- Email: [email protected]
Role of central prostaglandin E2 in the regulation of gastric acid secretion in the rat
European Journal of Pharmacology, 209 ( 1991) 1-7
1
© 1991 Elsevier Science Publishers B.V. All rights reserved 0014-2999/91/$03.50
EJP 52171
Role of central prostaglandin E 2 in the regulation of gastric acid secretion in the rat Esteban Saperas, Gordon Kauffman ~ and Yvette Tach6 Center for Ulcer Research and Education, VA Wadsworth Medical Center, Department of Medicine and Brain Research Institute, UCLA, Los Angeles, CA, U.S.A. and l Division of General Surgery, The Milton Hershey Medical Center, The Pennsyh,ania State Unit,ersity, Hershey, PA, U.S.A.
Received 10 September 1991, accepted 17 September 1991
The central action of prostaglandin E 2 (PGE2) on gastric acid secretion was investigated in rats by comparing the effects of intracisternal (i.ci.) and i.v. administration of PGE 2 and the influence of i.ci. injection of indomethacin on acid secretion and PGE 2 generation in the brain and stomach. I.ci. injections of PGE 2 (1-10 /xg) or the stable analog, 16,16-dimethyl PGE2, (0.01-0.1 /xg) induced a dose dependent inhibition of baclofen-stimulated gastric acid secretion by 0-82% and by 7-87% respectively. I.v. infusion of PGE 2 also induced a dose related inhibition of baclofen-stimulated acid secretion, but 10 fold higher doses were required. I.ci. or i.v. injection of indomethacin in doses ranging from 50 to 500 /xg/rat, produced a similar dose dependent inhibition of the PGE 2 generation in both the gastric mucosa and brain cortex measured 1 h post injection. I.ci. injection of indomethacin (500 p.g) increased within 10 min acid secretion with a peak response at 20-30 rain; 60-120 rain post injection, when prostaglandin synthesis was inhibited by 90%, basal and baclofen-stimulated acid output were not altered. These results further establish that PGE 2 acts in the brain to inhibit vagally stimulated gastric acid secretion in rats, and do not support a tonic inhibitory influence of endogenous brain PGE 2 in the regulation of gastric acid secretion. In addition, these data showed that indomethacin injected i.ci. at 500/xg does not induce a selective inhibition of prostaglandin synthesis in the brain.
Prostaglandin E2; Indomethacin; Gastric acid secretion; Vagus; Baclofen
1. I n t r o d u c t i o n
Prostaglandin E 2 ( P G E 2) receptors are widely distributed in the central nervous system (CNS) (Cseh et al., 1978; M a t s u m u r a et al., 1990). Growing n e u r o p h a r macological evidence suggests that prostaglandins act in the brain to influence homeostasis and a u t o n o m i c functions. P G E 2 injected into the cerebrospinal fluid (CSF) or into specific brain sites elicits behavioral changes (Chiu and Richardson, 1985), analgesia (Horiguchi et al., 1986), fever (Amir and Schiavetto, 1990), wakefulness (Hayaishi, 1991), hyperinsulinemia (Cornell, 1989), A C T H release (Katsuura et al., 1990; Cornell, 1989), sympathetic activation (Gfillner, 1983), and increases blood pressure and heart rate (Otorii et al., 1985). Prostaglandins, along with peptides, also exert C N S actions that influence gastrointestinal secretory and m o t o r functions (Puurunen, 1983a; BuEno et al., 1985; Tach6, 1987; Sautereau and Roz6, 1988; S t a u m o n t et al., 1990). In particular, one g r o u p of investigators has established that P G E 2 acts in the
Correspondence to: Y. TachS, VA Wadsworth Medical Center, Building 115, Room 203, Wilshire and Sawtelle BI, Los Angeles, CA 90073, U.S.A. Tel. 1.213.206 5489, fax 1.213.824 6752.
brain to inhibit vagally stimulated acid secretion in rats (Puurunen, 1983a, b, c). W h e t h e r e n d o g e n o u s P G E 2 in the brain exerts a tonic inhibition on gastric secretion is still to be established. In the present study we further investigated the influence of central prostaglandins in the regulation of gastric acid secretion in the rat. Comparison of the dose-related inhibition of vagally stimulated acid secretion in response to CSF injection or i.v. infusion of P G E 2 or the stable P G E 2 analog, 16,16-dimethyl P G E 2, was performed. In addition, the influence of i.ci. injection of the prostaglandin synthesis inhibitor, indomethacin, on basal and stimulated acid secretion was m o n i t o r e d over time along with its inhibitory effect on e n d o g e n o u s prostaglandin generation in the brain and stomach.
