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Evidence for the involvement of corticotropin-releasing factor in the gastrointestinal disturbances induced by acoustic and cold stress in mice
Brain Research, 441 (1988) 1-4 Elsevier BRE 13259
Research Reports
Evidence for the involvement of corticotropin-releasing factor in the gastrointestinal disturbances induced by acoustic and cold stress in mice L. Bu6no and M. Gu6 Department of Pharmacology and Toxicology INRA, Toulouse (France) (Acceoted 28 July 1987)
Key words: Corticotropin-releasing factor; Acoustic stress; Cold stress; Stomach; Gastric emptying; Motility; Mouse
The influence of acoustic (AS) and cold (CS) stress on gastric emptying and intestinal transit were evaluated in mice using a radiolabelled 51chromium test meal. AS was produced by playing music through loudspeakers (<86 dB) in a confined box at room temperature (20 °C) and CS was obtained by exposure to 10 °C. Twenty minutes exposure to AS or CS caused a significant (P < 0.05) increase in gastric emptying in mice. Intracerebroventricular (i.c.v.) administration of 150 ng of rat corticotropin-releasing factor (rCRF), 30 min before the test meal, also increased gastric emptying but neither intraperitoneai (i.p.) administration of rCRF at the same dosage nor corticosterone (300 ng) and ACTH (375 FtU) wcr,z able to induce significant changes in gastric emptying. The increase in gastric emptying induced by AS and CS and by i.c.v, injection of rCRF were blocked by previous i.p. administration of an antiserum against rCRF. These findings strongly support the hypothesis that alterations in gastric emptying induced by AS and CS in mice are due to the release of CRF acting directly on central structures involved in the control of gastrointestinal motility.
INTRODUCTION Changes in gastrointestinal motility associated with stressful stimuli are well established in man ~'~' 3(I.31 dogs,5 and in rats ~'13'~7'24'32. However, the nature of the mediation and the mechanisms involved remain unclear; for example, naloxone or a combination of a- and fl-adrenergic blockers suppress the inhibition of gastric motility induced by cold pain in man but have no effect on the migrating duodenal burst of activity induced by labyrinthine stimulation 25.26. F u r t h e r m o r e they are unable to block the acoustic stress (AS)-induced inhibition of gastric motility in fasted dogs I'~. Evidence for humoral pathways is supported by the release of fl-endorphin and catechoiamines into the peripheral circulation during stress 1"~'~6"~s. However, it has also been shown that various types of
stress cause a release of corticotropin-releasing factor (CRF) and that intracerebroventricular (i.c.v.) administration of C R F mimics the motor, behavioral, metabolic and hemodynamic responses to stress in animal.s 5.23'27. Consequently, these experiments were performed (i) to evaluate the effects of AS and CS on gastric emptying ( G E ) in mice and (ii) to establish the role of the central release of C R F in the genesis of the gastrointestinal m o t o r alterations responsible for changes in G E by using treat',nent with antiserum against rat (r) CRF. MATERIALS AND METHODS
Gastric emptying and transit evaluation G E and intestina'i transit of a test meal were evaluated in male N M R I mice (20-30 g) that had been fasted for 12 h using the technique described by Por-
Correspondence: L. Bu6no, Department of Pharmacology INRA, 180 chemin de Tournefeuille, F-3130(IToulouse, France. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
