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Developmental changes in the response of rat isolated duodenum to nicotine
European Journal of Pharmacology, 251 (1994) 75-81
75
© 1994 Elsevier Science B.V. All rights reserved 0014-2999/94/$07.00
EJP 53450
Developmental changes in the response of rat isolated duodenum to nicotine K a o r u Irie *, K y o k o F u r u k a w a , T e r u k o N o m o t o , E m i k o Fujii a n d T a k a m u r a M u r a k i Department of Pharmacology, Tokyo Women's Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162, Japan Received 13 September 1993, revised MS received 12 October 1993, accepted 15 October 1993
Developmental changes in the response to ganglionic stimulants, nicotine and dimethylphenylpiperazinium, were investigated in rat isolated duodenum by recording isotonic mechanical activity. The duodenal response to nicotine/dimethylphenylpiperazinium (3 x 10 - 7 to 10 -3 M) in neonatal rats was contraction, which was blocked by hexamethonium, tetrodotoxin and hyoscine. The response to nicotine/dimethylphenylpiperazinium (10 - 6 to 10 - 4 M) in the adult duodenum was relaxation, which was blocked by tetrodotoxin and hexamethonium, but by neither guanethidine nor hyoscine. The transition of the response to nicotine/dimethylphenylpiperazinium from contraction to relaxation occurred at around the 3rd postnatal week. Nicotineinduced relaxation of adult duodenum was significantly inhibited by preincubation with a-chymotrypsin, a proteolytic enzyme, and a combination of nucleotide pyrophosphatase and 8-phenyltheophylline, a P1 purinoceptor antagonist. Nicotine-induced relaxation was desensitized by a,/3-methylene ATP, a stable P2x purinoceptor agonist. These results suggest that the contractile response of isolated duodenum to nicotine is mediated through cholinergic transmission in neonatal rats and the relaxant response is mediated through non-adrenergic, non-cholinergic transmission, which involves both peptidergic and purinergic transmission, in adult rats. Nicotine; Peptidergic transmission; Purinergic transmission; NANC (non-adrenergic, non-cholinergic) nerve; Developmental pharmacology
1. Introduction
We have been studying the developmental changes in the responses of isolated duodenum to various neurotransmitters. For example, contractile responses were induced in the neonatal duodenum by thyrotropin releasing hormone (TRH), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), ATP and y-aminobutyric acid (GABA), while relaxant responses to these biologically active substances were induced in the adult duodenum of rats (Tonoue et al., 1981; Furukawa et al., 1989, 1990; Furukawa and Nomoto, 1988, 1989). The contractions of neonatal duodenum induced by T R H , C G R P and VIP were suggested to be mediated through cholinergic transmission, because of blockade of the contraction by hyoscine (Tonoue et al., 1981; Furukawa and Nomoto, 1988; Furukawa et al., 1989). The relaxations induced by T R H , C G R P and VIP in the adult duodenum, however, were probably due to direct stimulation of smooth muscle because of their tetrodotoxin-resistant property
* Corresponding author. Tel. 03-3353-8111 ext. 22513, fax 03-52697417.
SSDI 0 0 1 4 - 2 9 9 9 ( 9 3 ) E 0 7 5 9 - L
(Tonoue et al., 1979; Furukawa and Nomoto, 1988; Furukawa et al., 1989). Manzini et al. (1985) have reported that dimethylphenylpiperazinium mimics non-adrenergic, non-cholinergic (NANC) relaxation induced by electrical field stimulation in rat isolated duodenum. Our previous studies suggest that ganglionic stimulants may again contract neonatal rat duodenum. We now studied the effects of nicotine and dimethylphenylpiperazinium on the isolated duodenum from 1-11-week-old rats, and found that the responses of duodenum to nicotine and dimethylphenylpiperazinium were contraction in neonatal rats and relaxation in adults. The mechanism of the response to nicotine was also investigated in both neonatal and adult rats. Preliminary reports have appeared (Irie et al., 1990a,b).
2. Materials and methods 2.1. Preparations and measurement of mechanical
activity of duodenum Male Wistar-Imamichi rats aged 1, 3, 4 and 9-11 weeks were used. The rats were housed in an air-con-
76 ditioned room (22 + 1°C and 55 + 5% humidity) with controlled light-dark cycle (6:00-20:00 h light on) and free access to standard chow and tap water. Pups were removed from the mother on the 22nd postnatal day. Neonatal (1 week old) and young (3 and 4 weeks old) rats were killed by decapitation and adult rats (9-11 weeks old) were stunned by a blow to the head and bled. Each duodenum was dissected out, and the proximal portion but not the bulb (1.5 cm for neonatal rats and 2.2 cm for young and adult rats) was set up in an organ bath containing 5 ml of modified Locke solution (composition in mM: NaC1 154, KCI 4.02, CaCI z 1.36, MgC12 0.9, N a H C O 3 2.97, glucose 5.56, p H 7.0) saturated with 95% 0 2 and 5% CO 2 and maintained with continuous bubbling of air at 32 °C. Mechanical activity of the duodenal segments under a load of 0.5 g (neonates), 0.75 g (young), and 1 g (adult) was recorded through an isotonic transducer (Nihonkohden TD112S) on a polygraphic recorder (Nihonkohden WT645G). After 1-h equilibration, the duodenal segments were exposed to nicotine/dimethylphenylpiperazinium for 2 min and allowed a 30-min recovery period for subsequent exposures to n i c o t i n e / d i m e t h y l p h e n y l piperazinium or various drug pretreatments. The medium was changed every 5th to 10th min during equilibration and recovery. The concentration-response curve was obtained from serial exposures to increasing concentrations of nicotine at 30-min intervals. The values presented are means + S.E.M. Student's t-test was used for statistical analysis. 2.2. Drugs The following drugs were used: nicotine tartrate (Wako, Osaka), hyoscine hydrochloride (scopolamine hydrochloride), adenosine 5'-triphosphate (ATP) disodium salt, a,fl-methyleneadenosine 5'-triphosphate (a,/3-methylene ATP) lithium salt, dimethylphenylpiperazinium iodide, a-chymotrypsin, nucleotide pyrophophatase and 8-phenyltheophylline (Sigma), guanethidine sulfate (Ciba-Geigy), hexamethonium chloride (Tokyo Kasei), tetrodotoxin (kindly supplied by Sankyo, Tokyo). 8-Phenyltheophylline was dissolved in dimethyl sulfoxide and a 5-~1 aliquot was added to the organ bath. O t h e r drugs were dissolved in physiological saline and a 30- or 100-/~1 aliquot was added to the organ bath.
3. Results
3.1. Mechanical response to nicotine Nicotine (3 × 10 -7 to 10 - 4 M ) elicited contraction of isolated duodenum in a concentration-dependent manner in neonatal rats (fig. 1). The p D 2 value, which
lW
Nc 10-6M
3x 106M
1(~-5M
3 x 10"5M
10"4M 1 min
100 " o~ 80" 60" 40" (-~ 20"
0
, , 3x10 7 106
, 3x106 Nicotine
, , 102 3x10 s
10"
(M)
Fig. 1. Response of duodenum isolated from neonatal rat to nicotine (10 -6
to
10 -4
M)
and
concentration-response
curve.