2. M a t e r i a l s a n d m e t h o d s 2.1. A n i m a l s
Male S p r a g u e - D a w l e y rats, weighing 2 0 0 - 2 8 0 g (Simonsen Laboratories, Gilroy, California) were fasted with free access to water 24 h before the experiment.
2.2. Drugs and treatment P G E 2 and 16,16-dimethyl PGE 2 (Upjohn Co., Kalamazoo, Michigan)were dissolved in absolute ethanol (10 mg ml-~) and stored at - 2 0 ° C. The stock solution was diluted in sterile 0.9% saline immediately prior to i.ci. injection (10 /xl) or i.v. infusion (0.5 ml/h). /3-(pChlorophenyl)-¢-aminobutyric acid (baclofen, Lioresal; Geigy Pharmaceutical, Summit, New Jersey) was dissolved in saline and injected s.c. in 1 ml volume. Indomethacin (Sigma Chemical, St. Louis, Missouri) was dissolved in 1% sodium bicarbonate and injected i.ci. (10/xl), i.v. (1 ml kg -~) or i.p. (1 ml).
2.3. Measurement of gastric acid secretion Gastric acid secretion was measured as previously reported (Tach6 et al., 1985). In rats under urethane anesthesia (1.25 g / k g i.p.), the pylorus was ligated and a double-lumen cannula was acutely implanted in the forestomach. The inlet and outlet tubes of the gastric cannula were continuously perfused with a perislatic pump at flow rate of 3.5 m l / m i n with normal saline adjusted to pH 5.5. Gastric acid output was recorded every 2 min by titration of the gastric perfusate to a pH 5.5 with 0.02 N NaOH, using an automatic titrator with a zero suppression adaptor (TOA Electronics, Tokyo, Japan). In one group of experiments, gastric acid secretion was measured every 10 min by flushing the gastric lumen with two 5 ml boli of 0.15 M NaC1 and 5 ml of air at the end of each 10 min period. Acid output was determined by titration of the flushed perfusate with 0.1 M N a O H to pH 7.0 on an automatic titrator (Radiometer, Copenhagen).
2.4. Experimental design 2. 4.1. Effect of PGE 2 administration Basal gastric acid secretion was determined every 2 min for 20 min then baclofen was injected s.c. (4 m g / k g ) and 80 min later, vehicle or various doses of P G E 2 or 16,16-dimethyl P G E 2 were injected i.ci. or infused i.v. over a 30 rain period. Incremental doses of P G E 2 (1, 3 and 10 p~g) or the P G E 2 analog (0.01, 0.03 and 0.1 /xg) were injected i.ci. at 30 min intervals. I.v. infusion of P G E 2 was maintained throughout the experiment and doses (10, 30 and 1 0 0 / x g / r a t per 30 min) were increased every 30 rain. 2. 4. 2. Effects of indomethacin administration Basal acid secretion was measured for 30 rain before and 100 min after i.ci. injection of vehicle or indomethacin (500/xg) using the 10 min flush technique. In another experiment, indomethacin (500 p.g) was injected i.ci. and 1 h later basal acid secretion was measured every 2 min for 30 min, then baclofen was
injected s.c. (4 mg/kg) and acid secretion was measured for the following 90 min period.
2.5. Measurement of PGE 2 generation Under light ether anesthesia the animals were injected i.ci., i.v. through the jugular vein or i.p. with vehicle or indomethacin. Conscious rats were killed 1 h later by CO 2 inhalation and the corpus mucosa was stripped and brain tissue specimens were collected, weighed and processed for ex vivo P G E 2 generation as previously d'escribed (Whittle et al., 1980). The tissue was chopped with scissors for 45 s in microfuge plastic tubes containing 1 ml of phosphate buffer (100 raM, pH 7.4) and centrifuged in a fixed-speed bench centrifuge at 15000 rpm for 30 s. After discarding the supernatant, the tissue was suspended in 1 ml of phosphate buffer and mixed by vortex for 1 min at room temperature. Then, 10/xg (10/xl) of indomethacin was added to each sample to inhibit any further formation of prostaglandins. The samples were centrifuged for 1 min and the supernatants were stored at - 2 0 °C until assayed. P G E 2 was determined by a modification of a radioimmunoassay technique previously described (Bauminger, Zor and Lindner, 1973) using [3H]PGE2 as tracer, specific P G E 2 antiserum, and standard. The assay medium was 0.1 M phosphate buffer saline (0.9%) with gelatin (0.1%), pH 7.4. The radioimmunoassay was performed as previously published (Ligumsky et al., 1983). The generative capacity is expressed as nanograms of PGE 2 per gram wet weight of tissue per min (ng g J min-l).
