Vol. 181, No. 2, 1991 December 16. 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 8894393
ENDOGENOUS NITRK: MORPHINE-INDUCED A. Calignano,
OXIDE MODULATES CONSTIPATION
S. Moncada*
and M. Di Rosa
Department of Experimental Pharmacology, University of Naples “Federico If”, via D. Montesano 49, 80131 Naples, Italy *Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, U.K.
Received
October
26,
1991
SUMMARY: Administration of morphine in mice causes inhibition of the constipation in mice gastrointestinal transit of a charcoal meat. Morphine-induced seems to depend predominantly on action(s) on the central nervous system since Nmethyl morphine, a quaternary derivative, inhibits intestinal transit only when administered intracerebroventriculady (i.c.v.). L- but not D-arginine, given intraperitoneally, reversed the constipation induced by both morphine and its quaternary analogue. L-arginine was ineffective when given i.c.v. and did not reverse atropine-induced constipation. These results suggest that L-arginine preferentially modulates opioid-induced constipation through a stereospecific and peripheral action(s). It is possible that the effect of L-arginine is achieved by increasing the amount of nitric oxide released by non-adrenergic, non-cholinergic nerves in the gut. Thus, L-arginine may represent a useful agent for the treatment of undesirable constipation associated with the use of narcotic analgesics. 0 1991Academic Press, Inc.
Morphine
and other opioids cause inhibition of the propulsive activity of the gut
which may result in undesirable analgesics
constipation
complicating
(1). This effect of opioids on gastrointestinal
pain relief by narcotic motor function has been
attributed both to actions within the central nervous system (2) and to direct action on peripheral
receptors within the enteric nervous system (3). The relative importance
of the two sites of action, as well as the mechanism propulsive
activity of the gut, remain unclear (4).
Intestinal released
(s) by which opioids affect the
peristalsis
is coordinated
from the enteric nervous
by a complex
system
889
(4).
array of neurotransmitters
Non-adrenergic,
non-cholinergic
ooo6-291x191 $1.50 Copyright 0 1991 by Academic Press, far. All rights of reproduction in any form reserved.
Vol.
181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(NANC) neurones have been recognized in many parts of the gastrointestinal and have been found to mediate gastrointestinal
relaxation in several species via the
release of nitric oxide (NO), which acts either as a neurotransmitter of neurotransmission
or as a modulator
(6-8).
In order to investigate whether this putative neurotransmitter opioid-induced
tract (5)
plays a role in the
inhibition of the propulsive activity of the gut, we have studied the effect
of L-arginine, the physiological
precursor of NO (9), on morphine-induced
constipation
in mice. MATERIALS
AND METHODS
Adult male Swiss mice (20-25 g) were used after four days acclimatization to They were fasted for 12 h before experiments, with water housing conditions. available ad libitum. All animals received orally 0.1 ml of an aqueous suspension of 10% charcoal in 5% acacia gum. Animals were sacrificed 20 min after the charcoal meal and the intestine and stomach were removed. The length travelled by the charcoal was calculated as a percentage of the intestinal length (10) and the results were expressed as means f S.E.M. Means were compared by use of Student’s t test. Drugs under investigation were dissolved in saline and administered intraperitoneally (i.p. 0.5 ml/mouse), subcutaneously (s.c. 0.1 ml/mouse) or i.c.v. (10 @/mouse) at the doses indicated in the results section. Morphine and N-methyl morphine were administered 30 min before the charcoal meal, whereas atropine, Llysine, D- and L-arginine (hydrochloride) were administered 60 min before. Morphine hydrochloride was obtained from SALARS (Italy), N-methyl morphine was synthetized in our laboratory, and all other drugs were obtained from SIGMA (USA). RESULTS In preliminary propulsive
experiments
we established
effect, whereas when injected the propulsive
L-arginine
morphine
on the
meal.
