not involve acetylcholine release ( T a k a y a n a g i a n d Kizawa, 1983; T a k a y a n a g i et al., 1984; Kizawa a n d Takayanagi, 1984). I n order to o b t a i n additional evidence a b o u t the m e c h a n i s m of action of

* To whom all correspondence should be addressed. 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.

Rabbit bronchus

Tetrodotoxin

Cholinergic nerve

n i c o t i n e on bronchial muscle, we have studied the action of nicotine o n the m a i n bronchial preparation of rabbit•

2. Materials and methods 2.1. M e c h a n i c a l r e s p o n s e

Male rabbits ( 2 - 3 kg in b o d y weight) were killed by a blow on the head a n d exsanguinated. Their m a i n b r o n c h i were dissected free from lung tissue a n d cut helically. The bronchial preparations (about 30 m m long a n d 2 m m wide) were suspended in a 20 ml organ bath filled with K r e b s solution (NaCI 118, KCI 4.72, CaC12 2.56, MgSO 4 • 7 H 2 0 0.16, K H z P O 4 1.20, N a H C O 3 25.0 a n d dextrose 10.0 m M , p H 7.41), kept at 3 7 ° C a n d gassed with carbogen. Responses were recorded isotonically u n d e r a tension of 0.5 g. In some experiments two p l a t i n u m electrodes (2 x 35 ram) were set at intervals of 5 m m a n d field s t i m u l a t i o n

352 of the bronchial preparation was carried out by passing a rectangular pulse of 1 ms duration, supramaximal voltage and a frequency of 5 Hz between two electrodes for 10 s. The experiments were started after the preparations had been allowed to develop spontaneous tone for 90 min. All agonists were applied to the bronchial preparation at intervals of 60 min. To study the effects of antagonists, the preparation was preincubated with antagonists for th~ time shown in table 1. 2.2. Estimation o f acetylchofine concentration released

Acetylcholine release from the bronchial preparation was estimated by the method of Kilbinger et al. (1982). The bronchial preparation was suspended in a 5 ml organ bath with two electrodes set at 5 mm intervals, filled with Krebs solution, kept at 37 °C and gassed with carbogen. The preparation was under a tension of 0.5 g and was allowed to equilibrate for 45 min then incubated for 90 min in Krebs solution containing 8 × 10 5 C i / m l [3H]choline. The total concentration of labelled and nonlabelled choline was 10 6 M. During the 90 rain incubation with [3H]choline, field stimulation was carried out by passing rectangular pulses of 2 ms duration, supramaximal voltage and a frequency of 0.2 Hz between two electrodes for 30 rain to increase the uptake of [3H]choline. The mechanical responses to field stimulation and to nicotine were simultaneously recorded isotonically. After the 90 min incubation, the preparation was washed with Krebs solution at a flow rate of 2 m l / m i n for 60 min. The preparation was then constantly superfused with Krebs solution in the 5 ml organ bath at a rate of 2 m l / m i n . The composition of Krebs solution used for superfusion was the same as that described above, except that it contained hemicholinium-3 (10 5 M) in order to prevent reuptake of choline. Field stimulation or application of nicotine was done at 20 and 50 min after 60 min washing with Krebs solution. Field stimulation was carried out under the same experimental conditions as described in the series of experiments involving mechanical responses. To measure the increased efflux of tritium evoked by field stimulation or by