Mechanical
activity of the duodenum was recorded isotonically under a load of 0.5 g. A duodenal segment was exposed to nicotine for 2 rain at 30-min intervals. Values are means :t: S.E.M. (n = 6).
was obtained from the concentration-response curve, was 5.8 _+ 0.14 (n = 6). In contrast to neonatal rat duodenum, nicotine elicited concentration-dependent relaxation, which was transient at concentrations under 3 × 10 -4 M but became sustained at a higher concentration (10 -3 M), in adult rat duodenum (fig. 2). The p D 2 value of nicotine for transient relaxation was 5.4 _+ 0.04 (n -- 4).
Adult
Nc 106M
3x10~1
10-5M
3x10-SM
I0-4M
3xIO'4M 10-aM
J
, , , , , 106 3x106 10 s 3x10 s 10 ~ Nicotine (M)
Fig. 2. Response of duodenum isolated from adult rat to nicotine (10 -6 tO 10 -3 M) and concentration-response curve for transient
relaxant phase. Mechanical activity of the duodenum was recorded isotonically under a load of 1 g. A duodenal segment was exposed to nicotine for 2 rain at 30-min intervals. Values are means :t: S.E.M. (n = 4).
77
3.2. Mechanical response to dimethylphenylpiperazinium Dimethylphenylpiperazinium, like nicotine, elicited contraction of the d u o d e n u m in neonatal rats and relaxation in adults (fig. 3). Sustained relaxation was m u c h m o r e m a r k e d in the case of d i m e t h ylphenylpiperazinium-induced relaxation than nicotineinduced relaxation. In duodenum from 3-week-old rats, the first or several exposure(s) to dimethylphenylpiperazinium (even at a higher concentration of 3 × 10 -5 M) usually elicited contraction and then subsequent exposures to dimethylphenylpiperazinium elicited relaxation solely or as a biphasic response, i.e., relaxation followed by a contraction (fig. 3). This response variability may be due to tachyphylaxis for contractile responses produced by repeated exposures to dimethylphenylpiperazinium (see section 3.3). T h e response of duodenum to dimethylphenylpiperazinium was consistently relaxation in 4-week-old rats (fig. 3). A similar transition from contraction to relaxation was also observed in the duodenal response to nicotine in 3-4-week-old rats (data not shown).
1W
3xlO~M
3x10~
10"6M
3xlO-eM
10"5M
lO~
lo~
IO-'M
3W
j 3xIO'SM
4W
3x I 0"5M
I 0"~M
Adult
3"~1o~
lO~M
3x 1 0 " 6 M
"~,,~1~ lO'~
__11
Since the duodenal responses to nicotine and dimethylphenylpiperazinium were basically the same, nicotine was used for further studies on mechanisms.
3.3. Mechanism of nicotine-induced neonatal rat duodenum
contraction
of
R e p e a t e d exposures to nicotine at 30-min intervals led to tachyphylaxis of the contractile response to nicotine in neonatal rat duodenum (fig. 4a and b). The duodenum, however, recovered from tachyphylaxis after several exposures to nicotine, and thereafter consistently responded to repeated exposures to nicotine (fig. 4a and b). Therefore, the mechanism of nicotineinduced contraction of neonatal duodenum was examined after the tachyphylaxis of the contractile response disappeared. Nicotine (3 × 10 -6 to 10 -5 M)-induced contraction was inhibited by 30-min pretreatment with hexamethonium ( 4 × 10 -4 M, fig. 4a), a nicotinic ganglionic blocker. The contractile response to nicotine (3 × 10 -6 M) was abolished by 15-min pretreatment with tetrodotoxin (10 -6 M, fig. 4b), a Na + channel blocker, while tetrodotoxin produced only a partial inhibition of contraction elicited by a higher concentration of nicotine (10 -5 M, fig. 4d). The responses to nicotine were greater in saline pretreatments than those in previous exposures to nicotine in fig. 4a and b. These marked responses to nicotine with saline pretreatment may have resulted from a long incubation (20-35 min) of duodenum in unchanged medium, while the normal response to nicotine was examined 5 min after the baths were filled with fresh medium. Spontaneous activity was also getting more marked during a longer incubation period in unchanged medium (data not shown). Fifteen-minute pretreatment with hyoscine (2.5 X 10 -7 M) completely blocked nicotine (3 × 10 -6 M)-induced contraction of neonatal duodenum (fig. 4c) and the tetrodotoxin-resistant contraction to nicotine (10 -5 M) was also completely blocked by subsequent addition of hyoscine (fig. 4d), indicating cholinergic nerve mediation in the nicotine-induced contraction of neonatal duodenum. Inhibition by hexamethonium, tetrodotoxin and hyoscine was also confirmed for dimethylphenylpiperazinium-induced contraction (data not shown).
3.4. Mechanism of nicotine-induced relaxation of adult rat duodenum
lrn~
• DIdPP Fig. 3. Response of isolated duodenum to dimethylphenylpiperazinium (DMPP) in 1-, 3- and 4-week (W)-old rats and adult rat. Mechanical activity of the duodenum was recorded isotonically under a load of 0.5 g for 1 W, 0.75 g for 3 and 4 W, and 1 g for adult. A duodenal segment was exposed to dimethylphenylpiperazinium for 2 rain at 30-rain intervals.
R e p e a t e d exposure to nicotine (10 -5 M) did not lead to tachyphylaxis of the relaxant response of adult duodenum but elicited response of a constant magnitude, although the initial response was rather small (data not shown). Relaxation of duodenum elicited by nicotine (10 -5 M) was significantly inhibited by pre-
78
treatment with hexamethonium (4 × 10 - 4 M, 30 min) and tetrodotoxin ( 1 0 - 6 M , 15 min) (table 1). Nicotineinduced relaxation was not affected by pretreatment with a combination of guanethidine (6.4 × 10 - 6 M , 30 min) and hyoscine (2.5 x 10 - 7 M , 15 min) (table 1), indicating the mediation of NANC transmission. Transient relaxation induced by dimethylphenylpiperazinium was also inhibited by hexamethonium and tetrodotoxin, and was resistant to hyoscine and guanethidine (data not shown). We examined whether nicotine-induced relaxation of the duodenum was mediated by either peptidergic or purinergic neurons. Preincubation with a-chymotrypsin (3 units/ml, 15 min), a proteolytic enzyme,
significantly inhibited the nicotine (10 -5 M)-induced relaxation of duodenum (table 1). The concentration of a-chymotrypsin used (3 units/ml, 15 min) did not affect the resting tension or spontaneous activity of duodenum. ~-Chymotrypsin did not inhibit ATP ( 1 0 - 6 M)-induced relaxation (105 + 7.1% of control, n = 3), indicating that the inhibitory effect of a-chymotrypsin on nicotine-induced relaxation is not non-specific but due to its proteolytic effect. Nicotine (10 -5 M)-induced relaxation was also inhibited by preincubation with a combination of nucleotide pyrophosphatase (0.5 unit/ml, 15 min) and 8 - p h e n y l t h e o p h y l l i n e ( 1 0 - 6 M , 5 min), a P1 purinoceptor antagonist (table 1). This indicates the involvement
1W (a)
salinet 15 min Cs 4x104M, 30 min
3xlO'6M
3x10"SM
3xlO-SM
3xlO~M
3x10"¢%!