(2.5 mg/kg s.c.) had no
i.c.v. (0.06 mg/kg) it was as potent as morphine
in
given up to 30 mg/kg i.p. or up to 3 mg/kg i.c.v. on its own had no activity for it did not affect the length travelled
However, this amino
administration
abolished
ineffective when given i.c.v (3 mg/kg). by L-arginine
morphine
action of the gut (see table I).
effect on the gut propulsive charcoal
of morphine
activity of the mouse gut was 2.5 mg f 0.3 mg/kg when injected S.C. and
0.06 f 0.001 mg/kg when injected i.c.v. N-methyl inhibiting
that the ED,
was dose-related
acid given i.p. (30 mg/kg)
the opioid-induced
30 min before
constipation,
The reversal of morphine-induced
(Fig. 1). Furthermore, 890
L-arginine
by the
while it was constipation
given orally at 30
Vol.
181, No. 2, 1991
Table I.
BIOCHEMICAL
A+JD BIOPHYSICAL
RESEARCH COMMUNICATIONS
Effect of L-arginine on morphine-inducedconstipation
Drugs/Route
i.p. saline
i.c.v. saline
L-arg
saline1s.c.
8Of3 (12)
81f4 (12)
saline/i.c.v.
81*3 (12)
8M5 (12)
M0FVs.c.
37s (12)**
8of7” (12),,
M0Wi.c.v.
42&8 (6)”
N-M0Ws.c. N-M0Wi.c.v.
L-arg
(6)
81f7 (6)
n-t.
n.t.
4M4 (6)
42f8 (6)
81s (6)“O
n.t.
n.t.
8W8 (6)
85*5 (6)
n.t.
n.t.
37s (6)”
87s (6)
n.t.
n.t.
~2
Values are mean f S.E.M. of the intestinal length travelled by the charcoal meal expressed as percentage of the intestinal length. Numbersin brackets refer to the numbers of animals. Morphine (MCR) or N-methyl morphine (N-MOR) were administered(2.5 mg/kg S.C.or 0.06 mg/kg i.c.v.) 30 min before the charcoal meal; Larginine (L-arg) was given (30 mg/kg i.p. or 3 mgikg i.c.v.) 60 min before the charcoal meal. p < 0.05, ** p c 0.001 vs saline; O0c 0.001 x MOR. l
mgikg also abolished morphine-induced constipation (n=6, data not shown). Atropineinduced constipation (0.1 mgikg i.p.) was, however, unaffected by L-arginine given i.p. up to 30 mg/kg (see fig. 1).
-
morphine-,
L-arginine
Fioure 1 Effect of L-arginine on morphine- and atropine-inducedconstipation in mice. Each column represents the mean value f S.E.M. (n=6-8) of the length travelled by the charcoal meal, expressed as a percentage of the intestinal length. C = control. Morphine (2.5 mg/kg s.c.) was given 30 min before the charcoal meat; atropine (0.1 mg kg i.p.) and L-argininewere given i.p. 60 min before it at the dose (mgikg) indicated below the columns. p < 0.05, ** p < 0.01 s morphine alone. l
891
Vol.
181, No. 2, 1991
D-arginine propulsive
BtOCHEMtCAL
AND BtOPHYStCAL
RESEARCH COMMUNtCATtONS
or L-lysine, given i.p. at doses up to 50 mg/kg, had no effect on gut
activity nor did they modify morphine-induced
constipation
(n=6, data not
shown).
DISCUSSION Our results show that morphine has a constipating to depend predominantly ED,
(2.5 mg/kg)
interpretation, supported
on action(s) within the central nervous system since the i.p.
is about 40 times greater than that i.c.v. (0.06 mg/kg).
which is in agreement
by experiments
with previous
showing that N-methyl
of morphine caused constipation
L-arginine
or its quaternary
was ineffective in modifying
or atropine-induced
analogue
inhibition
i.c.v.
morphine.
In
constipation.
