Pergamon Press
Life Sciences Vol. 18, pp . 803-810 Printed is the U.S .A.
UNEXPECTED REVERSAL EFFECTS OF NALOXONE ON THE GUINEA PIG ILEUM Jan M . Vaa Nueten, Paul A , J. Janssen and Jeanine Fontaiae l Department of Pharmacology, Research Laboratories Janssen Pharmaceutics, B-2340 Beerae, Belgium (Received in final form March 5, 1976) Summary Distension of the guinea pig ileum segment elicits peristaltic activity . If the distension is maintained the peristaltic activity disappears gradually ; naloxoae restores normal activity in such "fatigued" preparations, The bath solution surrounding a fatigued preparation inhibits peristaltic reflex activity in non-fatigued segments ; this inhibitory effect is reversed by naloxoae, The latter also antagonizes the inhibitory effects of adenine-nucleotides, These results indicáte that during fatigue a substance is liberated which blocks peristalsis, They further suggest that naloocone-induced reversal of inhibition is the guinea pig ileum does not necessarily demonstrate that the inhibition ie caused by a direct action on morphine receptors, In the isolated guinea pig ileum stimulated electrically at low frequencies, morphine-like narcotics, adenosine, adenosine monophoaphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP) inhibit both the release of acetylcholiae from the poatganglionic nerve endings and the contractile response (1-5) . Morphine-like narcotica and adenine-nucleotiden also inhibit the peristaltic reflex activity ia the guinea pig ileum (6-10) . Naloxone and other "narcotic antagonists" reverse the inhibitory effects of all known morphine-like narcotics on the guinea pig ileum whether activated by electrical stimulation (11-13) or reflexly by pressure-induced dis tension (8), Naloxone has also been shown to antagonize the inhibitory effect of enkephalia, a peptide found in the brain of many species and in the guinea pig ileum; thin prompted the suggestion that enkephalin ie an agoRiat for opiate receptor sites (14, 15) . The present experiments demonstrate that naloxoae also can restore normal peristaltic reflex activity in fatigued preparations or after inhibition by adenine nucleotides, 1 Laboratoire de Pharmacologie, Institut de Pharmacie, Free University of Brussels, Belgium, 803
Naloaone on the Guinea Pig Ileum
804
Vol . 18, No . 8
Methods peristaltic reflex is studied on the isolated guinea pig ileum using a modification of the method of Trendelenburg, as described before (16) . Briefly, an ileum segment is suspended vertically in Tyrode solution of 37° C at pH7 .4 with a gas mixture 95 f°o 02, 5 °fo C02, The open aboral end is connected to perspex tubing linked up to a pressure bottle, and the closed oral end Yo a Grass FT-03 C force transducer, Pressure is increased in the lumen of the segment from -5 to 20 mmH20 by raising the pressure bottle gradually over a period of 50 sec, and is maintained at this level for the duration of the experiment . This pressure elicits repetitive peristaltic reflex activity, i,e, coordinated contractions of both muscle layers, leading to peristaltic waves travelling in an aboral direction and to expulsion of the intraluminal fluid, parameters recorded are the activity of the longitudinal muscle, measured as tension changes with the force transducer, the activity of the circular muscle, measured as development of intraluminal pressure with the aid of a Statham P23 GB pressure transducer, and the fluid volume displacement measured via an ultrasonic transit-time meter, based on the pulse principle, The resting tension is 2 g in the experiments, in which "fatigue" is to be elicited, and 1 g in the other experiments, All drugs are dissolved in distilled water and added in a volume of 0, 25 ml to the 100 ml organ bath 2-3 min following induction of the peristaltic reflex, Fig, 1 shows the original recording of an experiment performed as described, and illustrates the abolition of peristaltic reflex activity by morphine and its reversal by naloxone,
v
" NALO%ONE 10 nOhnl
MORPHINE 0.16 p0~ml
1 min.
