European Journal ~ff'Pharrnacology. 229 (1992) 273 276
273
~, 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$115.00
EJP 21157
Short communication
Involvement of endogenous nitric oxide in the regulation of rat intestinal motility in vivo A n t o n i o C a l i g n a n o ~, B r e n d a n J.R. Whittle, M a s s i m o Di R o s a ;1 and S a l v a d o r M o n c a d a Welleome Research Laboratories, Beckenham, Kent BR3 3BS, UK and " Department ~ff"Experimental Pharrnaeology. Utm'ersity of Naples, Naples. Italy Received 6 October 1992, accepted 13 October 1992
The effect of the nitric oxide (NO) synthase inhibitor, NC;-nitro-L-arginine methyl ester (L-NAME) on the motility of the small intestine in an acute model in the anaesthetised rat was determined by changes in jejunal intraluminal pressure. L-NAME (0.5-10 mg kg ~ i.v.) caused a dose-dependent increase in intraluminal pressure and initiated phasic intestinal contractions. These responses were inhibited by concurrent administration of L-arginine (200 mg kg i i.v.) but not by D-arginine (200 mg kg 1). The increase in jejunal motility induced by L-NAME was attenuated by atropine (4 mg kg 1), although even high doses of atropine (16 mg kg ~) did not abolish these responses. This indicates that although there are interactions between NO and muscarinic cholinergic mechanisms, other processes arc also involved in these contractile events following administration of L-NAME. These observations in the rat suggest that endogenous NO plays a role in the modulation of intestinal motility in vivo. Nitric oxide (NO); Intestinal motility; L-NAME (N C}-nitro-L-arginine methyl ester); Cholinergic mechanisms
1. Introduction
2. Materials and m e t h o d s
Nitric oxide (NO), formed from L-arginine by a constitutive NO synthase in endothelial cells (Moncada et al., 1991), plays an important role in the regulation of gastro-intestinal blood flow (Pique et al., 1989; Pizcueta et al., 1992). From studies in vitro, NO has also been proposed as the mediator of non-adrenergic, non-cholinergic (NANC) relaxation of the guinea-pig ileum and stomach (Gustafsson et al., 1990; Desai et al., 1991), of the rat stomach, colon, duodenum and ileum (Li and Rand, 1990; Hata et al., 1990; Irie et al., 1991; Kanada et al., 19921 and of the canine ileo-cecal junction, ileum, duodenum and colon (Boeckxstaens et al., 1990; Toda et al., 1990; Dalziel et al., 19911. Using the inhibitor of NO synthase (Rees et al., 1990), NG-nitro-L-arginine methyl ester (L-NAME), the role of endogenous NO in the modulation of intestinal motility in vivo has now been investigated in an acute model in the anesthetised rat.
Male Wistar rats (200-250 g) fasted for 18-24 h but allowed water, were anaesthetised with sodium pentobarbitone (60 mg kg i i.p.) and the trachea was cannulated to facilitate respiration. Systemic arterial blood pressure (BP) was recorded from a catheter inserted into a carotid artery and connected to a pressure transducer and chart recorder. Rectal temperature was maintained at 37°C by thermistor-controlled radiant heat. A 25 gauge butterfly needle was inserted into the tail vein for the administration of drugs (1 ml kg ~" flushed in with 0.25 ml of isotonic saline). Following a midline incision to expose the small intestine, a thin, saline-filled balloon, made from silicone rubber (Rhodosil; Rhone-Poulenc, France) and attached to a polythene catheter, was introduced into the jejunum via a small incision and tied in place avoiding large blood vessels. The volume in the balloon was adjusted to given an initial resting pressure of 5 mmHg, which was not sufficient to cause active distension of the gut wall, and after allowing the preparation to rest for 20-30 min, intestinal pressure and the number and amplitude of the jejunal contractions were recorded on a chart-recorder (Grass Medical Instrument, RPS 76 8) via a Gould Statham pressure transducer (P23 Db).
Correspondence to: B.J.R. Whittle, Department of Pharmacology, Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, UK. Tel. (44) 81-658 2211, fax (44) 81-663 6176.