reca and Burks 22. The test meal consisted of 0.5 ml of reconstituted milk (1 g of milk powder in 3 ml of water containing 1 ~tCi/ml of 5tCr sodium chromate)• Thirty minutes after the test meal the animals were sacrificed by cervical dislocation and the stomach and small bowel removed. These organs were placed on a ruled template and the intestine was cut into 10 segments of equal length• Then, the stomach, the intestinal segments and the proximal colon were placed into individual test tubes and counted in a gamma radiation counter for 2 min. GE was calculated from the counter values as the percent of total counts found in the small intestine and the colon. Intestinal transit was evaluated by the described geometric center (GC) technique 22 according to the following equation: GC = Y- [(fraction of counts in each segment) × (segment number)]
Experimentalprocedure In a first series of experiments, 10 min after the test meal (0.5 mi of milk), the animals were placed in a closed box equipped with loudspeakers and were subjected either to AS produced by continuously playing taped music (~<90 dB) with the box at room temperature (20 _+ 2 °C) or to CS by lowering of the box temperature to 10 + 1 °C. A similar procedure, but without playing music and at room temperature, was used for the controls. Each group of animals (6 subjects) was sacrificed 30 min after ~he test meal. In a second series of experiments, 30 min before the test meal and under light ether anesthesia, one group received i.c.v, rCRF (5 ~g/kg) diluted in distilled water 21. Another control group was injected i.c.v, with water alone. The 4 other groups were injected i.p. 10-15 min before the test meal with rCRF (5 ~g/kg), ACTH (12.5/~U/kg), corticosterone (10 Flg/kg) or their vehicle (0.1% alcohol solution). All the animals were sacrificed 30 min after the test meal. Values of GE and GC were compared by analysis of variance followed by paired t-test when ANOVA was significant. In a third series of experiments, 30 min before the meal, the animals were injected i.p. with 5 ~1 of diluted (1/10) rabbit antiserum against (human/rat) CRF provided from CRB (Cambridge, U.K.). Then
the animals were submitted as in the first series to AS, CS or to the i.c.v, administration of rCRF as in the second series. RESULTS In control (no stress) mice at room temperature (20 _+ 2 °C), the volume of the meal emptied after 30 min represented 42.5 +_ 6.5% of the total meal (Fig. 1) and the value of the intestinal GC was 6.03 + 0.74. When AS was applied from 10 to 30 min after the meal the volume emptied was 62.8 _ 15.5%, i.e. a 47.7% increase in GE compared to controls. Similarly exposure to CS also increased the rate of GE by 65.6% (Fig. 1) and there was a significant (P < 0.01) aborai displacement of the intestinal GC. Intracerebroventricular administration of rCRF (5 /~g/kg) increased the GE 30 min after the test meal by 52.1% compared to the controls (Fig. 2) without affecting the intestinal GC (Fig. 2). Such an effect was not reproduced by i.p. administration of rCRF at the same dosage (5 ~tg/kg). Furthermore, neither A C T H (12.5/~U/kg), nor corticosterone (10/~g/kg) injected i.p. affected the colume of meal emptied after 30 min (Table I). Anti-CRF serum (5 l~! of 1/10 dilution) injected i.p. 30 rain before the test meal, did not significantly change the GE (44.9 + 8.7 vs 42.5 + 6.5%) and the
I ANTISERUM I
I~ONTROLI
L ANTI-CRF I
80. ~60. n :s ,
¢ -~r ",<
uJ 40, , Ira
20.
./ /
O.
eol 4oi 2oi
oJ
fill
['-'INo stress ~"~lAcousttc s t r e s s ~ Cold stress * P~O05
Fig. 1. Influence of pretreatment with antiserum against rCRF on the effects of AS and CS on GE in mice (mean + S.D,, n = 6), paired t-test. GE was measured 30 min after the test meal consisting of 0.5 ml of reconstituted milk. Note that the increase in GE induced by AS and CS is blocked by pretreatment with antiserum administered i.p.
ANTISERUM
[CONTROLI
A
# 8o.
ANTI-CRF i
80.
T
Q
I
~F.-6 0 .
q
60.
l
w 40
1
40
0 o.
~" o~
20.
2O . O. I---]Vehicle ICV
0 ~
I,l
rCRF(5~Jg.kg -~ ICY]
Fig. 2. Effects of i.c.v, administration of rCRF and its blockade by treatment with antiserum anti-CRF (mean ± S.D., n = 6).