L
b
FIG . 1
Morphine block of peristaltic reflex activity of the guinea pig ileum and complete reversal by naloxone, a, longitudinal muscle tension; b, volume displacement ; c, circular muscle activity, measured ae intraluminal pressure, Results Distension of the guinea pig ileum segment by a pressure of
~Vol . 18, No . 8
Naloxone on the Guinea Pig Ileum
805
20 mmH20 elicits regular peristaltic reflex activity, This induced reflex activity remains surprisingly regular for several minutes, although, if the distension is prolonged, the activity eventually slows down, is interrupted in as intermittent way and may cease completely after 15-60 minutes (fig, 2),
NALOXONE 1D~ml
1 mln :
u~uJ~*1~Mr - UI~u~I~M~
e
J FIG . 2
Peristaltic reflex activity of a guinea pig ileum segment submitted to continuous distension of the lumen at a constant preload of 2 g, "Fatigue", expressed ae repeated interruptions of the reflex, appears within 30 minutes (~~, a period of 25 min), Naloxane abolishes the effect of "fatigue", and the longitudinal muscle develops more tension than before, a, b and c as in fig, 1, This phenomenon of "fatigue" (Ermtidung) (6) is reversed by 10 ng~ml of naloxone (fig, 2), a concentration which does not interfere with normal peristalsis ; in all of our 15 experiments regular reflex activity is restored within 30 seconds by the addition of naloxone, In control experimental conditions the solvent used for naloxone has no effect, When the tyrode solution bathing a "fatigued" segment is transferred to another bath in which a "non-fatigued" segment is suspended, a rapid inhibition of peristalsis is observed in all experiments (n=5) . Subsequent addition of 10 ng~ml of naloxone reverses this inhibition (fig, 3), A dose-related inhibition of the peristaltic reflex activity is observed with adenosine, AMP, ADP and ATP from doses as low as 0,63 to 2 .5 ug~ml, Nalaxoae antagonizes the inhibitory effect of all these adenosine nucleotides and restores peristaltic reflex activity (n=5, for each concentration of the nucleotidee), A typical example is shown for adenosine in fig, 4 . piecuesion Prolonged distension of a guinea pig ileum segment Leads to inhibition of the peristaltic reflex activity (fatigue), The bath solution surrounding a fatigued preparation inhibits peristalsis in a non-fatigued segment, Theee findings indicate that during fatigue a substance is released which inhibits peri-
806
Naloxone on the Guinea Pig Ileum
Vol . 18, No . 8
NALOXONE Xlnp/ml
F 1 ml yn
e
FIG . 3
Transfer (TR) of the solution bathing a "fatigued" guinea pig ileum segment to a bath in which a "non-fatigued" segment was suspended, inhibits the peristaltic activity of the latter, Nalaxone reverses this inhibition, a, b and c as in fig . 1 .
NALOXONE f0 nglml
ADENOSINE 1.25 ~p/ml
- 1~~^"
e
FIG . 4
Adenosine induced inhibition of peristaltic reflex activity of the guinea pig ileum and reversal by aaloxoae, a, b and c as in fig . 1 . stalaie . Naloxone reverses both the "fatigue" and the inhibitory effect produced by the endogenous substance liberated during "fatigue" . Adenosine, AMP, ADP and ATP inhibit peristaltic reflex activity and this
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Vol . 18, No . 8
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Naloaone on the Guinea Pig Ileum
Vol . 18, No . 8
inhibitory effect is reversed by naloxone, A variety of other substances inhibit peristalsis of the guinea pig ileum, Table 1 illustrates that the inhibitory effect of narcotic analgesics is reversed by naloxone and nalorphine, but also that naloxone antagonizes the inhibition caused by a number of substances un related to morphine, The fact that an inhibitory effect such as that caused by adenine-nucleotides is antagonized by naloxone therefore does not necessarily indicate a direct interaction with morphine receptors . An alternative explanation could be that adenine-nucleotides (and possibly other agents as well) produce inhibition of peristalsis by releasing an endogenous substance, the effect of which ie antagonized by naloxone, At least part of inhibitory effects on the peristaltic reflex is due to decrease of acetylcholine release from nerve endings innervating the longitudinal muscle (17) ; since naloxone antagonizes these effects, it may act on the de pressed guinea pig ileum when its rate of acetylcholine release ie abnormally low, Thie then could mean that the endogenous substance which is liberated during "fatigue", and possibly by adenine-nucleotides, may diminish the rate of acetylcholine release, The identity of the endogenous substance is not known, but there are a number of candidates that should be considered among which enkephalin appears the most likely . Acknowled¢mente We thank Mr, L. Helsen for technical assistance and Dr, Paul M, Vanhoutte for helpful suggestions, References 1, W, SCHAUMANN, Br . ~. Pharmacol . ~j, 115-118 (1957) . 