274
NG-Nitro-L-arginine methyl ester (L-NAME), Larginine hydrochloride, D-arginine hydrochloride, atropine sulphate and angiotensin I I were obtained from Sigma Chemical Company (Poole, Dorset, U.K.) and dissolved freshly in isotonic saline. Results, shown as the changes in resting intraluminal pressure (.1 m m H g ) and the amplitude (A m m H g ) and frequency (number min ~) of the jejunal motility responses are shown as the means + S.E.M. of In) studies. The difference between groups was evaluated by Student's t-test for paired or unpaired data, and P < 0.05 was taken as significant.
3. Results
Under resting conditions, stable levels of intraluminal pressure (3 + 1 mmHg; n = 30) were maintained in the rat jejunum, with minimal spontaneous contractions in this acute model (fig. 1). Intravenous administration of L - N A M E (0.5-10 mg kg-1) caused a dosedependent significant (P < 0.05) increase in jejunal intraluminal pressure (fig. 2). This effect on resting tone was apparent within 1 min of administration of these doses of L-NAME, reaching the maximal response after 2 - 3 min, which then diminished over the following 10-30 min. Furthermore, the administration of L - N A M E (1-10 mg kg 1 i.v.) also produced frequent intestinal muscular contractions, observed by inspection of the intestinal loops, in doses causing increases in jejunal tone. Both the amplitude and frequency of these contractions induced by L-NAME, which were
recorded as rapid changes in jejunal intraluminal pressure, were dose-related (fig. 2). Concurrent administration of L-arginine (100-200 mg kg ~ i.v.) reduced these motility responses induced by L-NAME. Thus, L-arginine (200 mg kg ~ i.v.) abolished I n = 5 , P < 0 . 0 0 1 ) the increase in intraluminal pressure and inhibited the frequency and amplitude by 51 _+ 7 and 74 + 8%. respectively (n = 5: P < 0.01) of the motility responses induced by L-NAME (10 mg k g t i.v.), whereas D-arginine (200 mg kg ~ i.v.) did not significantly inhibit these responses (n = 4). L-NAME (1. 5 and 10 mg kg ~ i.v.) induced a dose-dependent significant (P < 0.01) increase in systemic arterial blood pressure from its resting value (115 + 10 mmHg, n = 30), by A 26 + 4, .1 33 + 3 and d 35 + 2 m m H g respectively. Concurrent administration of L-arginine (200 mg kg ~ i.v.) reduced the hypertensive response to L - N A M E ( 1 0 mg kg J i.v.) by 87 + 20% (n = 5, P < 0.05), whereas D-arginine had no significant action (n - 4). As shown in fig. 1, a lower dose of L-arginine (100 mg kg ~ i.v.) substantially reduced the frequency of the motility responses to L-NAME (l0 mg kg 1) yet had minimal action on the increase in systemic BP, suggesting these motility changes are independent of changes in BP. Furthermore, intravenous infusion of angiotensin It (2 /~g kg I rain ~) increased BP (by 25 + 6 mmHg, n = 3) but did not significantly affect resting intralumina[ pressure (n = 3). Administration of atropine (4 mg kg ~ i.v.) significantly reduced the intraluminal pressure changes induced by L - N A M E ( I - 1 0 mg kg t ) a s shown in fig. 2 and also reduced the BP response (by 11 _+ 2 m m H g for the intermediate dose of L-NAME). The effect of
2001 mmHg /
0 ~
I
J
1rain
]
15-
mmHg
o
INTRALUMINAL PRESSURE
( L-NAME 10mg kg"1 i.v.
L-ARGININE 100mg kg"1 i.v.
Fig. 1. Effect of intravenous administration of L-NAME (10 mg kg i) on jejunal resting intraluminal pressure (A mmttg) and on systemic arterial blood pressure (BP, mmHg) in the anaesthetised rat. The chart-recording of the experiment also shows the inhibitory effect on motility of subsequent administration of L-arginine (ll)(I mg kg 1 i.v.).