2.6. Statistical analysis The results are presented as means _+ S.E.M. Statistical probabilities were calculated by Student's t-test or one-way analysis of variance followed by multiple comparisons with Dunnett's t-test.
3. Results
3.1. Effect of i.ci. injection or i.v. infusion of PGE 2 on baclofen-stimulated gastric acid secretion Baclofen (4 m g / k g s.c.) significantly stimulated the low basal acid secretion (0.2_+ 0.1 /xmol/2 rain) in urethane-anesthetized rats. A plateau response was observed within 58 + 3 min and maintained at this level for 90 min in rats injected i.ci. with vehicle (1% ethanol in 0.9% saline) every 30 min starting 80 min post baclofen injection (fig. 1). I.ci. injections of 16,16-dimethyl P G E 2 at 0.01, 0.03 and 0.1 p,g every 30 min, dose dependently inhibited baclofen-stimulated acid secretion by 7, 41 and 87% respectively. Thirty minutes
16,16 d m P G E 2 Dose (ng/rat)
A
Intravenous 30
10
Intraclsternal
I00 20,
'E ,o
i
~ 15,
g0-
Z
~
!..... ~?.. -y
20-
o Ix¢ nIJJ Z UJ (3
-o
10,
5'
LI,Ie4 o
•
20
•
0
20
40
80
60 TIME
100
120
140
160
(3
-
18'0
(min)
0
Fig. 1. Dose-related inhibition of baclofen-stimulated gastric acid secretion by i.ci. injection of 16,16-dimethyl PGE 2 in urethaneanesthetized rats. After 20 min basal period, baclofen was injected s.c. and 80 min later vehicle (©) or the PGE 2 analog (e) was injected i.ci. at various doses every 30 min. Each point represents the mean + S.E.M. of four rats. There is a significant (P < 0.05) dose-related inhibition of acid secretion by 16,16-dimethyl PGE 2 compared with vehicle-treated rats.
after i.ci. injection of 16,16-dimethyl PGE 2 at 0.1 /xg, acid secretion was completely inhibited (fig. 1). Likewise, PGE 2 injected i.ci. every 30 rain at step dose increments (1, 3 and 10 /xg), dose dependently inhibited baclofen-stimulated gastric acid output by 0, 45 and 82% respectively (fig. 2). The gastric inhibitory effect was observed within 3.7_+ 0.5 rain after i.ci. injection of PGE 2 or 16,16-dimethyl PGE 2. I.v. infusion of PGE 2 in doses ranging from 10 to 100 /xg/rat per 30 min every 30 min inhibited dose dependently baclofen-stimulated gastric acid output by 17-86% in urethane-anesthetized rats (fig. 2).
50
100
500
INDOMETHACIN
0 DOSE
50
100
500
(~tg/rat)
Fig. 3. Inhibition of PGE 2 generation in the brain cortex by i.v. or i.ci. injection of indomethacin. Rats fasted for 24 h were injected i.v. or i.ci. under light ether anesthesia with vehicle or indomethacin (50, 100 or 500 /xg/rat). Animals were killed I h later and brain cortex collected for PGE 2 generation. Each column represents the mean + S.E.M. of four rats. * P < 0.01 compared with vehicle-treated group.
3.2. Effect of i.ci. and i.u. injection of indomethacin on PGE2-generative capacity in L,arious tissues
I.v. or i.ci. injection of indomethacin (50-500 /zg) significantly suppressed PGE2-generative capacity in both the brain cortex and gastric mucosa as assessed 1 h after indomethacin injection (figs. 3 and 4). The inhibition of PGE 2 syntheses in the gastric mucosa in response to i.ci. or i.v. injection of indomethacin was dose-related (fig. 4). The percentages of inhibition produced by indomethacin in doses ranging from 50 to 500 /xg/rat were similar in the cortex after the i.ci. or i.v. route of administration (fig. 3). At the 500 /xg dose, indomethacin injected i.ci. inhibited PGE 2 generation 300
120,
Intraclaternal
A
E
10o.
o E
eo-
l--
60.