values of the GC measured at t = 30 min but it abolished the effects of both AS and CS. Antiserum treatment also completely abolished the increase in GE induced by i.c.v, administration of rCRF (5 ~g/kg) (Fig. 2). DISCUSSION
'~everal findings have led us to speculate that the central release of CRF is involved in the gastrointestinal motor alterations associated with stressful stimuli: in dogs, i.c.v, administration of CRF 2t and AS both 15 suppressed the cyclic gastric migrating myoelectric complex; these motor alterations induced by AS are blocked by diazepam I't which is thought to have a direct central inhibitory effect on stress-induced CRF release in dogs 2°. The related drug,
TABLE I
Comparative influence of A CTH corticosterone and rCRF administered i.p. on gastric emptying and intestinal transit of a milk test meal in mice Values are means _+ S.D., n = 6: no significant (P 1> 0.05) difference compared to control paired t-test,
Gastric emptying Geometric center (% test meal) (arbitrao' units) Vehicle (0.1 ml/i.p.) A C T H ( 12.5 mU/kg) Corticosterone (10pg/kg) rCRF (5 Itg/kg)
45.3 40.6 47.1 39.2
+ + + +
4.2 7.1 6.8 6.3
5.66 5.29 4.32 5.66
+ + + ±
0.61 I).84 0.45 1.2
chlordiazepoxide, affects various stress responses in which CRF has been implicated and suppresses acoustic startle, a reflex which is potentiated by exogenous CRF 2's, and attenuated the CRF-induced response suppression in the conflict test 3. The present experiments show that the stress-induced alterations of G E in mice are abolished by immunoneutralization of circulating CRF and consequently demowtrate that CRF is involved in the gastrointestinal motor alterations induced by both AS and CS. The efficiency of the immunoneutralization technique used is confirmed by blockade of the effects of exogenous rCRF on GE. Furthermore, CRF seems to act directly on the central structures controlling the gastric and(or) intestinal motility and not through a stimulation of the hypothalamo-pituitary-adrenal system as systemic administration of ACTH or corticosterone do not increase GE. Absence of alterations in GE following systemic administration of rCRF reinforces the hypothesis of a CNS origin. This result is also in agreement with previous data showing that in the dog, the gastrointestinal motor effects induced by picomolar doses of CRF administered centrally are not reproduced by i.v. or i.c.v. A C T H or cortisol'~'"L Several other findings have also suggested that the behavioral effects of intracerebral C R F are independent of its effects on the pituitary• The inhibition of gastric acid secretion by CRF also persisted after hypophysectomy or adrenalectomy2'2'L Several peptides administered centrally are able to affect gastrointestinal motility in both dogs and rats: these effects depend on the digestive status and are mediated through different neural or hormonal pathways 7"s'l'. Central administration of CRF modulates the parasympathetic outflow from the brain regulating gastric functions~': similarly stressful stimuli are known to produce vagal excitation and both the i.c.v. CRF- and AS-induced inhibition of gastric motility in dogs 'L"~ and the central CRF-induced inhibition of gastric secretion in rats 2"2~are blocked by bilateral vagotomy. All this information supports a major role of CRF to initiate centrally the gastric motor disturbances observed in a stressful situation• Finally, this report is the first to demonstrate that a hypothalamic factor (CRF) released in response to a stressful stimulus is directly responsible for alterations in gastrointestinal motility.
ACKNOWLEDGEMENTS The authors are indebted to Dr. M.J. Fargeas and
REFERENCES 1 Bortz, W.M., Angwin, P., Mefford, I.N., Boarder, M.R., Noyce, N. and Barchas, J.D., Catecholamines, dopamine and endprphin levels during extreme exercise, N. Engl. J. Med., I J05 (1982) 466-467. 2 Britton, D.R., Varela, M., Garcia, A. and Rivier, J., Behavioral effects of intracerebral CRF are independent of effects at the pituitary, Soc. Neurosci. Abstr., 10 (1984) 178. 3 Britton, K.T., Morgan, J., Rivier, J., Vale, W. and Koob, G.F., Chlordiazepoxide attenuates CRF-induced response suppression in the conflict test, Psychopharmacology, 86 (1985) 170-174. 4 Brodie, D.A., ! Tlceration of the stomach produced by restraint in rats, Gastroenterology, 43 (1962) 107-109. 5 Brown, M.C., Fisher, L.A., Spiess, J., Rivier, C. and Vale, W., Corticotropin-releasing factor: effects on the sympathetic nervous system and oxygen consumption, Life Sci., 30 (1982) 207-210. 6 Brown, M.R., Fischer, L.A., Spiess, J., Rivier, C., Rivier, J. and Wale, W., Corticotropin-releasing factor: actions on the sympathetic nervous system and metabolism, Endocrinology, 111 (1982) 928-932. 7 Bu6no, L. and Ferr6, J.P., Central regulation of intestinal motility by somatostatin and cholecystokinin octapeptide, Science, 216 (1982) 1427-1429. 8 Bu~no, L., Fioramonti, J., Hond~, C., Fargeas, M.J. and Primi, M.P., Central and peripheral control of gastrointestinal and colonic motility by endogenous opiates in conscious dogs, Gastroenterology, 88 (1985)549-556. 9 Bu6no, L. and Fioramonti, J., Effects of corticotropin-releasing factor, corticotropin and cortisol on gastrointestinal motility in dogs, Peptides, 7 (1986) 73-77. 10 Bu6no, L,, Fargeas, M.J., Gu6, M., Peeters, T.L., Bormans, V. and Fioramonti, J., Effects of corticotropin-releasing factor on plasma motilin and somatostatin levels and gastrointestinal motility in dogs, Gastroenterology, 91 (1986) 884-889. 11 Cohen, M., Pickar, D., Dubois, M., Roth, Y.F., Naber, D. and Bunney, W.E., Surgical stress and endorphins, Lancet, i (1981) 213-214. 12 Fargeas, M.J., Fioramonti, J. and Bu6no, L., Prostaglandin E2: a neuromodulator in the central of gastrointestinal motility and feeding behavior by calcitonin, Science, 225 (1984) 1050-1052. 13 Fioramonti, J. and Bu6no, L., Gastrointestinal myoelectric activity disturbances in gastric ulcer disease in rats and dogs, Dig. Dis. Sci., 25 (1980) 900-975. 14 Gu6, M. and Bu6no, L., Diazepam and muscimol blockade of the gastrointestinal motor disturbances induced by acoustic stress in dogs, Eur. J. Pharmacol., 131 (1986) 123-127. 15 Gu6, M., Fioramonti, J., Frexinos, J., Alvinerie, M. and Bu6no, L., Influence of acoustic stress by noise on gastrointestinal motility in dogs, Dig. Dis. Sci., in press. 16 Kalin, N.H., Behavioral effects of ovine corticotropin-releasing factor administered to rhesus monkey, Fed. Proc.,
C. Dargelos for their skillful technical assistance. This work was supported in part by a grant from the Institut National de la R e c h e r c h e A g r o n o m i q u e .