nicotine, one ml of the superfusate was collected for 1 min and added to 10 ml of a Triton-toluenescintillator which contained Triton X-100 500 ml, 2,5-diphenyloxalate (PPO) 4 g, 1,4-bis[2-(4-methyl5-phenyloxazolyl)benzene (POPOP) 0.1 g in one litre of toluene and radioactivity was measured with a liquid scintillator spectrometer. In control experiments, where a test drug (tetrodotoxin) was not used, the efflux of tritium was found to decline during repeated stimulations (field stimulation or nicotine). In a series of test experiments, the first stimulation was applied in the absence of tetrodotoxin and the second stimulation in the presence of tetrodotoxin. The ratio was calculated of the efflux of tritium during the second stimulation in the presence of tetrodotoxin to the efflux during the first stimulation in the absence of tetrodotoxin. The effect of tetrodotoxin was determined by comparing the ratio in the test experiments with that in control experiments. Drugs used: nicotine bitartrate (Nakarai), acetylcholine chloride (Daiichi), hexamethonium dibromide (Tokyo-Kasei), physostigmine salicylate (Merck), atropine sulfate (Sigma), d-tubocurarine chloride (Nakarai), tetrodotoxin (Sankyo), chlorpheniramine maleate (Sankyo), indomethacin (Sigma), prazosin hydrochloride (Pfizer), methysergide dimaleate (Sandoz), 2-(1,2-benzisoxazol-3yl)-3-[2-(2-piperidinoethoxy)phenyl]acrylonitrile (SX-284, Dainippon; Takayanagi et al., 1982), hemicholinium-3 hydrate(Aldrich) and [methyl3H]choline chloride (specific a c t i v i t y = 8 0 C i / mmol; New England Nuclear), all in powder form. All drugs were used as solutions in double-distilled water. Other chemicals used were of analytical grade.

3. Results

3.1. Mechanical response

Contractile responses to nicotine were reproducible under the experimental conditions used. Nicotine produced a phasic contraction in the rabbit bronchial preparation (fig. 1), though a tonic contraction had been observed in the guineapig bronchial preparation (Takayanagi and

353

~

mm

° lOmin i

/ 0 •/!1 10-5

Pap 10-4M

Nc 3x10-SM

Nc 10-4M

Nc 3x10-4M

Nc 10-3M

\

Fig. 1. Mechanical response of rabbit bronchial preparation to nicotine(Nc) and papaverine(Pap). Note that nicotine induced only a contractile response concentration dependently.

Kizawa, 1983; Takayanagi et al., 1984; Kizawa and Takayanagi, 1984). A concentration-response curve for nicotine was obtained at concentrations of 3 × 1 0 - 5 - 1 0 -3 M and the maximum response to nicotine was 51.2_+ 4.3% (mean_+ S.E. of 14 experiments) of that to acetylcholine. No inhibitory response to nicotine (3 x 1 0 - s - 1 0 -3 M) was observed in any preparation while all were greatly relaxed by papaverine (10 -4 M) (fig. 1). Hexamethonium (10 -6 M) produced a parallel shift to the right of the concentration-response curve for nicotine (fig. 2). The action of nicotine was inhibited non-competitively by atropine (10 9 M), suggesting that the response to nicotine was mediated through the release of acetylcholine from cholinergic nerves. Equieffective concentrations of nicotine (3 × 10 4 M) and acetylcholine (3 x 10 -6 M) were used as agonists in the experiments to be described next. The response to nicotine was considerably inhibited by hexamethonium (10 6 M) or pentolinium (10 -6 M) and was abolished by application of either drug at 10 s M. The response to nicotine was abolished by atropine (10 7 M) as was the acetylcholine-induced contraction (3 × 10 6 M). Atropine (10 7 M) also almost completely inhibited the response to field stimulation

16 4

10-3

16 2

Nicotine (M)

Fig. 2. Effects of hexamethonium on concentration-action curve of nicotine in a rabbit bronchial preparation. • nicotine alone; O with hexamethonium 10 -6 M. Ordinate: % of the contraction of the bronchial preparation by acetylcholine 10 -3 M and abscissa: concentration(M) of nicotine. Each value is presented as a mean + S.E. (bar) of 4 experiments.