O. k..~ 0
40-
a <
0
1
i; 3
Intravenous
Intravenous
o
10
PGE 2
0 DOSE
0
30
100
50
a0o
Intraclstemal
500
0
so
100
5oo
INDOMEI'HACINDOSE 0ag/rat)
(gg/rat)
Fig. 2. Dose-related inhibition of baclofen-stimulated gastric acid secretion by i.ci. injection or i.v. infusion of PGE 2 in urethaneanesthetized rats. After 20 min basal period, baclofen (4 m g / k g s.c.) was injected and 80 min later vehicle or PGE 2 was injected i.ci. every 30 min or infused i.v. for 30 min at various doses. Each column represents the mean + S.E.M. of four rats; * P < 0.05 and * * P < 0.01 compared with vehicle-treated group (©).
Fig. 4. Dose-related inhibition of PGE 2 generation in the gastric mucosa induced by i.v. or i.ci. injection of indomethacin. Under light ether anesthesia rats fasted for 24 h received a single i.v. or i.ci. injection of vehicle (1% sodium bicarbonate) or indomethacin (50, 100 or 500 ~ g / r a t ) . Fundic mucosa was stripped and collected for measurement of prostaglandin generation 1 h later. Each column represents the m e a n + S.E.M. of four rats. ~ P < 0.05 and * P < 0.01 compared with vehicle-treated group.
I•
4o,
~
[]
TABLE 1
Control Indomethacin
Effect of indomethacin pretreatment on basal and baclofen-stimulated gastric acid secretion in urethane-anesthetized rats.
3o.
Treatment ;~
F-
2o.
w Z ul
Vehicle Indomethacin
10.
~' Rats fasted for 24 h were anesthetized with urethane and double lumen was implanted in the forestomach, lndomethacin (500 p.g) or vehicle was injected i.ci. and stomach was constantly perfused. One hour after the injection, gastric acid output was measured for 30 min under basal conditions and for 90 min following baclofen injection (4 m g / k g s.c.), b Results are expressed as means_+S.E.M, of four rats in each group; acid output is expressed as 30 min periods before and 60 min after baclofen injection, c p < 0.05 compared with respective basal values.
Z <
Cerebral Cortex
Brain Stem
Hypothalamus Area
Cerebral Cortex
Brain Stem
Hypothalamus Area
Fig. 5. Effect of i.p. (left) or i.ci. (right) injection of indomethacin on PGE z generation in several brain areas. Rats were injected with vehicle or indomethacin i.p. (10 mg/kg), or i.ci. (500 Ixg/rat). Animals were killed 1 h later and cerebral cortex, brain stem and hypothalamus area were collected for prostaglandin generation. Each column represents the mean _+S.E.M. of four rats. * P < 0.01 compared to control groups.
by 96% in the gastric mucosa and by 80% in the brain cortex and when injected i.v. by 89 and 72% respectively. However, indomethacin injected i.ci. at a lower dose (50 /xg) induced a higher inhibitory effect in the brain (85%) than in the gastric mucosa (26%) (figs. 3 and 4). The inhibition of P G E 2 synthesis in several brain areas including the cerebral cortex, brain stem and hypothalamus was similar after i.p. (10 mg/kg) or i.ci. (500 txg/rat) injection of indomethacin (fig. 5).
3.3. Effect of i. ci. injection of indomethacin on basal and baclofen-stimulated gastric acid secretion I.ci. injection of indomethacin (500 /zg) increased gastric acid output (fig. 6). The onset was observed within 10 rain and a peak response with values three fold over basal occurred 20-30 min post injection;
o
V II-
g
"
-~o
"
~
"
2b
TIME
(mln)
"
~i~
"
6b
"
~
"
16o
Fig. 6. Effect of i.ci. injection of indomethacin (500 p~g) on basal acid output in urethane-anesthetized rats. Each point represents the mean _+S.E.M. of six rats. In the indomethacin-treated group, the acid output for the 60 min period was significantly increased (P < 0.01) compared with saline-treated group. Vehicle ©; indomethacin I.
Gastric acid output 0 z m o l / 3 / / m i n ) b Basal
Baclofen
14+2 16 + 2
78_+ 4 ~ 88 + 11 ~
thereafter acid secretion gradually decreased to reach basal level at 60 min (fig. 6). I.ci. injection of indomethacin (500/xg) 1 h before the experiment, modified neither basal nor baclofen-stimulated acid output (table 1). Indomethacin injected i.ci. at higher doses (750-1000 /xg/rat) induced acute severe vascular injury in the lung and lethality within 10 rain. Gross anatomic examination revealed numerous punctate and mainly confluent hemorrhagic spots throughout the lungs.
4.