2 . W,D .M, PATON, ~r_ T, Pharmacol . ~, 119-127 (1957) . 3. M, R . FENNESSY, R . L, H. HEIMANS, and M, J . RAND, ~, ~. Pharmacol . 436-449 (1969) . 4 . K, TAK~SGI, aad I, TAKAYANAGI, ,~,,L, Pharmacol . Z,Z, 33-36 (1972), 5 . M . MORI, S, YAMADA, S, TAKAMURA, and E, HAYASHI, , ~a .,i . harmacol. ~ 124 (abetr) (1973), s Arch , EXA . thol ~har6. P, TRENDELENBURG, Naunva-Schmiedebergmakol . 81 55-129 (1917), 7 . O, SCH~.UMANN, M, GIOVANNINI, and K. JOCHUM, hLauaya-Schmiede ber¢s Arch .Exp , thol Pharmakol . ~1,~ 406-468 (1952) . 8, E,A, GYANG, H,W . KOSTERLITZ, and G,M, LEES, Naunyn-Schmiede ber¢s Arch , Exp , Pathol .pharmakol . ~8 231-246 (1964), ei 1 Pharmacol. 9, F,K. OKWUASABA, and J.T, HAMILTON, an .,j, 972-977 (1975) . rma10 . J. FONTAINE, J. J . REUSE, and J, M. VANNUETEN, Arc nt, codyn , her, ~ 376-380 (1973), 11 . H. W . KOSTERLITZ, and A, J, WATT, ,~,~ . Pharmacol. ~ 266-276 (1968) . 12, A, L . COWIE, H, W, . Z{OSTERLITZ, and A . J . WATT, at re ~,~,Q 10401042 (1968) . 13 . S, EHRENPRELS, I . LIGHT, and G,H. SCHONBUCH, In Drua Addiction: Exyerimental Pharmacolo¢y . J . M. Singh, L . Miller and H. Lal (Eds), pp 319-342, Futura, Mt Kieco, New York (1972) . 14 . J. HUGHES, rain es 88 295-308 (1975) .
Vol . 18, No . 8
Naloxone oa the Guinea Pig Ileum
809
15 . J . HUGHES, T . SMITH, B . MORGAN, aad L . FOTHERGII~L, i e í~çi~ 1~ 1753-1758 (1975) . 16 . J . M. VAN NUETEN, H . LEIVERS, J. FONTAINE, aad P .A, J, JANSSEN, Arc ~t . Pharmacodva . Ther , ~} 411-414 (1973) . 17 . H . W . KOSTERLITZ, aad G . M . LEES, Pharmacol . ev 1~ 301 -339 (1964) . 18, J. M . VAN NUETEN, P.A, J . JANSSEN, aad J . FONTAINE, Arch , nt Pharmacodya, her (abstr), in press (1976) .
Life Sciences Vol. 18, pp . 803-810 Printed is the U.S .A.
UNEXPECTED REVERSAL EFFECTS OF NALOXONE ON THE GUINEA PIG ILEUM Jan M . Vaa Nueten, Paul A , J. Janssen and Jeanine Fontaiae l Department of Pharmacology, Research Laboratories Janssen Pharmaceutics, B-2340 Beerae, Belgium (Received in final form March 5, 1976) Summary Distension of the guinea pig ileum segment elicits peristaltic activity . If the distension is maintained the peristaltic activity disappears gradually ; naloxoae restores normal activity in such "fatigued" preparations, The bath solution surrounding a fatigued preparation inhibits peristaltic reflex activity in non-fatigued segments ; this inhibitory effect is reversed by naloxoae, The latter also antagonizes the inhibitory effects of adenine-nucleotides, These results indicáte that during fatigue a substance is liberated which blocks peristalsis, They further suggest that naloocone-induced reversal of inhibition is the guinea pig ileum does not necessarily demonstrate that the inhibition ie caused by a direct action on morphine receptors, In the isolated guinea pig ileum stimulated electrically at low frequencies, morphine-like narcotics, adenosine, adenosine monophoaphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP) inhibit both the release of acetylcholiae from the poatganglionic nerve endings and the contractile response (1-5) . Morphine-like narcotica and adenine-nucleotiden also inhibit the peristaltic reflex activity ia the guinea pig ileum (6-10) . Naloxone and other "narcotic antagonists" reverse the inhibitory effects of all known morphine-like narcotics on the guinea pig ileum whether activated by electrical stimulation (11-13) or reflexly by pressure-induced dis tension (8), Naloxone has also been shown to antagonize the inhibitory effect of enkephalia, a peptide found in the brain of many species and in the guinea pig ileum; thin prompted the suggestion that enkephalin ie an agoRiat for opiate receptor sites (14, 15) . The present experiments demonstrate that naloxoae also can restore normal peristaltic reflex activity in fatigued preparations or after inhibition by adenine nucleotides, 1 Laboratoire de Pharmacologie, Institut de Pharmacie, Free University of Brussels, Belgium, 803
Naloaone on the Guinea Pig Ileum
804
Vol . 18, No . 8