(b)
3x IO-SM
3x 10SM
TTX lOSM, saline, 30 rain 15 rain / / ~
/ 3x lO'Sld
3xlO-SM
3xlO-SM
(c)
3xlO'SM
3xlO-SM
IO-SM
\
3x IO-SM
(d) TTX 106M, 15 rain hyoscine 2.5x 107M, 15 min 3; IO'eM
3xlO-SM
lO-SM
lO-SM
hyoscine2.5x 10-7M
Ilrnm llV=n
• nicotine Fig. 4. Effects of repeated exposures to nicotine and various drugs on nicotine-induced contraction of neonatal duodenum. A duodenal segment was exposed to nicotine for 2 min and a 30-min recovery period was allowed for subsequent exposure to nicotine or drug pretreatment. Concentrations and preincubation period of hexamethonium (C6), tetrodotoxin (TTX) and hyoscine are shown. Hyoscine was added to the bath at the peak of nicotine-induced contraction in (d).
79 TABLE 1
with a , / 3 - m e t h y l e n e A T P (10 -5 M), a stable Pax p u r i n o c e p t o r agonist (fig. 5a). a , / 3 - M e t h y l e n e A T P (10 -5 M ) significantly i n h i b i t e d t h e r e l a x a n t r e s p o n s e c a u s e d by a , 3 - m e t h y l e n e A T P (10 -5 M ) itself to 17 + 7.5% (n = 4) b u t n o t t h a t c a u s e d by A T P (10 -5 M), a n o n - s p e c i f i c P2 p u r i n o c e p t o r a g o n i s t (fig. 5b). R e l a x a t i o n i n d u c e d by A T P even at a lower c o n c e n t r a t i o n (10 - 6 M ) was n o t i n h i b i t e d at all (109 + 2.7% o f control, n = 4) by p r e t r e a t m e n t with a , 3 - m e t h y l e n e A T P (10 -5 M). In s t u d i e s using a d u l t d u o d e n u m , p r e i n c u b a t i o n with saline for 1 5 - 3 0 min as c o n t r o l e x p e r i m e n t s did not p r o d u c e a m a r k e d r e s p o n s e to nicotine, in c o n t r a s t to n e o n a t a l d u o d e n u m ( t a b l e 1 a n d see section 3.3).
Effects of various drugs on the nicotine (10 -s M)-induced relaxation of adult duodenum. The response to nicotine was examined before and after pretreatment with various drugs. The former response was used as the control. Values are means + S.E.M. Pretreatment
Relaxation by n nicotine (% of control)
Saline, 30 min Hexamethonium 4 × 10-4, 30 rain Guanethidine 6.4× 10 - 6 M, 30 min + hyoscine 2.5 X 10-7 M, 15 min
95 + 7.6 17 + 7.7 a 104 ± 9.9
Saline, 15 min Tetrodotoxin 10 -6 M, 15 min
ce-Chymotrypsin 3 units/ml M, 15 min Saline, 15 min + dimethyl sulfoxied, 5 min Nucleotide pyrophosphatase 0.5 unit/ml, 15 min + 8-phenyltheophylline 10 - 6 M, 5 min
4 4 4
96 ± 5.7 5+4.8 b 40±8.8 b
7 4 5
99 ± 7.0
4
52±9.9 c
4
4. Discussion T h e g a n g l i o n i c stimulants, n i c o t i n e a n d d i m e t h ylphenylpiperazinium, elicited contraction of neonatal r a t d u o d e n u m a n d r e l a x a t i o n o f a d u l t rat d u o d e n u m in this study. I n h i b i t i o n o f n i c o t i n e - i n d u c e d c o n t r a c t i o n by h e x a m e t h o n i u m a n d t e t r o d o t o x i n suggests t h a t t h e c o n t r a c t i o n is nicotinic r e c e p t o r - m e d i a t e d a n d o f n e u r o g e n i c origin. T e t r o d o t o x i n has b e e n r e p o r t e d to inhibit n e r v e c o n d u c t i o n b u t has no effect at p r e s y n a p t i c n e r v e t e r m i n a l s to r e l e a s e t r a n s m i t t e r s ( N a r a h a s h i , 1974). Since c o n t r a c t i o n c a u s e d by a h i g h e r c o n c e n t r a tion o f n i c o t i n e was p a r t i a l l y r e s i s t a n t to t e t r o d o t o x i n in this study, the nicotinic r e c e p t o r m a y t h e r e f o r e exist o n nerve t e r m i n a l s as well as on ganglionic soma. N i c o t i n e is r e p o r t e d to act on p o s t g a n g l i o n i c nerve t e r m i n a l s in r a b b i t iris s p h i n c t e r a n d o t h e r tissues (see H i s a y a m a et al., 1988). C o m p l e t e b l o c k a d e by hyoscine o f n i c o t i n e - i n d u c e d c o n t r a c t i o n , i n c l u d i n g the t e t r o d o t o x i n - r e s i s t a n t p a r t of the c o n t r a c t i o n , suggests that
ap
< 0.01 vs. saline 30 rain. b p < 0.01 VS. saline 15 min. c p < 0.01 vs. saline 15 min + dimethyl su|foxide 5 rain.
o f p u r i n e r g i c n e u r o n s in n i c o t i n e - i n d u c e d relaxation. 8 - P h e n y l t h e o p h y l l i n e was a d d e d to t h e m e d i u m to inhibit a p o s s i b l e r e l a x a t i o n d u e to A M P , which is a p r o d u c t o f A T P d e g r a d a t i o n by n u c l e o t i d e p y r o p h o s p h a t a s e . P r e t r e a t m e n t with n u c l e o t i d e p y r o p h o s p h a t a s e e l i c i t e d a slight d e c r e a s e in resting t o n u s d u e to t h e n o n - s p e c i f i c effect, b e c a u s e n o r e p i n e p h r i n e i n d u c e d r e l a x a t i o n o f t h e d u o d e n u m was i n h i b i t e d by 17 + 6 % (n = 3) a f t e r 15-min p r e t r e a t m e n t with nuc l e o t i d e p y r o p h o s p h a t a s e at 1 u n i t / m l c o n c e n t r a t i o n (not shown). N i c o t i n e (10 -5 M ) - i n d u c e d r e l a x a t i o n was significantly r e d u c e d to 55 + 11.8% o f c o n t r o l (n = 4, P < 0.05 vs. saline p r e t r e a t m e n t ) by 5 - m i n p r e i n c u b a t i o n
Adult
(a) Nc 10SM
Nc ~0"SM
mATP 105M
(b) ATP 10"SM
o~Aw
lo~
mATP loSM o
ATP 1051vl •
1 mln
Fig. 5. Effect of pretreatment with a,/3-methylene ATP (mATP) on relaxation induced by nicotine (Nc) (a), ce,fl-methylene ATP itself and ATP (b).