44 (1985) 249-253. 17 Koo, M., Ogle, C. and Cho, C., The effect of cold-restraint stress on gastric emptying in rats, Pharmacol. Biochem. Behay., 23 (1985) 969-972. 18 Kopin, K.J., Lake, R.C. and Ziegler, M., Plasma levels of norepinephrine, Ann. Int. Med., 88 (1978)671-680. 19 McRae, S., Younger, K., Thompson, D.G. and Wingate, D.L., Sustained mental stress alters human jejunal motor activity, Gut, 23 (1982) 404-409. 20 Ninan, D.J., Insel, T.M., Cohen, R.M., Cook, J.M., Skolnick, P. and Paul, S.M., Benzodiazepine receptor-mediated experimental 'anxiety' in primates, Science, 218 (1982) 1332-1334. 21 Pedigo, N.W., Dewey, W.L. and Harris, L.S., Determination and characterization of the antinociceptive activity of intraventri~ularly administered acetylcholine in mice, J. Pharmacol. Exp. Ther., 193 (1975) 845-852. 22 Porreca, F. and Burks, T.F., The spinal cord as a site of opioid effects on gastrointestinal transit in the mouse, J. Pharmacol. Exp. Ther., 227 (1983) 22-27. 23 Rivier, C., Rivier, J. and Vale, W., Inhibition of adrenocorticotropic hormone secretion in the rat by immunoneutralisation of corticotropin releasing factor, Science, 218 (1982) 377- 379. 24 Sato, Y. and Terui, N., Changes in duodenal motility produced by noxious mechanical stimulation of the skin in rats, Neurosci. Lett., 2 (1976) 189-193. 25 Stanghellini, V., Malagelada, J.R., Zinsmeister, A.R., Go, V.L. and Kao, P.C., Stress-induced gastroduodenal motor disturbances in humans: possible humoral mechanisms, Gastroenterology, 85 (1983)83-91. 26 Stanghellini, V., Malagelada, J.R., Zinsmeister, A.R., Go, V.L. and Kao, P.C., Effects of opiate and adrenergic blockers on the gut motor response to centrally acting stimuli, Gastroenterology, 87 (1984) 1104-1113. 27 Sutton, R.E., Koob, G.G., Lemoal, M., Rivier, J. and Vale, W., Corticotropin releasing factor produces behavioral activation in rats, Nature (London), 297 (1982) 331-333. 28 Swerdlow, N., Geyer, M., Vale, W. and Koob, G.F., Corticotropin-releasing factor (CRF) potentiates acoustic startle reflex (ASR) in rats: blockade by chlordiazepoxide, Soc. Neurosci. Abstr., 11 (1985) 620. 29 Tache, Y., Goto, Y., Gunion, M., Rivier, J. and Debas, H., Inhibition of gastric acid secretion in rat~ and dogs by corticotropin releasing factor, Gastroentero'ogy, 86 (1984) 281-286. 30 Thompson, D.G., Richeison, J.R. and Malagelada, J.R., Perturbation of upper gastrointestinal function by cold stress, Gut, 24 (1983) 277-283. 31 Valori, R.M., Kumar, D. and Wingate, D., Effects of different types of stress and of 'prokinetic' drugs on the control of the fasting motor complex in humans, Gastroenterology, 90 (1986) 1890-1900. 32 Watanabe, K., Some pharmacological factors involved in formation and prevention of stress ulceration in rats, Chem. Pharmacol. Bull., 14 (1966) 101-107.