D-
activity nor were they able to modulates
opioid-induced
and operates by peripheral
mechanism(s).
in peristalsis
by relaxing the gut (14).
evidence suggests that NANC neurones possess nicotinic cholinergic
(15) and that presynaptic
acetylcholine
N-methyl
atropine-induced
preferentially
It is well known that NANC nerves participate receptors
derivative
constipation.
an effect that is stereospecific
Pharmacological
is further
activity, was effective in reversing the constipation
These results suggest that L-arginine constipation,
a quatemary
This
i.p., but not i.c.v., which on its own
arginine and L-lysine had no effect on gut propulsive modify morphine
(ll-13),
at the doses used only when administered
had no effect on gut propulsive induced in mice by morphine
findings
morphine,
We have shown that L-arginine administered
contrast,
effect in mice which seems
inhibition
by morphine
results in a reduced
release at both muscarinic and nicotinic receptor sites (4,16). Thus, the
of gut propulsive activity induced by morphine
both actions of acetylcholine muscarinic
on gastrointestinal
receptors) and relaxation
may result from prevention of
smooth muscle:
i.e. contraction
(via the nicotinic receptors of NANC nerves).
Recently it has been shown that relaxation of gastrointestinal by NO synthase
inhibitors
muscle following NANC
stimulation
is abolished
suggesting
that NO is released by NANC nerves (6,6). A conceivable
our results is that L-arginine
(via
and restored
could reverse the constipating
by L-arginine, explanation
of
effect of morphine
by
increasing the levels of NO released by NANC nerves, creating a relaxing tone which enables the reduced levels of acetylcholine peristalsis via stimulation
of muscarinic receptors. This hypothesis is supported by the
results showing that L-arginine depends
on the blockade
mechanism(s)
released to be effective in promoting
had no effect on atropine-induced
of muscarinic
by which L-arginine
receptors.
abolishes 892
constipation
which
Although the elucidation
of the
morphine-induced
constipation
in mice
Vol.
181, No. 2, 1991
deserves
BIOCHEMICAL
further investigations,
our findings
represent a useful agent for the treatment with therapeutic
administration
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
also suggest
of undesirable
or with compulsive
that L-arginine
constipation
could
often associated
use of narcotic analgesics.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Jaffe, J.H. and Martin, W.R. (1990). In: The Pharmacological Basis of Therapeutics, eds. Goodman Gillman A, Rail, T.W., Nies, A.S. & Taylor, P. Pergamon Press, 485521. Porreca, F. and Burks, T.F. (1983). J. Pharmacol. Exp. Ther., 227. 22-27. Tavani, A., Bianchi, G., Cerretti, P. and Manara, L. (1980). Life Sci., 27. 221 l2217. Kromer, W. (1988). Pharmacol. Rev., 40. 121-126. Abrahamsson, H. (1986). Arch. Int. Pharmacodyn. Ther., 280, 50-61. Boeckxstaens, G.E., Pelckmans, P.A., Bult, H., De Man, J.G, Costerbosch, L., Herman, A.G., Van Maercke, Y.M. (1990). J. Pharmacol. Exp. Ther., 256,441447. Gustafsson, L.E., Wiklund, C.U., Wiklund, N.P., Persson, M.G. and Moncada, S. (1990). Biochem. Biophys. Res. Commun., 173, 106-l 10. Desai, K.M., Sessa, W.C. and Vane, JR. (1991). Nature, 351. 477-479. Palmer, R.M.J., Ashton, D.S. and Moncada, S. (1988). Nature, 333,664666. Ramabadran, K., Bansinath, M., Turndorf, H. and Puig, M.M. (1988). Eur. J. Pharmacol., 155. 394-331. Schulz, R., Wuster, M. and Herz, A. (1979). Naunyn. Schmied. Arch. Pharmacol., 308.255-260. Margolin, S. (1963). Proc. Sot. Exp. Biol. Med., 112, 31 l-315. Parolaro, D., Sala, M. and Gori, E. (1977). Eur. J. Pharmacol., 46, 329338. Costa, M., Fumess, J.B. and Humphreys, C.M.S. (1986). Naunyn. Schmied. Arch. Pharmacol., 332, 79-88. Burnstock, G., Campbell, G. and Rand, M. J. (1966). J. Physiol., 182. 504-526. Gillian, M.G.C. and Pollock, D. (1976). Br. J. Pharmacol., 57. 444-445.