275
~=
I~
3
+At[opine
¢-~:2 "~E
~
o 5 4
-a3
273
~, 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$115.00
EJP 21157
Short communication
Involvement of endogenous nitric oxide in the regulation of rat intestinal motility in vivo A n t o n i o C a l i g n a n o ~, B r e n d a n J.R. Whittle, M a s s i m o Di R o s a ;1 and S a l v a d o r M o n c a d a Welleome Research Laboratories, Beckenham, Kent BR3 3BS, UK and " Department ~ff"Experimental Pharrnaeology. Utm'ersity of Naples, Naples. Italy Received 6 October 1992, accepted 13 October 1992
The effect of the nitric oxide (NO) synthase inhibitor, NC;-nitro-L-arginine methyl ester (L-NAME) on the motility of the small intestine in an acute model in the anaesthetised rat was determined by changes in jejunal intraluminal pressure. L-NAME (0.5-10 mg kg ~ i.v.) caused a dose-dependent increase in intraluminal pressure and initiated phasic intestinal contractions. These responses were inhibited by concurrent administration of L-arginine (200 mg kg i i.v.) but not by D-arginine (200 mg kg 1). The increase in jejunal motility induced by L-NAME was attenuated by atropine (4 mg kg 1), although even high doses of atropine (16 mg kg ~) did not abolish these responses. This indicates that although there are interactions between NO and muscarinic cholinergic mechanisms, other processes arc also involved in these contractile events following administration of L-NAME. These observations in the rat suggest that endogenous NO plays a role in the modulation of intestinal motility in vivo. Nitric oxide (NO); Intestinal motility; L-NAME (N C}-nitro-L-arginine methyl ester); Cholinergic mechanisms
1. Introduction
2. Materials and m e t h o d s
Nitric oxide (NO), formed from L-arginine by a constitutive NO synthase in endothelial cells (Moncada et al., 1991), plays an important role in the regulation of gastro-intestinal blood flow (Pique et al., 1989; Pizcueta et al., 1992). From studies in vitro, NO has also been proposed as the mediator of non-adrenergic, non-cholinergic (NANC) relaxation of the guinea-pig ileum and stomach (Gustafsson et al., 1990; Desai et al., 1991), of the rat stomach, colon, duodenum and ileum (Li and Rand, 1990; Hata et al., 1990; Irie et al., 1991; Kanada et al., 19921 and of the canine ileo-cecal junction, ileum, duodenum and colon (Boeckxstaens et al., 1990; Toda et al., 1990; Dalziel et al., 19911. Using the inhibitor of NO synthase (Rees et al., 1990), NG-nitro-L-arginine methyl ester (L-NAME), the role of endogenous NO in the modulation of intestinal motility in vivo has now been investigated in an acute model in the anesthetised rat.
Male Wistar rats (200-250 g) fasted for 18-24 h but allowed water, were anaesthetised with sodium pentobarbitone (60 mg kg i i.p.) and the trachea was cannulated to facilitate respiration. Systemic arterial blood pressure (BP) was recorded from a catheter inserted into a carotid artery and connected to a pressure transducer and chart recorder. Rectal temperature was maintained at 37°C by thermistor-controlled radiant heat. A 25 gauge butterfly needle was inserted into the tail vein for the administration of drugs (1 ml kg ~" flushed in with 0.25 ml of isotonic saline). Following a midline incision to expose the small intestine, a thin, saline-filled balloon, made from silicone rubber (Rhodosil; Rhone-Poulenc, France) and attached to a polythene catheter, was introduced into the jejunum via a small incision and tied in place avoiding large blood vessels. The volume in the balloon was adjusted to given an initial resting pressure of 5 mmHg, which was not sufficient to cause active distension of the gut wall, and after allowing the preparation to rest for 20-30 min, intestinal pressure and the number and amplitude of the jejunal contractions were recorded on a chart-recorder (Grass Medical Instrument, RPS 76 8) via a Gould Statham pressure transducer (P23 Db).
Correspondence to: B.J.R. Whittle, Department of Pharmacology, Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, UK. Tel. (44) 81-658 2211, fax (44) 81-663 6176.