80 nicotine contracted the duodenum through the release of acetylcholine from cholinergic neurons in neonatal duodenum. Tachyphylaxis of the contractile response was observed when the duodenum was repeatedly exposed to nicotine. This may be due to depletion of transmitters. It is very interesting that the contractile response to nicotine appeared again several hours later. This phenomenon can be explained by the suggestion that exposure to nicotine stimulated the synthesis of transmitters, and several hours later the pool of transmitters was replenished and recovered from tachyphylaxis. In contrast to neonatal duodenum, nicotine elicited relaxation of the duodenum from adult rats. The relaxation was transient at concentrations under 3 × 10 - 4 M, and was blocked by hexamethonium and tetrodotoxin, indicating a nicotinic receptor-mediated and neurogenic response. The result that transient relaxation was resistant to both guanethidine and hyoscine shows that it is mediated through the NANC nerve. Thus the effect of nicotine on adult duodenum is similar to that of dimethylphenylpiperazinium in relaxing the duodenum through the NANC pathway (Manzini et al., 1985). The peptidergic neuron is a candidate for the NANC nerves (H6kfelt et al., 1980; Furness and Costa, 1982; Grider and Rivier, 1990). Partial inhibition of nicotine-induced relaxation by a-chymotrypsin suggests that peptidergic transmitters are involved in the nicotineinduced relaxation. Manzini et al. (1985), however, doubted the role of peptides, because of their result that dimethylphenylpiperazinium-induced relaxation was not significantly inhibited by a-chymotrypsin. A lower concentration of a-chymotrypsin (2 units/ml) which they used may produce insufficient inhibition. Many neuropeptides are localized in the gastro-intestinal neuronal system (H6kfelt et al., 1980; Furness and Costa, 1982). Previously we reported that neuropeptides, such as [MetS]enkephalin, TRH, CGRP and VIP, relax the adult rat duodenum by a direct effect on smooth muscle (Tonoue et al., 1979; Furukawa et al., 1980, 1989; Furukawa and Nomoto, 1988). The relaxations induced by [MetS]enkephalin, TRH and CGRP were transient in duration (Tonoue et al., 1979; Furukawa et al., 1980; Furukawa and Nomoto, 1988) and similar to relaxation induced by nicotine in this study. We suspect that any of these peptides may at least partly mediate the effect of nicotine. The purinergic neuron is another candidate for the NANC nerves, although there is controversy in this regard (Burnstock, 1972, 1981; Manzini et al., 1985; Serio et al., 1990). Inhibition of nicotine-induced relaxation by a combination of nucleotide pyrophosphatase with 8-phenyltheophylline and a,/3-methylene ATP suggests that the purinergic mechanism is included in nicotine-induced relaxation of rat duodenum, consis-
tent with the observation about dimethylphenylpiperazinium by Manzini et al. (1985). Inhibition by a,/3methylene ATP of nicotine-induced relaxation indicates P2x purinoceptor mediation of nicotine-induced relaxation, a,/3-Methylene ATP revealed that relaxation induced by ATP was different from that induced by nicotine and may be mediated through the P2v purinoceptor subtype, as suggested by Nicholls et al. (1990). Therefore, nicotine-releasable endogenous purines may be different from ATP, but be ATP analogues. We have previously observed involvement of nitric oxide (NO) in nicotine-induced relaxation of adult duodenum by using nitro-L-arginine (Irie et al., 1991), an inhibitor for NO synthase. In contrast to this, we found that endogenous NO was not involved in tetrodotoxinresistant relaxation of duodenum induced by ATP, ~,/3-methylene ATP, TRH, CGRP and VIP (Irie et al., 1991, 1992). Both neuropeptides and purine nucleotides may be released together with NO by nicotine stimulation, and all these transmitters are involved in relaxation of duodenum. The hypothesis that endogenous NO is an inhibitory neurotransmitter was proposed by Bult et al. (1990) and supported by others (Boeckxstaens et al., 1991; Sanders and Ward, 1992). Another possible role of endogenous NO in nicotineinduced relaxation is as a neuromodulator in releasing purine nucleotides and neuropeptides, thereby relaxing the smooth muscle of the duodenum, although further investigation is required to test this hypothesis. Prast and Philippu (1992), using a push-pull cannula and superfusion, noted that NO stimulates the release of acetylcholine in the forebrain. It is interesting to consider the reason why responses of duodenum to nicotine and dimethylphenylpiperazinium change during development. One possible explanation is differential development of enteric nerves containing different neurotransmitters innervating the duodenum. The cholinergic neurons may be predominant in neonatal rats, while inhibitory NANC neurons may become predominant in adult duodenum. Stimulatory cholinergic responses are reported to be present before birth in rat intestine, whereas an inhibitory noradrenergic response becomes obvious by 12 days postnatally (Miyazaki et al., 1982). The relaxant component in the response to nicotine appeared in 2-week-old rats and completely replaced the contractile component in 4-week-old rats in this study. Responses to TRH, CGRP, VIP and ATP also changed from contraction to relaxation during the 2-3 weeks after birth in our previous studies (Tonoue et al., 1981; Furukawa et al., 1989; Furukawa and Nomoto, 1988, 1989). The transition of duodenal responses to these neurotransmitters to the adult pattern may be one of the physiological maturations that occur during the weaning period in rats (see Henning, 1981).
81
In conclusion, our results clearly show that nicotine elicits cholinergic nerve-mediated contraction in neonatal duodenum and NANC-mediated relaxation in adult duodenum. Both peptidergic and purinergic mechanisms and endogenous NO are involved in nicotine-induced relaxation of adult duodenum. The cholinergic nerves may be predominant in neonatal duodenum and replaced by the inhibitory NANC nerves in the adult rat duodenum.
References Boeckxstaens, G.E., P.A. Pelckmans, H. Bult, J.G. De Man, A.G. Herman and Y.M. Van Maercke, 1991, Evidence for nitric oxide as mediator of non-adrenergic, non-cholinergic relaxations induced by ATP and GABA in the canine gut, Br. J. Pharmacol. 102, 434. Bult, H., G.E. Boeckxstaens, P.A. Pelckmans, F.H. Jordaens, Y.M. Van Maercke and A.G. Herman, 1990, Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter, Nature 345, 346. Burnstock, G., 1972, Purinergic nerves, Pharmacol. Rev. 24, 509. Burnstock, G., 1981, Neurotransmitters and trophic factors in the autonomic nervous system, J. Physiol. 313, 1. Furness, J.B. and M. Costa, 1982, Identification of gastrointestinal neurotransmitters, in: Mediators and Drugs in Gastrointestinal Motility. Handbook of Experimental Pharmacology, Vol. 59/1, ed. G. Bertaccini (Springer-Verlag, Berlin) p. 383. Furukawa, K. and T. Nomoto, 1988, Transition from neurogenic to myogenic response to calcitonin gene-related peptide (CGRP) in the duodenum of developing rat, Jpn. J. Pharmacol. 46 (Suppl.), 313p. Furukawa, K. and T. Nomoto, 1989, Postnatal changes in response to adenosine and adenine nucleotides in rat duodenum, Br. J. Pharmacol. 97, 1111. Furukawa, K., T. Nomoto and T. Tonoue, 1980, Myogenic receptivity for met-enkephalin in the rat duodenum: comparison with the guinea-pig duodenum, Eur. J. Pharmacol. 66, 129. Furukawa, K., K. Irie and T. Nomoto, 1989, Postnatal ontogenesis of the response to vasoactive intestinal peptide (VIP) in rat duodenum, Jpn. J. Pharmacol. 49 (Suppl.), 281p. Furukawa, K., K. lrie, T. Muraki and T. Nomoto, 1990, Developmental changes in reseponse to y-aminobutyric acid, 3-aminopropane sulphonic acid and baclofen in rat duodenum, Eur. J. Pharmacol. 183, 2201. Grider, J.R. and J.R. Rivier, 1990, Vasoactive intestinal peptide (VIP) as transmitter of inhibitory motor neurons of the gut:
evidence from the use of selective VIP antagonists and VIP antiserum, J. Pharmacol. Exp. Ther. 253, 738. Henning, S.J., 1981, Postnatal development: coordination of feeding, digestion, and metabolism, Am. J. Physiol. 241, G199. Hisayama, T., M. Shinkai, I. Takayanagi, S.-I. Morimoto and K. Ishida, 1988, Mechanism of action of nicotine in isolated iris sphincter preparations of rabbit, Br. J. Pharmacol. 95, 459. H6kfelt, T., O. Johansson, A. Ljungdahl, J.M. Lundberg and M. Schultzberg, 1980, Peptidergic neurones, Nature 284, 515. Irie, K., K. Furukawa, T. Muraki and T. Nomoto, 1990a, Postnatal changes in response to dimethylphenylpiperazinium (DMPP) in rat duodenum, Jpn. J. Pharmacol. 52 (Suppl. 1), 277p. Irie, K., T. Nomoto, T. Muraki and K. Furukawa, 1990b, Postganglionic neurons in duodenum are non-adrenergic, non-cholinergic in adult rats, while they are cholinergic in neonates, Eur. J. Pharmacol. 183, 2193. Irie, K., T. Muraki, K. Furukawa and T. Nomoto, 1991, L-NO-Nitro arginine inhibits nicotine-induced relaxation of isolated rat duodenum, Eur. J. Pharmacol. 202, 285. Irie, K., T. Muraki, K. Furukawa and T. Nomoto, 1992, Effect of L-NG-nitro-arginine (L-NOARG) on the relaxation induced by y-aminobutyric acid (GABA) and vasoactive intestinal peptide (VIP) in rat isolated duodenum, Jpn. J. Pharmacol. 58 (Suppl. 2), 317p. Manzini, S., C.A. Maggi and A. Meli, 1985, Further evidence for involvement of adenosine-5'-triphosphate in non-adrenergic noncholinergic relaxation of the isolated rat duodenum, Eur. J. Pharmacol. 113, 399. Miyazaki, H., A. Ohga and K. Saito, 1982, Development of motor response to intramural nerve stimulation and to drugs in rat small intestine, Br. J. Pharmacol. 76, 531. Narahashi, T., 1974, Chemicals as tools in the study of excitable membranes, Physiol. Rev. 54, 813. Nicholls, J., S.M.O. Hourani and I. Kitchen, 1990, The ontogeny of purinoceptors in rat urinary bladder and duodenum, Br. J. Pharmacol. 100, 874. Prast, H. and A. Philippu, 1992, Nitric oxide releases acetylcholine in the basal forebrain, Eur. J. Pharmacol. 216, 139. Sanders, K.M. and S.M. Ward, 1992, Nitric oxide as a mediator of nonadrenergic noncholinergic neurotransmission, Am. J. Physiol. 262, G379. Serio, R., F. Mul6, E.B. Adamo and A. Postorino, 1990, Evidence against purines being neurotransmitters of non-adrenergic, noncholinergic nerves in rat duodenum, Eur. J. Pharmacol. 182, 487. Tonoue, T., K. Furukawa and T. Nomoto, 1979, The direct influence of thyrotropin-releasing hormone (TRH) on the smooth muscle of rat duodenum, Life Sci. 25, 2011. Tonoue, T., K. Furukawa and T. Nomoto, 198l, Transition from neurogenic to myogenic receptivity for thyrotropin-releasing hormone (TRH) in the duodenum of the neonatal rat, Endocrinology 108, 723.
75
© 1994 Elsevier Science B.V. All rights reserved 0014-2999/94/$07.00
EJP 53450
Developmental changes in the response of rat isolated duodenum to nicotine K a o r u Irie *, K y o k o F u r u k a w a , T e r u k o N o m o t o , E m i k o Fujii a n d T a k a m u r a M u r a k i Department of Pharmacology, Tokyo Women's Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162, Japan Received 13 September 1993, revised MS received 12 October 1993, accepted 15 October 1993
Developmental changes in the response to ganglionic stimulants, nicotine and dimethylphenylpiperazinium, were investigated in rat isolated duodenum by recording isotonic mechanical activity. The duodenal response to nicotine/dimethylphenylpiperazinium (3 x 10 - 7 to 10 -3 M) in neonatal rats was contraction, which was blocked by hexamethonium, tetrodotoxin and hyoscine. The response to nicotine/dimethylphenylpiperazinium (10 - 6 to 10 - 4 M) in the adult duodenum was relaxation, which was blocked by tetrodotoxin and hexamethonium, but by neither guanethidine nor hyoscine. The transition of the response to nicotine/dimethylphenylpiperazinium from contraction to relaxation occurred at around the 3rd postnatal week. Nicotineinduced relaxation of adult duodenum was significantly inhibited by preincubation with a-chymotrypsin, a proteolytic enzyme, and a combination of nucleotide pyrophosphatase and 8-phenyltheophylline, a P1 purinoceptor antagonist. Nicotine-induced relaxation was desensitized by a,/3-methylene ATP, a stable P2x purinoceptor agonist. These results suggest that the contractile response of isolated duodenum to nicotine is mediated through cholinergic transmission in neonatal rats and the relaxant response is mediated through non-adrenergic, non-cholinergic transmission, which involves both peptidergic and purinergic transmission, in adult rats. Nicotine; Peptidergic transmission; Purinergic transmission; NANC (non-adrenergic, non-cholinergic) nerve; Developmental pharmacology
1. Introduction
We have been studying the developmental changes in the responses of isolated duodenum to various neurotransmitters. For example, contractile responses were induced in the neonatal duodenum by thyrotropin releasing hormone (TRH), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), ATP and y-aminobutyric acid (GABA), while relaxant responses to these biologically active substances were induced in the adult duodenum of rats (Tonoue et al., 1981; Furukawa et al., 1989, 1990; Furukawa and Nomoto, 1988, 1989). The contractions of neonatal duodenum induced by T R H , C G R P and VIP were suggested to be mediated through cholinergic transmission, because of blockade of the contraction by hyoscine (Tonoue et al., 1981; Furukawa and Nomoto, 1988; Furukawa et al., 1989). The relaxations induced by T R H , C G R P and VIP in the adult duodenum, however, were probably due to direct stimulation of smooth muscle because of their tetrodotoxin-resistant property
* Corresponding author. Tel. 03-3353-8111 ext. 22513, fax 03-52697417.
SSDI 0 0 1 4 - 2 9 9 9 ( 9 3 ) E 0 7 5 9 - L
(Tonoue et al., 1979; Furukawa and Nomoto, 1988; Furukawa et al., 1989). Manzini et al. (1985) have reported that dimethylphenylpiperazinium mimics non-adrenergic, non-cholinergic (NANC) relaxation induced by electrical field stimulation in rat isolated duodenum. Our previous studies suggest that ganglionic stimulants may again contract neonatal rat duodenum. We now studied the effects of nicotine and dimethylphenylpiperazinium on the isolated duodenum from 1-11-week-old rats, and found that the responses of duodenum to nicotine and dimethylphenylpiperazinium were contraction in neonatal rats and relaxation in adults. The mechanism of the response to nicotine was also investigated in both neonatal and adult rats. Preliminary reports have appeared (Irie et al., 1990a,b).