893
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 8894393
ENDOGENOUS NITRK: MORPHINE-INDUCED A. Calignano,
OXIDE MODULATES CONSTIPATION
S. Moncada*
and M. Di Rosa
Department of Experimental Pharmacology, University of Naples “Federico If”, via D. Montesano 49, 80131 Naples, Italy *Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, U.K.
Received
October
26,
1991
SUMMARY: Administration of morphine in mice causes inhibition of the constipation in mice gastrointestinal transit of a charcoal meat. Morphine-induced seems to depend predominantly on action(s) on the central nervous system since Nmethyl morphine, a quaternary derivative, inhibits intestinal transit only when administered intracerebroventriculady (i.c.v.). L- but not D-arginine, given intraperitoneally, reversed the constipation induced by both morphine and its quaternary analogue. L-arginine was ineffective when given i.c.v. and did not reverse atropine-induced constipation. These results suggest that L-arginine preferentially modulates opioid-induced constipation through a stereospecific and peripheral action(s). It is possible that the effect of L-arginine is achieved by increasing the amount of nitric oxide released by non-adrenergic, non-cholinergic nerves in the gut. Thus, L-arginine may represent a useful agent for the treatment of undesirable constipation associated with the use of narcotic analgesics. 0 1991Academic Press, Inc.
Morphine
and other opioids cause inhibition of the propulsive activity of the gut
which may result in undesirable analgesics
constipation
complicating
(1). This effect of opioids on gastrointestinal
pain relief by narcotic motor function has been
attributed both to actions within the central nervous system (2) and to direct action on peripheral
receptors within the enteric nervous system (3). The relative importance
of the two sites of action, as well as the mechanism propulsive
activity of the gut, remain unclear (4).
Intestinal released
(s) by which opioids affect the
peristalsis
is coordinated
from the enteric nervous
by a complex
system
889
(4).
array of neurotransmitters
Non-adrenergic,
non-cholinergic
ooo6-291x191 $1.50 Copyright 0 1991 by Academic Press, far. All rights of reproduction in any form reserved.
Vol.
181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(NANC) neurones have been recognized in many parts of the gastrointestinal and have been found to mediate gastrointestinal
relaxation in several species via the
release of nitric oxide (NO), which acts either as a neurotransmitter of neurotransmission
or as a modulator
(6-8).
In order to investigate whether this putative neurotransmitter opioid-induced
tract (5)
plays a role in the
inhibition of the propulsive activity of the gut, we have studied the effect
of L-arginine, the physiological
precursor of NO (9), on morphine-induced
constipation
in mice. MATERIALS
AND METHODS
Adult male Swiss mice (20-25 g) were used after four days acclimatization to They were fasted for 12 h before experiments, with water housing conditions. available ad libitum. All animals received orally 0.1 ml of an aqueous suspension of 10% charcoal in 5% acacia gum. Animals were sacrificed 20 min after the charcoal meal and the intestine and stomach were removed. The length travelled by the charcoal was calculated as a percentage of the intestinal length (10) and the results were expressed as means f S.E.M. Means were compared by use of Student’s t test. Drugs under investigation were dissolved in saline and administered intraperitoneally (i.p. 0.5 ml/mouse), subcutaneously (s.c. 0.1 ml/mouse) or i.c.v. (10 @/mouse) at the doses indicated in the results section. Morphine and N-methyl morphine were administered 30 min before the charcoal meal, whereas atropine, Llysine, D- and L-arginine (hydrochloride) were administered 60 min before. Morphine hydrochloride was obtained from SALARS (Italy), N-methyl morphine was synthetized in our laboratory, and all other drugs were obtained from SIGMA (USA). RESULTS In preliminary propulsive
experiments
we established
effect, whereas when injected the propulsive
L-arginine
morphine
on the
meal.