274
NG-Nitro-L-arginine methyl ester (L-NAME), Larginine hydrochloride, D-arginine hydrochloride, atropine sulphate and angiotensin I I were obtained from Sigma Chemical Company (Poole, Dorset, U.K.) and dissolved freshly in isotonic saline. Results, shown as the changes in resting intraluminal pressure (.1 m m H g ) and the amplitude (A m m H g ) and frequency (number min ~) of the jejunal motility responses are shown as the means + S.E.M. of In) studies. The difference between groups was evaluated by Student's t-test for paired or unpaired data, and P < 0.05 was taken as significant.
3. Results
Under resting conditions, stable levels of intraluminal pressure (3 + 1 mmHg; n = 30) were maintained in the rat jejunum, with minimal spontaneous contractions in this acute model (fig. 1). Intravenous administration of L - N A M E (0.5-10 mg kg-1) caused a dosedependent significant (P < 0.05) increase in jejunal intraluminal pressure (fig. 2). This effect on resting tone was apparent within 1 min of administration of these doses of L-NAME, reaching the maximal response after 2 - 3 min, which then diminished over the following 10-30 min. Furthermore, the administration of L - N A M E (1-10 mg kg 1 i.v.) also produced frequent intestinal muscular contractions, observed by inspection of the intestinal loops, in doses causing increases in jejunal tone. Both the amplitude and frequency of these contractions induced by L-NAME, which were
recorded as rapid changes in jejunal intraluminal pressure, were dose-related (fig. 2). Concurrent administration of L-arginine (100-200 mg kg ~ i.v.) reduced these motility responses induced by L-NAME. Thus, L-arginine (200 mg kg ~ i.v.) abolished I n = 5 , P < 0 . 0 0 1 ) the increase in intraluminal pressure and inhibited the frequency and amplitude by 51 _+ 7 and 74 + 8%. respectively (n = 5: P < 0.01) of the motility responses induced by L-NAME (10 mg k g t i.v.), whereas D-arginine (200 mg kg ~ i.v.) did not significantly inhibit these responses (n = 4). L-NAME (1. 5 and 10 mg kg ~ i.v.) induced a dose-dependent significant (P < 0.01) increase in systemic arterial blood pressure from its resting value (115 + 10 mmHg, n = 30), by A 26 + 4, .1 33 + 3 and d 35 + 2 m m H g respectively. Concurrent administration of L-arginine (200 mg kg ~ i.v.) reduced the hypertensive response to L - N A M E ( 1 0 mg kg J i.v.) by 87 + 20% (n = 5, P < 0.05), whereas D-arginine had no significant action (n - 4). As shown in fig. 1, a lower dose of L-arginine (100 mg kg ~ i.v.) substantially reduced the frequency of the motility responses to L-NAME (l0 mg kg 1) yet had minimal action on the increase in systemic BP, suggesting these motility changes are independent of changes in BP. Furthermore, intravenous infusion of angiotensin It (2 /~g kg I rain ~) increased BP (by 25 + 6 mmHg, n = 3) but did not significantly affect resting intralumina[ pressure (n = 3). Administration of atropine (4 mg kg ~ i.v.) significantly reduced the intraluminal pressure changes induced by L - N A M E ( I - 1 0 mg kg t ) a s shown in fig. 2 and also reduced the BP response (by 11 _+ 2 m m H g for the intermediate dose of L-NAME). The effect of
2001 mmHg /
0 ~
I
J
1rain
]
15-
mmHg
o
INTRALUMINAL PRESSURE
( L-NAME 10mg kg"1 i.v.
L-ARGININE 100mg kg"1 i.v.
Fig. 1. Effect of intravenous administration of L-NAME (10 mg kg i) on jejunal resting intraluminal pressure (A mmttg) and on systemic arterial blood pressure (BP, mmHg) in the anaesthetised rat. The chart-recording of the experiment also shows the inhibitory effect on motility of subsequent administration of L-arginine (ll)(I mg kg 1 i.v.).
275
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I~
3
+At[opine
¢-~:2 "~E
~
o 5 4
-a3