2. Materials and methods 2.1. Preparations and measurement of mechanical
activity of duodenum Male Wistar-Imamichi rats aged 1, 3, 4 and 9-11 weeks were used. The rats were housed in an air-con-
76 ditioned room (22 + 1°C and 55 + 5% humidity) with controlled light-dark cycle (6:00-20:00 h light on) and free access to standard chow and tap water. Pups were removed from the mother on the 22nd postnatal day. Neonatal (1 week old) and young (3 and 4 weeks old) rats were killed by decapitation and adult rats (9-11 weeks old) were stunned by a blow to the head and bled. Each duodenum was dissected out, and the proximal portion but not the bulb (1.5 cm for neonatal rats and 2.2 cm for young and adult rats) was set up in an organ bath containing 5 ml of modified Locke solution (composition in mM: NaC1 154, KCI 4.02, CaCI z 1.36, MgC12 0.9, N a H C O 3 2.97, glucose 5.56, p H 7.0) saturated with 95% 0 2 and 5% CO 2 and maintained with continuous bubbling of air at 32 °C. Mechanical activity of the duodenal segments under a load of 0.5 g (neonates), 0.75 g (young), and 1 g (adult) was recorded through an isotonic transducer (Nihonkohden TD112S) on a polygraphic recorder (Nihonkohden WT645G). After 1-h equilibration, the duodenal segments were exposed to nicotine/dimethylphenylpiperazinium for 2 min and allowed a 30-min recovery period for subsequent exposures to n i c o t i n e / d i m e t h y l p h e n y l piperazinium or various drug pretreatments. The medium was changed every 5th to 10th min during equilibration and recovery. The concentration-response curve was obtained from serial exposures to increasing concentrations of nicotine at 30-min intervals. The values presented are means + S.E.M. Student's t-test was used for statistical analysis. 2.2. Drugs The following drugs were used: nicotine tartrate (Wako, Osaka), hyoscine hydrochloride (scopolamine hydrochloride), adenosine 5'-triphosphate (ATP) disodium salt, a,fl-methyleneadenosine 5'-triphosphate (a,/3-methylene ATP) lithium salt, dimethylphenylpiperazinium iodide, a-chymotrypsin, nucleotide pyrophophatase and 8-phenyltheophylline (Sigma), guanethidine sulfate (Ciba-Geigy), hexamethonium chloride (Tokyo Kasei), tetrodotoxin (kindly supplied by Sankyo, Tokyo). 8-Phenyltheophylline was dissolved in dimethyl sulfoxide and a 5-~1 aliquot was added to the organ bath. O t h e r drugs were dissolved in physiological saline and a 30- or 100-/~1 aliquot was added to the organ bath.
3. Results
3.1. Mechanical response to nicotine Nicotine (3 × 10 -7 to 10 - 4 M ) elicited contraction of isolated duodenum in a concentration-dependent manner in neonatal rats (fig. 1). The p D 2 value, which
lW
Nc 10-6M
3x 106M
1(~-5M
3 x 10"5M
10"4M 1 min
100 " o~ 80" 60" 40" (-~ 20"
0
, , 3x10 7 106
, 3x106 Nicotine
, , 102 3x10 s
10"
(M)
Fig. 1. Response of duodenum isolated from neonatal rat to nicotine (10 -6
to
10 -4
M)
and
concentration-response
curve.
Mechanical
activity of the duodenum was recorded isotonically under a load of 0.5 g. A duodenal segment was exposed to nicotine for 2 rain at 30-min intervals. Values are means :t: S.E.M. (n = 6).
was obtained from the concentration-response curve, was 5.8 _+ 0.14 (n = 6). In contrast to neonatal rat duodenum, nicotine elicited concentration-dependent relaxation, which was transient at concentrations under 3 × 10 -4 M but became sustained at a higher concentration (10 -3 M), in adult rat duodenum (fig. 2). The p D 2 value of nicotine for transient relaxation was 5.4 _+ 0.04 (n -- 4).
Adult
Nc 106M
3x10~1
10-5M
3x10-SM
I0-4M
3xIO'4M 10-aM
J
, , , , , 106 3x106 10 s 3x10 s 10 ~ Nicotine (M)
Fig. 2. Response of duodenum isolated from adult rat to nicotine (10 -6 tO 10 -3 M) and concentration-response curve for transient
relaxant phase. Mechanical activity of the duodenum was recorded isotonically under a load of 1 g. A duodenal segment was exposed to nicotine for 2 rain at 30-min intervals. Values are means :t: S.E.M. (n = 4).
77
3.2. Mechanical response to dimethylphenylpiperazinium Dimethylphenylpiperazinium, like nicotine, elicited contraction of the d u o d e n u m in neonatal rats and relaxation in adults (fig. 3). Sustained relaxation was m u c h m o r e m a r k e d in the case of d i m e t h ylphenylpiperazinium-induced relaxation than nicotineinduced relaxation. In duodenum from 3-week-old rats, the first or several exposure(s) to dimethylphenylpiperazinium (even at a higher concentration of 3 × 10 -5 M) usually elicited contraction and then subsequent exposures to dimethylphenylpiperazinium elicited relaxation solely or as a biphasic response, i.e., relaxation followed by a contraction (fig. 3). This response variability may be due to tachyphylaxis for contractile responses produced by repeated exposures to dimethylphenylpiperazinium (see section 3.3). T h e response of duodenum to dimethylphenylpiperazinium was consistently relaxation in 4-week-old rats (fig. 3). A similar transition from contraction to relaxation was also observed in the duodenal response to nicotine in 3-4-week-old rats (data not shown).
1W
3xlO~M
3x10~
10"6M
3xlO-eM
10"5M
lO~
lo~
IO-'M
3W
j 3xIO'SM
4W
3x I 0"5M
I 0"~M
Adult
3"~1o~
lO~M
3x 1 0 " 6 M
"~,,~1~ lO'~
__11
Since the duodenal responses to nicotine and dimethylphenylpiperazinium were basically the same, nicotine was used for further studies on mechanisms.
3.3. Mechanism of nicotine-induced neonatal rat duodenum
contraction
of
R e p e a t e d exposures to nicotine at 30-min intervals led to tachyphylaxis of the contractile response to nicotine in neonatal rat duodenum (fig. 4a and b). The duodenum, however, recovered from tachyphylaxis after several exposures to nicotine, and thereafter consistently responded to repeated exposures to nicotine (fig. 4a and b). Therefore, the mechanism of nicotineinduced contraction of neonatal duodenum was examined after the tachyphylaxis of the contractile response disappeared. Nicotine (3 × 10 -6 to 10 -5 M)-induced contraction was inhibited by 30-min pretreatment with hexamethonium ( 4 × 10 -4 M, fig. 4a), a nicotinic ganglionic blocker. The contractile response to nicotine (3 × 10 -6 M) was abolished by 15-min pretreatment with tetrodotoxin (10 -6 M, fig. 4b), a Na + channel blocker, while tetrodotoxin produced only a partial inhibition of contraction elicited by a higher concentration of nicotine (10 -5 M, fig. 4d). The responses to nicotine were greater in saline pretreatments than those in previous exposures to nicotine in fig. 4a and b. These marked responses to nicotine with saline pretreatment may have resulted from a long incubation (20-35 min) of duodenum in unchanged medium, while the normal response to nicotine was examined 5 min after the baths were filled with fresh medium. Spontaneous activity was also getting more marked during a longer incubation period in unchanged medium (data not shown). Fifteen-minute pretreatment with hyoscine (2.5 X 10 -7 M) completely blocked nicotine (3 × 10 -6 M)-induced contraction of neonatal duodenum (fig. 4c) and the tetrodotoxin-resistant contraction to nicotine (10 -5 M) was also completely blocked by subsequent addition of hyoscine (fig. 4d), indicating cholinergic nerve mediation in the nicotine-induced contraction of neonatal duodenum. Inhibition by hexamethonium, tetrodotoxin and hyoscine was also confirmed for dimethylphenylpiperazinium-induced contraction (data not shown).