Research Reports
Evidence for the involvement of corticotropin-releasing factor in the gastrointestinal disturbances induced by acoustic and cold stress in mice L. Bu6no and M. Gu6 Department of Pharmacology and Toxicology INRA, Toulouse (France) (Acceoted 28 July 1987)
Key words: Corticotropin-releasing factor; Acoustic stress; Cold stress; Stomach; Gastric emptying; Motility; Mouse
The influence of acoustic (AS) and cold (CS) stress on gastric emptying and intestinal transit were evaluated in mice using a radiolabelled 51chromium test meal. AS was produced by playing music through loudspeakers (<86 dB) in a confined box at room temperature (20 °C) and CS was obtained by exposure to 10 °C. Twenty minutes exposure to AS or CS caused a significant (P < 0.05) increase in gastric emptying in mice. Intracerebroventricular (i.c.v.) administration of 150 ng of rat corticotropin-releasing factor (rCRF), 30 min before the test meal, also increased gastric emptying but neither intraperitoneai (i.p.) administration of rCRF at the same dosage nor corticosterone (300 ng) and ACTH (375 FtU) wcr,z able to induce significant changes in gastric emptying. The increase in gastric emptying induced by AS and CS and by i.c.v, injection of rCRF were blocked by previous i.p. administration of an antiserum against rCRF. These findings strongly support the hypothesis that alterations in gastric emptying induced by AS and CS in mice are due to the release of CRF acting directly on central structures involved in the control of gastrointestinal motility.
INTRODUCTION Changes in gastrointestinal motility associated with stressful stimuli are well established in man ~'~' 3(I.31 dogs,5 and in rats ~'13'~7'24'32. However, the nature of the mediation and the mechanisms involved remain unclear; for example, naloxone or a combination of a- and fl-adrenergic blockers suppress the inhibition of gastric motility induced by cold pain in man but have no effect on the migrating duodenal burst of activity induced by labyrinthine stimulation 25.26. F u r t h e r m o r e they are unable to block the acoustic stress (AS)-induced inhibition of gastric motility in fasted dogs I'~. Evidence for humoral pathways is supported by the release of fl-endorphin and catechoiamines into the peripheral circulation during stress 1"~'~6"~s. However, it has also been shown that various types of
stress cause a release of corticotropin-releasing factor (CRF) and that intracerebroventricular (i.c.v.) administration of C R F mimics the motor, behavioral, metabolic and hemodynamic responses to stress in animal.s 5.23'27. Consequently, these experiments were performed (i) to evaluate the effects of AS and CS on gastric emptying ( G E ) in mice and (ii) to establish the role of the central release of C R F in the genesis of the gastrointestinal m o t o r alterations responsible for changes in G E by using treat',nent with antiserum against rat (r) CRF. MATERIALS AND METHODS
Gastric emptying and transit evaluation G E and intestina'i transit of a test meal were evaluated in male N M R I mice (20-30 g) that had been fasted for 12 h using the technique described by Por-
Correspondence: L. Bu6no, Department of Pharmacology INRA, 180 chemin de Tournefeuille, F-3130(IToulouse, France. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
reca and Burks 22. The test meal consisted of 0.5 ml of reconstituted milk (1 g of milk powder in 3 ml of water containing 1 ~tCi/ml of 5tCr sodium chromate)• Thirty minutes after the test meal the animals were sacrificed by cervical dislocation and the stomach and small bowel removed. These organs were placed on a ruled template and the intestine was cut into 10 segments of equal length• Then, the stomach, the intestinal segments and the proximal colon were placed into individual test tubes and counted in a gamma radiation counter for 2 min. GE was calculated from the counter values as the percent of total counts found in the small intestine and the colon. Intestinal transit was evaluated by the described geometric center (GC) technique 22 according to the following equation: GC = Y- [(fraction of counts in each segment) × (segment number)]