(tables 1 and 2). A 30 min pretreatment with physostigmine (10 6 M) significantly potentiated the responses to nicotine, acetylcholine and field stimulation (tables 1 and 2). However, the response to nicotine was not influenced by tetrodotoxin (3 x 10 -6 M) (table 2) or by SX-284 (3 x 10 7 M), which inhibits acetylcholine release from parasympathetic nerves (Takayanagi et al., 1982; Sone and Takayanagi, 1983). The H 1a n t a g o n i s t , chlorpheniramine (10 6 M), the cyclooxygenase inhibitor, indomethacin (10 6 M), the al-adrenoceptor inhibitor, prazosin (10 7 M), and the antiserotonin agent, methysergide (10 7 M) all were without effect on the response to nicotine (table 1). The concentration (10 -6 M) of indomethacin was sufficient to block cyclooxygenase activity but not to influence the spontaneous tone of the smooth muscle (Anderson et al., 1979).

3.2. Estimation of acetylcholine release 3.2.1. Response to nicotine Nicotine (3 x 10 -4 M) was applied to the bronchial preparation for 3 min. In control experiments the ratio of the efflux of tritium in the first application to the efflux in the second was 0.78 + 0.10 (mean + S.E. of 4 experiments), while the mechanical response was not influenced by repeated applications of nicotine. Typical tracings

354 TABLE 1 Effects of some drugs on the contractile response to nicotine. Each value is presented as a mean + S.E. of the n u m b e r of experiment~ s h o w n in parentheses. * significant difference from the control value (100%) at P < 0.05. T r e a t m e n t (M)

I n c u b a t i o n time

N i c o t i n e (3 × 10 4) + hexamethonium (10- 6) + h e x a m e t h o n i u m ( 1 0 - 5) + p e n t o l i n i u m (10 6) + p e n t o l i n i u m (10 5) + atropine (10 7) + p h y s o s t i g m i n e (10 6) + t e t r o d o t o x i n (3 × 10 - 6 ) + S X - 2 8 4 ( 3 × 1 0 -7) + c h l o r p h e n i r a m i n e (10 6) + i n d o m e t h a c i n (10 - 6 ) + prazosin (10- 7) + m e t h y s e r g i d e ( 1 0 - 7)

% of c o n t r a c t i o n 100.0

5 rain

5 5 5 5 30 15 5 5 30 5 5

mm mm mln mln min min min mm min mm mln

31.6+ 3.4"(7) 0.0 * (6) 6 0 . 0 + 5.5 * (5) 0.0 * (6) 0.0 *

(6)

1 7 8 . 2 + 1 1 . 9 * (6) 9 9 . 7 + 3.1(10) 101.2+ 1.0 (101 95.6_+ 2.2 (7) 97.4_+ 1.7 (8) 9 8 . 6 + 2 . 1 (8) 105.4_+ 1.5 (8)

TABLE 2 Effects of some drugs on the responses to acetylcholine a n d to field stimulation. Each value is p r e s e n t e d as the mean_+ S.E. of the n u m b e r of e x p e r i m e n t s s h o w n in parentheses. * significant difference from the control value (100%) at P < 0.05. T r e a t m e n t (M) A c e t y l c h o l i n e (3 × 10 6) +atropine (10 -71

+physostigmine (10 -6) Field s t i m u l a t i o n +atropine(10 -7) + t e t r o d o t o x i n (3 × 10 - 6 ) + p h y s o s t i g m i n e ( 1 0 - 6)

2001 150

~ ~

1oo

so

M

o

,;"'

1"o 2;)

3'0

% of c o n t r a c t i o n

5 min 30 m i n

100.0 0.0 * (4) 173.0+_ 5.9 * (6)

5min 15 min 30 rain

100.0 0.6+_ 0 . 6 " ( 5 ) 0.0 * (4) 503.9 _+ 20.6 * (4)

are shown in fig. 3. In the p r e p a r a t i o n treated with t e t r o d o t o x i n (3 x 10 6 M) for 15 min the ratio was 0.90 + 0.10. T h e ratios were not significantly different from each other, i n d i c a t i n g that the efflux of tritium or acetylcholine evoked by nicotine

5 ~rain

e T.