Methods peristaltic reflex is studied on the isolated guinea pig ileum using a modification of the method of Trendelenburg, as described before (16) . Briefly, an ileum segment is suspended vertically in Tyrode solution of 37° C at pH7 .4 with a gas mixture 95 f°o 02, 5 °fo C02, The open aboral end is connected to perspex tubing linked up to a pressure bottle, and the closed oral end Yo a Grass FT-03 C force transducer, Pressure is increased in the lumen of the segment from -5 to 20 mmH20 by raising the pressure bottle gradually over a period of 50 sec, and is maintained at this level for the duration of the experiment . This pressure elicits repetitive peristaltic reflex activity, i,e, coordinated contractions of both muscle layers, leading to peristaltic waves travelling in an aboral direction and to expulsion of the intraluminal fluid, parameters recorded are the activity of the longitudinal muscle, measured as tension changes with the force transducer, the activity of the circular muscle, measured as development of intraluminal pressure with the aid of a Statham P23 GB pressure transducer, and the fluid volume displacement measured via an ultrasonic transit-time meter, based on the pulse principle, The resting tension is 2 g in the experiments, in which "fatigue" is to be elicited, and 1 g in the other experiments, All drugs are dissolved in distilled water and added in a volume of 0, 25 ml to the 100 ml organ bath 2-3 min following induction of the peristaltic reflex, Fig, 1 shows the original recording of an experiment performed as described, and illustrates the abolition of peristaltic reflex activity by morphine and its reversal by naloxone,
v
" NALO%ONE 10 nOhnl
MORPHINE 0.16 p0~ml
1 min.
L
b
FIG . 1
Morphine block of peristaltic reflex activity of the guinea pig ileum and complete reversal by naloxone, a, longitudinal muscle tension; b, volume displacement ; c, circular muscle activity, measured ae intraluminal pressure, Results Distension of the guinea pig ileum segment by a pressure of
~Vol . 18, No . 8
Naloxone on the Guinea Pig Ileum
805
20 mmH20 elicits regular peristaltic reflex activity, This induced reflex activity remains surprisingly regular for several minutes, although, if the distension is prolonged, the activity eventually slows down, is interrupted in as intermittent way and may cease completely after 15-60 minutes (fig, 2),
NALOXONE 1D~ml
1 mln :
u~uJ~*1~Mr - UI~u~I~M~
e
J FIG . 2
Peristaltic reflex activity of a guinea pig ileum segment submitted to continuous distension of the lumen at a constant preload of 2 g, "Fatigue", expressed ae repeated interruptions of the reflex, appears within 30 minutes (~~, a period of 25 min), Naloxane abolishes the effect of "fatigue", and the longitudinal muscle develops more tension than before, a, b and c as in fig, 1, This phenomenon of "fatigue" (Ermtidung) (6) is reversed by 10 ng~ml of naloxone (fig, 2), a concentration which does not interfere with normal peristalsis ; in all of our 15 experiments regular reflex activity is restored within 30 seconds by the addition of naloxone, In control experimental conditions the solvent used for naloxone has no effect, When the tyrode solution bathing a "fatigued" segment is transferred to another bath in which a "non-fatigued" segment is suspended, a rapid inhibition of peristalsis is observed in all experiments (n=5) . Subsequent addition of 10 ng~ml of naloxone reverses this inhibition (fig, 3), A dose-related inhibition of the peristaltic reflex activity is observed with adenosine, AMP, ADP and ATP from doses as low as 0,63 to 2 .5 ug~ml, Nalaxoae antagonizes the inhibitory effect of all these adenosine nucleotides and restores peristaltic reflex activity (n=5, for each concentration of the nucleotidee), A typical example is shown for adenosine in fig, 4 . piecuesion Prolonged distension of a guinea pig ileum segment Leads to inhibition of the peristaltic reflex activity (fatigue), The bath solution surrounding a fatigued preparation inhibits peristalsis in a non-fatigued segment, Theee findings indicate that during fatigue a substance is released which inhibits peri-
806
Naloxone on the Guinea Pig Ileum
Vol . 18, No . 8
NALOXONE Xlnp/ml
F 1 ml yn
e
FIG . 3
Transfer (TR) of the solution bathing a "fatigued" guinea pig ileum segment to a bath in which a "non-fatigued" segment was suspended, inhibits the peristaltic activity of the latter, Nalaxone reverses this inhibition, a, b and c as in fig . 1 .