3.4. Mechanism of nicotine-induced relaxation of adult rat duodenum
lrn~
• DIdPP Fig. 3. Response of isolated duodenum to dimethylphenylpiperazinium (DMPP) in 1-, 3- and 4-week (W)-old rats and adult rat. Mechanical activity of the duodenum was recorded isotonically under a load of 0.5 g for 1 W, 0.75 g for 3 and 4 W, and 1 g for adult. A duodenal segment was exposed to dimethylphenylpiperazinium for 2 rain at 30-rain intervals.
R e p e a t e d exposure to nicotine (10 -5 M) did not lead to tachyphylaxis of the relaxant response of adult duodenum but elicited response of a constant magnitude, although the initial response was rather small (data not shown). Relaxation of duodenum elicited by nicotine (10 -5 M) was significantly inhibited by pre-
78
treatment with hexamethonium (4 × 10 - 4 M, 30 min) and tetrodotoxin ( 1 0 - 6 M , 15 min) (table 1). Nicotineinduced relaxation was not affected by pretreatment with a combination of guanethidine (6.4 × 10 - 6 M , 30 min) and hyoscine (2.5 x 10 - 7 M , 15 min) (table 1), indicating the mediation of NANC transmission. Transient relaxation induced by dimethylphenylpiperazinium was also inhibited by hexamethonium and tetrodotoxin, and was resistant to hyoscine and guanethidine (data not shown). We examined whether nicotine-induced relaxation of the duodenum was mediated by either peptidergic or purinergic neurons. Preincubation with a-chymotrypsin (3 units/ml, 15 min), a proteolytic enzyme,
significantly inhibited the nicotine (10 -5 M)-induced relaxation of duodenum (table 1). The concentration of a-chymotrypsin used (3 units/ml, 15 min) did not affect the resting tension or spontaneous activity of duodenum. ~-Chymotrypsin did not inhibit ATP ( 1 0 - 6 M)-induced relaxation (105 + 7.1% of control, n = 3), indicating that the inhibitory effect of a-chymotrypsin on nicotine-induced relaxation is not non-specific but due to its proteolytic effect. Nicotine (10 -5 M)-induced relaxation was also inhibited by preincubation with a combination of nucleotide pyrophosphatase (0.5 unit/ml, 15 min) and 8 - p h e n y l t h e o p h y l l i n e ( 1 0 - 6 M , 5 min), a P1 purinoceptor antagonist (table 1). This indicates the involvement
1W (a)
salinet 15 min Cs 4x104M, 30 min
3xlO'6M
3x10"SM
3xlO-SM
3xlO~M
3x10"¢%!
(b)
3x IO-SM
3x 10SM
TTX lOSM, saline, 30 rain 15 rain / / ~
/ 3x lO'Sld
3xlO-SM
3xlO-SM
(c)
3xlO'SM
3xlO-SM
IO-SM
\
3x IO-SM
(d) TTX 106M, 15 rain hyoscine 2.5x 107M, 15 min 3; IO'eM
3xlO-SM
lO-SM
lO-SM
hyoscine2.5x 10-7M
Ilrnm llV=n
• nicotine Fig. 4. Effects of repeated exposures to nicotine and various drugs on nicotine-induced contraction of neonatal duodenum. A duodenal segment was exposed to nicotine for 2 min and a 30-min recovery period was allowed for subsequent exposure to nicotine or drug pretreatment. Concentrations and preincubation period of hexamethonium (C6), tetrodotoxin (TTX) and hyoscine are shown. Hyoscine was added to the bath at the peak of nicotine-induced contraction in (d).
79 TABLE 1
with a , / 3 - m e t h y l e n e A T P (10 -5 M), a stable Pax p u r i n o c e p t o r agonist (fig. 5a). a , / 3 - M e t h y l e n e A T P (10 -5 M ) significantly i n h i b i t e d t h e r e l a x a n t r e s p o n s e c a u s e d by a , 3 - m e t h y l e n e A T P (10 -5 M ) itself to 17 + 7.5% (n = 4) b u t n o t t h a t c a u s e d by A T P (10 -5 M), a n o n - s p e c i f i c P2 p u r i n o c e p t o r a g o n i s t (fig. 5b). R e l a x a t i o n i n d u c e d by A T P even at a lower c o n c e n t r a t i o n (10 - 6 M ) was n o t i n h i b i t e d at all (109 + 2.7% o f control, n = 4) by p r e t r e a t m e n t with a , 3 - m e t h y l e n e A T P (10 -5 M). In s t u d i e s using a d u l t d u o d e n u m , p r e i n c u b a t i o n with saline for 1 5 - 3 0 min as c o n t r o l e x p e r i m e n t s did not p r o d u c e a m a r k e d r e s p o n s e to nicotine, in c o n t r a s t to n e o n a t a l d u o d e n u m ( t a b l e 1 a n d see section 3.3).
Effects of various drugs on the nicotine (10 -s M)-induced relaxation of adult duodenum. The response to nicotine was examined before and after pretreatment with various drugs. The former response was used as the control. Values are means + S.E.M. Pretreatment
Relaxation by n nicotine (% of control)
Saline, 30 min Hexamethonium 4 × 10-4, 30 rain Guanethidine 6.4× 10 - 6 M, 30 min + hyoscine 2.5 X 10-7 M, 15 min
95 + 7.6 17 + 7.7 a 104 ± 9.9
Saline, 15 min Tetrodotoxin 10 -6 M, 15 min
ce-Chymotrypsin 3 units/ml M, 15 min Saline, 15 min + dimethyl sulfoxied, 5 min Nucleotide pyrophosphatase 0.5 unit/ml, 15 min + 8-phenyltheophylline 10 - 6 M, 5 min
4 4 4
96 ± 5.7 5+4.8 b 40±8.8 b
7 4 5
99 ± 7.0
4
52±9.9 c
4
4. Discussion T h e g a n g l i o n i c stimulants, n i c o t i n e a n d d i m e t h ylphenylpiperazinium, elicited contraction of neonatal r a t d u o d e n u m a n d r e l a x a t i o n o f a d u l t rat d u o d e n u m in this study. I n h i b i t i o n o f n i c o t i n e - i n d u c e d c o n t r a c t i o n by h e x a m e t h o n i u m a n d t e t r o d o t o x i n suggests t h a t t h e c o n t r a c t i o n is nicotinic r e c e p t o r - m e d i a t e d a n d o f n e u r o g e n i c origin. T e t r o d o t o x i n has b e e n r e p o r t e d to inhibit n e r v e c o n d u c t i o n b u t has no effect at p r e s y n a p t i c n e r v e t e r m i n a l s to r e l e a s e t r a n s m i t t e r s ( N a r a h a s h i , 1974). Since c o n t r a c t i o n c a u s e d by a h i g h e r c o n c e n t r a tion o f n i c o t i n e was p a r t i a l l y r e s i s t a n t to t e t r o d o t o x i n in this study, the nicotinic r e c e p t o r m a y t h e r e f o r e exist o n nerve t e r m i n a l s as well as on ganglionic soma. N i c o t i n e is r e p o r t e d to act on p o s t g a n g l i o n i c nerve t e r m i n a l s in r a b b i t iris s p h i n c t e r a n d o t h e r tissues (see H i s a y a m a et al., 1988). C o m p l e t e b l o c k a d e by hyoscine o f n i c o t i n e - i n d u c e d c o n t r a c t i o n , i n c l u d i n g the t e t r o d o t o x i n - r e s i s t a n t p a r t of the c o n t r a c t i o n , suggests that
ap
< 0.01 vs. saline 30 rain. b p < 0.01 VS. saline 15 min. c p < 0.01 vs. saline 15 min + dimethyl su|foxide 5 rain.