Experimentalprocedure In a first series of experiments, 10 min after the test meal (0.5 mi of milk), the animals were placed in a closed box equipped with loudspeakers and were subjected either to AS produced by continuously playing taped music (~<90 dB) with the box at room temperature (20 _+ 2 °C) or to CS by lowering of the box temperature to 10 + 1 °C. A similar procedure, but without playing music and at room temperature, was used for the controls. Each group of animals (6 subjects) was sacrificed 30 min after ~he test meal. In a second series of experiments, 30 min before the test meal and under light ether anesthesia, one group received i.c.v, rCRF (5 ~g/kg) diluted in distilled water 21. Another control group was injected i.c.v, with water alone. The 4 other groups were injected i.p. 10-15 min before the test meal with rCRF (5 ~g/kg), ACTH (12.5/~U/kg), corticosterone (10 Flg/kg) or their vehicle (0.1% alcohol solution). All the animals were sacrificed 30 min after the test meal. Values of GE and GC were compared by analysis of variance followed by paired t-test when ANOVA was significant. In a third series of experiments, 30 min before the meal, the animals were injected i.p. with 5 ~1 of diluted (1/10) rabbit antiserum against (human/rat) CRF provided from CRB (Cambridge, U.K.). Then
the animals were submitted as in the first series to AS, CS or to the i.c.v, administration of rCRF as in the second series. RESULTS In control (no stress) mice at room temperature (20 _+ 2 °C), the volume of the meal emptied after 30 min represented 42.5 +_ 6.5% of the total meal (Fig. 1) and the value of the intestinal GC was 6.03 + 0.74. When AS was applied from 10 to 30 min after the meal the volume emptied was 62.8 _ 15.5%, i.e. a 47.7% increase in GE compared to controls. Similarly exposure to CS also increased the rate of GE by 65.6% (Fig. 1) and there was a significant (P < 0.01) aborai displacement of the intestinal GC. Intracerebroventricular administration of rCRF (5 /~g/kg) increased the GE 30 min after the test meal by 52.1% compared to the controls (Fig. 2) without affecting the intestinal GC (Fig. 2). Such an effect was not reproduced by i.p. administration of rCRF at the same dosage (5 ~tg/kg). Furthermore, neither A C T H (12.5/~U/kg), nor corticosterone (10/~g/kg) injected i.p. affected the colume of meal emptied after 30 min (Table I). Anti-CRF serum (5 l~! of 1/10 dilution) injected i.p. 30 rain before the test meal, did not significantly change the GE (44.9 + 8.7 vs 42.5 + 6.5%) and the
I ANTISERUM I
I~ONTROLI
L ANTI-CRF I
80. ~60. n :s ,
¢ -~r ",<
uJ 40, , Ira
20.
./ /
O.
eol 4oi 2oi
oJ
fill
['-'INo stress ~"~lAcousttc s t r e s s ~ Cold stress * P~O05
Fig. 1. Influence of pretreatment with antiserum against rCRF on the effects of AS and CS on GE in mice (mean + S.D,, n = 6), paired t-test. GE was measured 30 min after the test meal consisting of 0.5 ml of reconstituted milk. Note that the increase in GE induced by AS and CS is blocked by pretreatment with antiserum administered i.p.
ANTISERUM
[CONTROLI
A
# 8o.
ANTI-CRF i
80.
T
Q
I
~F.-6 0 .
q
60.
l
w 40
1
40
0 o.
~" o~
20.
2O . O. I---]Vehicle ICV
0 ~
I,l
rCRF(5~Jg.kg -~ ICY]
Fig. 2. Effects of i.c.v, administration of rCRF and its blockade by treatment with antiserum anti-CRF (mean ± S.D., n = 6).
values of the GC measured at t = 30 min but it abolished the effects of both AS and CS. Antiserum treatment also completely abolished the increase in GE induced by i.c.v, administration of rCRF (5 ~g/kg) (Fig. 2). DISCUSSION
'~everal findings have led us to speculate that the central release of CRF is involved in the gastrointestinal motor alterations associated with stressful stimuli: in dogs, i.c.v, administration of CRF 2t and AS both 15 suppressed the cyclic gastric migrating myoelectric complex; these motor alterations induced by AS are blocked by diazepam I't which is thought to have a direct central inhibitory effect on stress-induced CRF release in dogs 2°. The related drug,
TABLE I
Comparative influence of A CTH corticosterone and rCRF administered i.p. on gastric emptying and intestinal transit of a milk test meal in mice Values are means _+ S.D., n = 6: no significant (P 1> 0.05) difference compared to control paired t-test,
Gastric emptying Geometric center (% test meal) (arbitrao' units) Vehicle (0.1 ml/i.p.) A C T H ( 12.5 mU/kg) Corticosterone (10pg/kg) rCRF (5 Itg/kg)
45.3 40.6 47.1 39.2
+ + + +
4.2 7.1 6.8 6.3
5.66 5.29 4.32 5.66
+ + + ±
0.61 I).84 0.45 1.2
chlordiazepoxide, affects various stress responses in which CRF has been implicated and suppresses acoustic startle, a reflex which is potentiated by exogenous CRF 2's, and attenuated the CRF-induced response suppression in the conflict test 3. The present experiments show that the stress-induced alterations of G E in mice are abolished by immunoneutralization of circulating CRF and consequently demowtrate that CRF is involved in the gastrointestinal motor alterations induced by both AS and CS. The efficiency of the immunoneutralization technique used is confirmed by blockade of the effects of exogenous rCRF on GE. Furthermore, CRF seems to act directly on the central structures controlling the gastric and(or) intestinal motility and not through