I n c u b a t i o n time

4o

fraction number

s'o

6'0

Fig. 3. A typical tracing of the efflux of tritium and c o n t r a c t i o n e v o k e d by nicotine in a rabbit bronchial preparation. The b r o n c h i a l p r e p a r a t i o n was i n c u b a t e d with [3H]choline and s u b s e q u e n t l y superfused with Krebs solution c o n t a i n i n 8 h e m i c h o l i n i u m - 3 at a rate of 2 m l / m i n . The superfusate was collected as 1 min samples and radioactivity was measured. The m e c h a n i c a l response was s i m u l t a n e o u s l y recorded isotonically. Superfusion with nicotine 3 × 10 -4 M is indicated by horizontal bars. Upper tracing: m e c h a n i c a l response and lower tracing: efflux of tritium, ordinate; efflux of tritium ( × 103 d p m / g tissue) and abscissa; fraction number. N o t e that the efflux of t r i t i u m was decreased by repeated a p p l i c a t i o n of nicotine.

355

was not influenced by tetrodotoxin. The ratio of the mechanical responses in the presence and absence of tetrodotoxin (3 x 10 6 M) was 0.90 + 0.05 in the same preparations.

3.2.2. Response to field stimulation In control experiments, the ratio of the efflux of tritium evoked by the first field stimulation to the efflux during the second stimulation was 0.83 + 0.07 ( m e a n _ S.E. of 4 experiments). The same ratio in the test experiments was 0.13 + 0.03. The ratios were significantly different from each other, indicating that the efflux of tritium or acetylcholine evoked by field stimulation was inhibited by tetrodotoxin (3 × 1 0 - 6 M). The mechanical response to field stimulation was also blocked by tetrodotoxin (3 × 1 0 - 6 M ) .

4. Discussion Nicotine induced only a contraction in the rabbit bronchial preparation. This contractile response to nicotine was abolished by atropine and ganglion blockers. These results suggest that the contractile response to nicotine was mediated through an action on the nicotinic receptor and was due to acetylcholine release. Furthermore, the present study (table 1) s aggests that the contractile response to nicotine was not due to release of prostaglandins, histamine, serotonin or noradrenaline. However, the mechanical response and the efflux of tritium or acetylcholine evoked by nicotine were not influenced by tetrodotoxin. In other tissues, tetrodotoxin was also reported not to inhibit the nicotine-induced response, e.g. in rat vas deferens, cat splenic strip and guinea-pig aorta (Su and Bevan, 1970; Jayasundar and Vohra, 1978; Ikushima et al., 1981) and dog cerebral artery (Muramatsu et al., 1980). On the other hand, tetrodotoxin abolished the responses to nicotine in tissues such as guinea-pig trachea, ileum and aorta, rat vas deferens and rabbit ear artery. These observations suggest that there are two mechanisms involved in the transmitter release induced by nicotine, one sodium action potential-dependent and the other potential-independent (Ikushima et

al., 1982). In the rabbit bronchial preparation, the nicotine-induced response could be mainly produced through the latter mechanism, the sodium action potential-independent process, as this was resistant to tetrodotoxin and SX-284. However, a possible contribution to acetylcholine release from the acetylcholine store in the smooth muscle could not be ruled out in the contractile mechanisms for nicotine. This mechanism for acetylcholine release by nicotine may be tetrodotoxin-resistant. Furthermore, endogenous prostaglandins seem not to regulate this mechanism, since indomethacin did not influence the nicotine-induced contraction (table 1). In previous reports (Takayanagi and Kizawa, 1983; Kizawa and Takayanagi, 1984; Takayanagi et al., 1984) the nicotine-induced contraction in the guinea-pig bronchial preparation was not influenced by atropine, tetrodotoxin, SX-284 or physostigmine, though it was abolished by hexamethonium and pentolinium. These findings suggest that in the guinea-pig bronchial preparation, nicotine did not bring about its effect by stimulation of cholinergic ganglion cells. The mechanism for nicotine-induced contraction in the bronchial preparation therefore, seems to vary with the species.

Acknowledgement This research was supported by a grant from the Japan Monopoly Corporation.

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