o f p u r i n e r g i c n e u r o n s in n i c o t i n e - i n d u c e d relaxation. 8 - P h e n y l t h e o p h y l l i n e was a d d e d to t h e m e d i u m to inhibit a p o s s i b l e r e l a x a t i o n d u e to A M P , which is a p r o d u c t o f A T P d e g r a d a t i o n by n u c l e o t i d e p y r o p h o s p h a t a s e . P r e t r e a t m e n t with n u c l e o t i d e p y r o p h o s p h a t a s e e l i c i t e d a slight d e c r e a s e in resting t o n u s d u e to t h e n o n - s p e c i f i c effect, b e c a u s e n o r e p i n e p h r i n e i n d u c e d r e l a x a t i o n o f t h e d u o d e n u m was i n h i b i t e d by 17 + 6 % (n = 3) a f t e r 15-min p r e t r e a t m e n t with nuc l e o t i d e p y r o p h o s p h a t a s e at 1 u n i t / m l c o n c e n t r a t i o n (not shown). N i c o t i n e (10 -5 M ) - i n d u c e d r e l a x a t i o n was significantly r e d u c e d to 55 + 11.8% o f c o n t r o l (n = 4, P < 0.05 vs. saline p r e t r e a t m e n t ) by 5 - m i n p r e i n c u b a t i o n
Adult
(a) Nc 10SM
Nc ~0"SM
mATP 105M
(b) ATP 10"SM
o~Aw
lo~
mATP loSM o
ATP 1051vl •
1 mln
Fig. 5. Effect of pretreatment with a,/3-methylene ATP (mATP) on relaxation induced by nicotine (Nc) (a), ce,fl-methylene ATP itself and ATP (b).
80 nicotine contracted the duodenum through the release of acetylcholine from cholinergic neurons in neonatal duodenum. Tachyphylaxis of the contractile response was observed when the duodenum was repeatedly exposed to nicotine. This may be due to depletion of transmitters. It is very interesting that the contractile response to nicotine appeared again several hours later. This phenomenon can be explained by the suggestion that exposure to nicotine stimulated the synthesis of transmitters, and several hours later the pool of transmitters was replenished and recovered from tachyphylaxis. In contrast to neonatal duodenum, nicotine elicited relaxation of the duodenum from adult rats. The relaxation was transient at concentrations under 3 × 10 - 4 M, and was blocked by hexamethonium and tetrodotoxin, indicating a nicotinic receptor-mediated and neurogenic response. The result that transient relaxation was resistant to both guanethidine and hyoscine shows that it is mediated through the NANC nerve. Thus the effect of nicotine on adult duodenum is similar to that of dimethylphenylpiperazinium in relaxing the duodenum through the NANC pathway (Manzini et al., 1985). The peptidergic neuron is a candidate for the NANC nerves (H6kfelt et al., 1980; Furness and Costa, 1982; Grider and Rivier, 1990). Partial inhibition of nicotine-induced relaxation by a-chymotrypsin suggests that peptidergic transmitters are involved in the nicotineinduced relaxation. Manzini et al. (1985), however, doubted the role of peptides, because of their result that dimethylphenylpiperazinium-induced relaxation was not significantly inhibited by a-chymotrypsin. A lower concentration of a-chymotrypsin (2 units/ml) which they used may produce insufficient inhibition. Many neuropeptides are localized in the gastro-intestinal neuronal system (H6kfelt et al., 1980; Furness and Costa, 1982). Previously we reported that neuropeptides, such as [MetS]enkephalin, TRH, CGRP and VIP, relax the adult rat duodenum by a direct effect on smooth muscle (Tonoue et al., 1979; Furukawa et al., 1980, 1989; Furukawa and Nomoto, 1988). The relaxations induced by [MetS]enkephalin, TRH and CGRP were transient in duration (Tonoue et al., 1979; Furukawa et al., 1980; Furukawa and Nomoto, 1988) and similar to relaxation induced by nicotine in this study. We suspect that any of these peptides may at least partly mediate the effect of nicotine. The purinergic neuron is another candidate for the NANC nerves, although there is controversy in this regard (Burnstock, 1972, 1981; Manzini et al., 1985; Serio et al., 1990). Inhibition of nicotine-induced relaxation by a combination of nucleotide pyrophosphatase with 8-phenyltheophylline and a,/3-methylene ATP suggests that the purinergic mechanism is included in nicotine-induced relaxation of rat duodenum, consis-
tent with the observation about dimethylphenylpiperazinium by Manzini et al. (1985). Inhibition by a,/3methylene ATP of nicotine-induced relaxation indicates P2x purinoceptor mediation of nicotine-induced relaxation, a,/3-Methylene ATP revealed that relaxation induced by ATP was different from that induced by nicotine and may be mediated through the P2v purinoceptor subtype, as suggested by Nicholls et al. (1990). Therefore, nicotine-releasable endogenous purines may be different from ATP, but be ATP analogues. We have previously observed involvement of nitric oxide (NO) in nicotine-induced relaxation of adult duodenum by using nitro-L-arginine (Irie et al., 1991), an inhibitor for NO synthase. In contrast to this, we found that endogenous NO was not involved in tetrodotoxinresistant relaxation of duodenum induced by ATP, ~,/3-methylene ATP, TRH, CGRP and VIP (Irie et al., 1991, 1992). Both neuropeptides and purine nucleotides may be released together with NO by nicotine stimulation, and all these transmitters are involved in relaxation of duodenum. The hypothesis that endogenous NO is an inhibitory neurotransmitter was proposed by Bult et al. (1990) and supported by others (Boeckxstaens et al., 1991; Sanders and Ward, 1992). Another possible role of endogenous NO in nicotineinduced relaxation is as a neuromodulator in releasing purine nucleotides and neuropeptides, thereby relaxing the smooth muscle of the duodenum, although further investigation is required to test this hypothesis. Prast and Philippu (1992), using a push-pull cannula and superfusion, noted that NO stimulates the release of acetylcholine in the forebrain. It is interesting to consider the reason why responses of duodenum to nicotine and dimethylphenylpiperazinium change during development. One possible explanation is differential development of enteric nerves containing different neurotransmitters innervating the duodenum. The cholinergic neurons may be predominant in neonatal rats, while inhibitory NANC neurons may become predominant in adult duodenum. Stimulatory cholinergic responses are reported to be present before birth in rat intestine, whereas an inhibitory noradrenergic response becomes obvious by 12 days postnatally (Miyazaki et al., 1982). The relaxant component in the response to nicotine appeared in 2-week-old rats and completely replaced the contractile component in 4-week-old rats in this study. Responses to TRH, CGRP, VIP and ATP also changed from contraction to relaxation during the 2-3 weeks after birth in our previous studies (Tonoue et al., 1981; Furukawa et al., 1989; Furukawa and Nomoto, 1988, 1989). The transition of duodenal responses to these neurotransmitters to the adult pattern may be one of the physiological maturations that occur during the weaning period in rats (see Henning, 1981).
81
In conclusion, our results clearly show that nicotine elicits cholinergic nerve-mediated contraction in neonatal duodenum and NANC-mediated relaxation in adult duodenum. Both peptidergic and purinergic mechanisms and endogenous NO are involved in nicotine-induced relaxation of adult duodenum. The cholinergic nerves may be predominant in neonatal duodenum and replaced by the inhibitory NANC nerves in the adult rat duodenum.
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