a stimulation of the hypothalamo-pituitary-adrenal system as systemic administration of ACTH or corticosterone do not increase GE. Absence of alterations in GE following systemic administration of rCRF reinforces the hypothesis of a CNS origin. This result is also in agreement with previous data showing that in the dog, the gastrointestinal motor effects induced by picomolar doses of CRF administered centrally are not reproduced by i.v. or i.c.v. A C T H or cortisol'~'"L Several other findings have also suggested that the behavioral effects of intracerebral C R F are independent of its effects on the pituitary• The inhibition of gastric acid secretion by CRF also persisted after hypophysectomy or adrenalectomy2'2'L Several peptides administered centrally are able to affect gastrointestinal motility in both dogs and rats: these effects depend on the digestive status and are mediated through different neural or hormonal pathways 7"s'l'. Central administration of CRF modulates the parasympathetic outflow from the brain regulating gastric functions~': similarly stressful stimuli are known to produce vagal excitation and both the i.c.v. CRF- and AS-induced inhibition of gastric motility in dogs 'L"~ and the central CRF-induced inhibition of gastric secretion in rats 2"2~are blocked by bilateral vagotomy. All this information supports a major role of CRF to initiate centrally the gastric motor disturbances observed in a stressful situation• Finally, this report is the first to demonstrate that a hypothalamic factor (CRF) released in response to a stressful stimulus is directly responsible for alterations in gastrointestinal motility.
ACKNOWLEDGEMENTS The authors are indebted to Dr. M.J. Fargeas and
REFERENCES 1 Bortz, W.M., Angwin, P., Mefford, I.N., Boarder, M.R., Noyce, N. and Barchas, J.D., Catecholamines, dopamine and endprphin levels during extreme exercise, N. Engl. J. Med., I J05 (1982) 466-467. 2 Britton, D.R., Varela, M., Garcia, A. and Rivier, J., Behavioral effects of intracerebral CRF are independent of effects at the pituitary, Soc. Neurosci. Abstr., 10 (1984) 178. 3 Britton, K.T., Morgan, J., Rivier, J., Vale, W. and Koob, G.F., Chlordiazepoxide attenuates CRF-induced response suppression in the conflict test, Psychopharmacology, 86 (1985) 170-174. 4 Brodie, D.A., ! Tlceration of the stomach produced by restraint in rats, Gastroenterology, 43 (1962) 107-109. 5 Brown, M.C., Fisher, L.A., Spiess, J., Rivier, C. and Vale, W., Corticotropin-releasing factor: effects on the sympathetic nervous system and oxygen consumption, Life Sci., 30 (1982) 207-210. 6 Brown, M.R., Fischer, L.A., Spiess, J., Rivier, C., Rivier, J. and Wale, W., Corticotropin-releasing factor: actions on the sympathetic nervous system and metabolism, Endocrinology, 111 (1982) 928-932. 7 Bu6no, L. and Ferr6, J.P., Central regulation of intestinal motility by somatostatin and cholecystokinin octapeptide, Science, 216 (1982) 1427-1429. 8 Bu~no, L., Fioramonti, J., Hond~, C., Fargeas, M.J. and Primi, M.P., Central and peripheral control of gastrointestinal and colonic motility by endogenous opiates in conscious dogs, Gastroenterology, 88 (1985)549-556. 9 Bu6no, L. and Fioramonti, J., Effects of corticotropin-releasing factor, corticotropin and cortisol on gastrointestinal motility in dogs, Peptides, 7 (1986) 73-77. 10 Bu6no, L,, Fargeas, M.J., Gu6, M., Peeters, T.L., Bormans, V. and Fioramonti, J., Effects of corticotropin-releasing factor on plasma motilin and somatostatin levels and gastrointestinal motility in dogs, Gastroenterology, 91 (1986) 884-889. 11 Cohen, M., Pickar, D., Dubois, M., Roth, Y.F., Naber, D. and Bunney, W.E., Surgical stress and endorphins, Lancet, i (1981) 213-214. 12 Fargeas, M.J., Fioramonti, J. and Bu6no, L., Prostaglandin E2: a neuromodulator in the central of gastrointestinal motility and feeding behavior by calcitonin, Science, 225 (1984) 1050-1052. 13 Fioramonti, J. and Bu6no, L., Gastrointestinal myoelectric activity disturbances in gastric ulcer disease in rats and dogs, Dig. Dis. Sci., 25 (1980) 900-975. 14 Gu6, M. and Bu6no, L., Diazepam and muscimol blockade of the gastrointestinal motor disturbances induced by acoustic stress in dogs, Eur. J. Pharmacol., 131 (1986) 123-127. 15 Gu6, M., Fioramonti, J., Frexinos, J., Alvinerie, M. and Bu6no, L., Influence of acoustic stress by noise on gastrointestinal motility in dogs, Dig. Dis. Sci., in press. 16 Kalin, N.H., Behavioral effects of ovine corticotropin-releasing factor administered to rhesus monkey, Fed. Proc.,
C. Dargelos for their skillful technical assistance. This work was supported in part by a grant from the Institut National de la R e c h e r c h e A g r o n o m i q u e .