(2.5 mg/kg s.c.) had no
i.c.v. (0.06 mg/kg) it was as potent as morphine
in
given up to 30 mg/kg i.p. or up to 3 mg/kg i.c.v. on its own had no activity for it did not affect the length travelled
However, this amino
administration
abolished
ineffective when given i.c.v (3 mg/kg). by L-arginine
morphine
action of the gut (see table I).
effect on the gut propulsive charcoal
of morphine
activity of the mouse gut was 2.5 mg f 0.3 mg/kg when injected S.C. and
0.06 f 0.001 mg/kg when injected i.c.v. N-methyl inhibiting
that the ED,
was dose-related
acid given i.p. (30 mg/kg)
the opioid-induced
30 min before
constipation,
The reversal of morphine-induced
(Fig. 1). Furthermore, 890
L-arginine
by the
while it was constipation
given orally at 30
Vol.
181, No. 2, 1991
Table I.
BIOCHEMICAL
A+JD BIOPHYSICAL
RESEARCH COMMUNICATIONS
Effect of L-arginine on morphine-inducedconstipation
Drugs/Route
i.p. saline
i.c.v. saline
L-arg
saline1s.c.
8Of3 (12)
81f4 (12)
saline/i.c.v.
81*3 (12)
8M5 (12)
M0FVs.c.
37s (12)**
8of7” (12),,
M0Wi.c.v.
42&8 (6)”
N-M0Ws.c. N-M0Wi.c.v.
L-arg
(6)
81f7 (6)
n-t.
n.t.
4M4 (6)
42f8 (6)
81s (6)“O
n.t.
n.t.
8W8 (6)
85*5 (6)
n.t.
n.t.
37s (6)”
87s (6)
n.t.
n.t.
~2
Values are mean f S.E.M. of the intestinal length travelled by the charcoal meal expressed as percentage of the intestinal length. Numbersin brackets refer to the numbers of animals. Morphine (MCR) or N-methyl morphine (N-MOR) were administered(2.5 mg/kg S.C.or 0.06 mg/kg i.c.v.) 30 min before the charcoal meal; Larginine (L-arg) was given (30 mg/kg i.p. or 3 mgikg i.c.v.) 60 min before the charcoal meal. p < 0.05, ** p c 0.001 vs saline; O0c 0.001 x MOR. l
mgikg also abolished morphine-induced constipation (n=6, data not shown). Atropineinduced constipation (0.1 mgikg i.p.) was, however, unaffected by L-arginine given i.p. up to 30 mg/kg (see fig. 1).
-
morphine-,
L-arginine
Fioure 1 Effect of L-arginine on morphine- and atropine-inducedconstipation in mice. Each column represents the mean value f S.E.M. (n=6-8) of the length travelled by the charcoal meal, expressed as a percentage of the intestinal length. C = control. Morphine (2.5 mg/kg s.c.) was given 30 min before the charcoal meat; atropine (0.1 mg kg i.p.) and L-argininewere given i.p. 60 min before it at the dose (mgikg) indicated below the columns. p < 0.05, ** p < 0.01 s morphine alone. l
891
Vol.
181, No. 2, 1991
D-arginine propulsive
BtOCHEMtCAL
AND BtOPHYStCAL
RESEARCH COMMUNtCATtONS
or L-lysine, given i.p. at doses up to 50 mg/kg, had no effect on gut
activity nor did they modify morphine-induced
constipation
(n=6, data not
shown).
DISCUSSION Our results show that morphine has a constipating to depend predominantly ED,
(2.5 mg/kg)
interpretation, supported
on action(s) within the central nervous system since the i.p.
is about 40 times greater than that i.c.v. (0.06 mg/kg).
which is in agreement
by experiments
with previous
showing that N-methyl
of morphine caused constipation
L-arginine
or its quaternary
was ineffective in modifying
or atropine-induced
analogue
inhibition
i.c.v.
morphine.
In
constipation.