NALOXONE f0 nglml
ADENOSINE 1.25 ~p/ml
- 1~~^"
e
FIG . 4
Adenosine induced inhibition of peristaltic reflex activity of the guinea pig ileum and reversal by aaloxoae, a, b and c as in fig . 1 . stalaie . Naloxone reverses both the "fatigue" and the inhibitory effect produced by the endogenous substance liberated during "fatigue" . Adenosine, AMP, ADP and ATP inhibit peristaltic reflex activity and this
Naloaone oa the Guinea Pig Ileum
Vol . 18, No . 8
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Naloaone on the Guinea Pig Ileum
Vol . 18, No . 8
inhibitory effect is reversed by naloxone, A variety of other substances inhibit peristalsis of the guinea pig ileum, Table 1 illustrates that the inhibitory effect of narcotic analgesics is reversed by naloxone and nalorphine, but also that naloxone antagonizes the inhibition caused by a number of substances un related to morphine, The fact that an inhibitory effect such as that caused by adenine-nucleotides is antagonized by naloxone therefore does not necessarily indicate a direct interaction with morphine receptors . An alternative explanation could be that adenine-nucleotides (and possibly other agents as well) produce inhibition of peristalsis by releasing an endogenous substance, the effect of which ie antagonized by naloxone, At least part of inhibitory effects on the peristaltic reflex is due to decrease of acetylcholine release from nerve endings innervating the longitudinal muscle (17) ; since naloxone antagonizes these effects, it may act on the de pressed guinea pig ileum when its rate of acetylcholine release ie abnormally low, Thie then could mean that the endogenous substance which is liberated during "fatigue", and possibly by adenine-nucleotides, may diminish the rate of acetylcholine release, The identity of the endogenous substance is not known, but there are a number of candidates that should be considered among which enkephalin appears the most likely . Acknowled¢mente We thank Mr, L. Helsen for technical assistance and Dr, Paul M, Vanhoutte for helpful suggestions, References 1, W, SCHAUMANN, Br . ~. Pharmacol . ~j, 115-118 (1957) . 2 . W,D .M, PATON, ~r_ T, Pharmacol . ~, 119-127 (1957) . 3. M, R . FENNESSY, R . L, H. HEIMANS, and M, J . RAND, ~, ~. Pharmacol . 436-449 (1969) . 4 . K, TAK~SGI, aad I, TAKAYANAGI, ,~,,L, Pharmacol . Z,Z, 33-36 (1972), 5 . M . MORI, S, YAMADA, S, TAKAMURA, and E, HAYASHI, , ~a .,i . harmacol. ~ 124 (abetr) (1973), s Arch , EXA . thol ~har6. P, TRENDELENBURG, Naunva-Schmiedebergmakol . 81 55-129 (1917), 7 . O, SCH~.UMANN, M, GIOVANNINI, and K. JOCHUM, hLauaya-Schmiede ber¢s Arch .Exp , thol Pharmakol . ~1,~ 406-468 (1952) . 8, E,A, GYANG, H,W . KOSTERLITZ, and G,M, LEES, Naunyn-Schmiede ber¢s Arch , Exp , Pathol .pharmakol . ~8 231-246 (1964), ei 1 Pharmacol. 9, F,K. OKWUASABA, and J.T, HAMILTON, an .,j, 972-977 (1975) . rma10 . J. FONTAINE, J. J . REUSE, and J, M. VANNUETEN, Arc nt, codyn , her, ~ 376-380 (1973), 11 . H. W . KOSTERLITZ, and A, J, WATT, ,~,~ . Pharmacol. ~ 266-276 (1968) . 12, A, L . COWIE, H, W, . Z{OSTERLITZ, and A . J . WATT, at re ~,~,Q 10401042 (1968) . 13 . S, EHRENPRELS, I . LIGHT, and G,H. SCHONBUCH, In Drua Addiction: Exyerimental Pharmacolo¢y . J . M. Singh, L . Miller and H. Lal (Eds), pp 319-342, Futura, Mt Kieco, New York (1972) . 14 . J. HUGHES, rain es 88 295-308 (1975) .
Vol . 18, No . 8
Naloxone oa the Guinea Pig Ileum
809
15 . J . HUGHES, T . SMITH, B . MORGAN, aad L . FOTHERGII~L, i e í~çi~ 1~ 1753-1758 (1975) . 16 . J . M. VAN NUETEN, H . LEIVERS, J. FONTAINE, aad P .A, J, JANSSEN, Arc ~t . Pharmacodva . Ther , ~} 411-414 (1973) . 17 . H . W . KOSTERLITZ, aad G . M . LEES, Pharmacol . ev 1~ 301 -339 (1964) . 18, J. M . VAN NUETEN, P.A, J . JANSSEN, aad J . FONTAINE, Arch , nt Pharmacodya, her (abstr), in press (1976) .