44 (1985) 249-253. 17 Koo, M., Ogle, C. and Cho, C., The effect of cold-restraint stress on gastric emptying in rats, Pharmacol. Biochem. Behay., 23 (1985) 969-972. 18 Kopin, K.J., Lake, R.C. and Ziegler, M., Plasma levels of norepinephrine, Ann. Int. Med., 88 (1978)671-680. 19 McRae, S., Younger, K., Thompson, D.G. and Wingate, D.L., Sustained mental stress alters human jejunal motor activity, Gut, 23 (1982) 404-409. 20 Ninan, D.J., Insel, T.M., Cohen, R.M., Cook, J.M., Skolnick, P. and Paul, S.M., Benzodiazepine receptor-mediated experimental 'anxiety' in primates, Science, 218 (1982) 1332-1334. 21 Pedigo, N.W., Dewey, W.L. and Harris, L.S., Determination and characterization of the antinociceptive activity of intraventri~ularly administered acetylcholine in mice, J. Pharmacol. Exp. Ther., 193 (1975) 845-852. 22 Porreca, F. and Burks, T.F., The spinal cord as a site of opioid effects on gastrointestinal transit in the mouse, J. Pharmacol. Exp. Ther., 227 (1983) 22-27. 23 Rivier, C., Rivier, J. and Vale, W., Inhibition of adrenocorticotropic hormone secretion in the rat by immunoneutralisation of corticotropin releasing factor, Science, 218 (1982) 377- 379. 24 Sato, Y. and Terui, N., Changes in duodenal motility produced by noxious mechanical stimulation of the skin in rats, Neurosci. Lett., 2 (1976) 189-193. 25 Stanghellini, V., Malagelada, J.R., Zinsmeister, A.R., Go, V.L. and Kao, P.C., Stress-induced gastroduodenal motor disturbances in humans: possible humoral mechanisms, Gastroenterology, 85 (1983)83-91. 26 Stanghellini, V., Malagelada, J.R., Zinsmeister, A.R., Go, V.L. and Kao, P.C., Effects of opiate and adrenergic blockers on the gut motor response to centrally acting stimuli, Gastroenterology, 87 (1984) 1104-1113. 27 Sutton, R.E., Koob, G.G., Lemoal, M., Rivier, J. and Vale, W., Corticotropin releasing factor produces behavioral activation in rats, Nature (London), 297 (1982) 331-333. 28 Swerdlow, N., Geyer, M., Vale, W. and Koob, G.F., Corticotropin-releasing factor (CRF) potentiates acoustic startle reflex (ASR) in rats: blockade by chlordiazepoxide, Soc. Neurosci. Abstr., 11 (1985) 620. 29 Tache, Y., Goto, Y., Gunion, M., Rivier, J. and Debas, H., Inhibition of gastric acid secretion in rat~ and dogs by corticotropin releasing factor, Gastroentero'ogy, 86 (1984) 281-286. 30 Thompson, D.G., Richeison, J.R. and Malagelada, J.R., Perturbation of upper gastrointestinal function by cold stress, Gut, 24 (1983) 277-283. 31 Valori, R.M., Kumar, D. and Wingate, D., Effects of different types of stress and of 'prokinetic' drugs on the control of the fasting motor complex in humans, Gastroenterology, 90 (1986) 1890-1900. 32 Watanabe, K., Some pharmacological factors involved in formation and prevention of stress ulceration in rats, Chem. Pharmacol. Bull., 14 (1966) 101-107.