D-
activity nor were they able to modulates
opioid-induced
and operates by peripheral
mechanism(s).
in peristalsis
by relaxing the gut (14).
evidence suggests that NANC neurones possess nicotinic cholinergic
(15) and that presynaptic
acetylcholine
N-methyl
atropine-induced
preferentially
It is well known that NANC nerves participate receptors
derivative
constipation.
an effect that is stereospecific
Pharmacological
is further
activity, was effective in reversing the constipation
These results suggest that L-arginine constipation,
a quatemary
This
i.p., but not i.c.v., which on its own
arginine and L-lysine had no effect on gut propulsive modify morphine
(ll-13),
at the doses used only when administered
had no effect on gut propulsive induced in mice by morphine
findings
morphine,
We have shown that L-arginine administered
contrast,
effect in mice which seems
inhibition
by morphine
results in a reduced
release at both muscarinic and nicotinic receptor sites (4,16). Thus, the
of gut propulsive activity induced by morphine
both actions of acetylcholine muscarinic
on gastrointestinal
receptors) and relaxation
may result from prevention of
smooth muscle:
i.e. contraction
(via the nicotinic receptors of NANC nerves).
Recently it has been shown that relaxation of gastrointestinal by NO synthase
inhibitors
muscle following NANC
stimulation
is abolished
suggesting
that NO is released by NANC nerves (6,6). A conceivable
our results is that L-arginine
(via
and restored
could reverse the constipating
by L-arginine, explanation
of
effect of morphine
by
increasing the levels of NO released by NANC nerves, creating a relaxing tone which enables the reduced levels of acetylcholine peristalsis via stimulation
of muscarinic receptors. This hypothesis is supported by the
results showing that L-arginine depends
on the blockade
mechanism(s)
released to be effective in promoting
had no effect on atropine-induced
of muscarinic
by which L-arginine
receptors.
abolishes 892
constipation
which
Although the elucidation
of the
morphine-induced
constipation
in mice
Vol.
181, No. 2, 1991
deserves
BIOCHEMICAL
further investigations,
our findings
represent a useful agent for the treatment with therapeutic
administration
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
also suggest
of undesirable
or with compulsive
that L-arginine
constipation
could
often associated
use of narcotic analgesics.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Jaffe, J.H. and Martin, W.R. (1990). In: The Pharmacological Basis of Therapeutics, eds. Goodman Gillman A, Rail, T.W., Nies, A.S. & Taylor, P. Pergamon Press, 485521. Porreca, F. and Burks, T.F. (1983). J. Pharmacol. Exp. Ther., 227. 22-27. Tavani, A., Bianchi, G., Cerretti, P. and Manara, L. (1980). Life Sci., 27. 221 l2217. Kromer, W. (1988). Pharmacol. Rev., 40. 121-126. Abrahamsson, H. (1986). Arch. Int. Pharmacodyn. Ther., 280, 50-61. Boeckxstaens, G.E., Pelckmans, P.A., Bult, H., De Man, J.G, Costerbosch, L., Herman, A.G., Van Maercke, Y.M. (1990). J. Pharmacol. Exp. Ther., 256,441447. Gustafsson, L.E., Wiklund, C.U., Wiklund, N.P., Persson, M.G. and Moncada, S. (1990). Biochem. Biophys. Res. Commun., 173, 106-l 10. Desai, K.M., Sessa, W.C. and Vane, JR. (1991). Nature, 351. 477-479. Palmer, R.M.J., Ashton, D.S. and Moncada, S. (1988). Nature, 333,664666. Ramabadran, K., Bansinath, M., Turndorf, H. and Puig, M.M. (1988). Eur. J. Pharmacol., 155. 394-331. Schulz, R., Wuster, M. and Herz, A. (1979). Naunyn. Schmied. Arch. Pharmacol., 308.255-260. Margolin, S. (1963). Proc. Sot. Exp. Biol. Med., 112, 31 l-315. Parolaro, D., Sala, M. and Gori, E. (1977). Eur. J. Pharmacol., 46, 329338. Costa, M., Fumess, J.B. and Humphreys, C.M.S. (1986). Naunyn. Schmied. Arch. Pharmacol., 332, 79-88. Burnstock, G., Campbell, G. and Rand, M. J. (1966). J. Physiol., 182. 504-526. Gillian, M.G.C. and Pollock, D. (1976). Br. J. Pharmacol., 57. 444-445.
893