Discussion
Gastric acid secretion is low in rats anesthetized with urethane. We reported that urethane anesthesiainduced low gastric acid secretion results from the enhanced expression and release of somatostatin in the stomach (Yang et al., 1990). Electrophysiological, surgical and pharmacological studies have established that peripheral injection of the GABA B receptor agonist, baclofen, stimulates gastric acid secretion through vagal pathways (Goto et al., 1984, 1985; Goto and Debas, 1983). In the present study, s.c. injection of baclofen in urethane-anesthetized rats induced within 1 h a plateau stimulation of gastric acid secretion which was maintained throughout the 2 h experimental period as reported in previous studies (Goto et al., 1985; Goto and Debas, 1983; Yang et al., 1989). PGE 2 and 16,16-dimethyl PGE2, injected i.ci. caused a dose-related inhibition of baclofen stimulated acid secretion in urethane-anesthetized rats. The minimal effective dose of 16,16-dimethyl PGE 2 was 0.03 /xg and 100 fold lower compared to that of PGE 2. This shows the potency of the stable PGE z analog to inhibit gastric secretion as demonstrated in other experimental conditions (Robert et al., 1979). I.v. infusion of P G E 2 also inhibited baclofen-stimulated gastric acid secretion. However, ten fold higher i.v. doses were required to produce a
significant effect compared to CSF injection. These results indicate that P G E 2 or P G E 2 analog injected i.ci. acts in the brain to inhibit vagally stimulated acid secretion and not in the periphery through leakage into the circulation. These findings further support Puurunen's previous report (Puurunen, 1983b) showing that PGE 2 injected i.c.v, dose dependently inhibited gastric acid secretion induced by chemical (insulin) or electrical vagal stimulation through a CNS-mediated action. Injection of P G E 2 into the lateral ventricle was also reported to inhibit acetylcholine- and pentagastrin-stimulated acid secretion (Puurunen, 1983b; Barocelli et al., 1991). Specific brain sites of P G E 2 to inhibit acid secretion stimulated by pentagastrin or pylorus ligation have been recently localized in the central amygdala, ventromedial hypothalamic nucleus, nucleus accumbens and preoptic area (Barocelli et al., 1991; Saperas et al., 1991). Although previous and present pharmacological studies provide consistent results on the central inhibitory effect of P G E 2 on stimulated acid secretion, it is still unclear whether endogenous prostaglandins play a physiological role in the central regulation of gastric acid secretion. Earlier studies indicate that i.c.v, injection of the prostaglandin synthetase inhibitor, indomethacin (400-500 p~g) significantly increased basal gastric acid secretion in 1 h pylorus-ligated conscious rats and insulin-stimulated gastric acid output in urethane-anesthetized rats (Puurunen, 1983a, c). These data were interpreted as suggesting a tonic inhibitory influence of central prostaglandins in the regulation of acid secretion (Puurunen, 1983a, c). By contrast, i.ci. injection of indomethacin (400 ~g) was reported to not modify basal gastric acid secretion measured 2 h after pylorus ligation in conscious rats (Guglietta et al., 1989). The present kinetic study indicates that i.ci. injection of indomethacin (500 /~g) stimulates gastric acid output. The response started within 10 min, with a peak occurring at 20-30 min, and thereafter values returned to basal levels. However, at 60-120 min post indomethacin injection, neither basal, nor baclofenstimulated gastric acid secretion were modified. The stimulation of gastric acid secretion immediately following i.ci. injection of indomethacin added to the vanishing of the secretory response 1 or 2 h later at a time when brain prostaglandin synthesis is inhibited, do not favor a tonic inhibitory influence of brain P G E 2 on gastric acid secretion. Indomethacin is not only known to inhibit prostaglandin synthesis but also calcium uptake (Burch et al., 1983; Northover, 1977). To what extent the initial secretory response to central injection of indomethacin may reflect alteration of calcium flux rather than prostaglandin synthesis inhibition remains to be further investigated. Likewise, the immediate increase in postprandial intestinal spike activity induced by CSF (cerebrospinal fluid) injection of
indomethacin (500 p,g) was shown to be unrelated to alterations of brain prostaglandin synthesis (Fargeas et al., 1985). Although there is no convincing evidence that brain P G E 2 exerts a tonic inhibition on gastric acid secretion in rats under basal conditions, brain P G E 2 may however be involved in pathophysiological conditions associated with enhanced release. For instance, interleukin-1/3 are known to increase P G E 2 production in several tissues including the brain (Sirko et al., 1989; Katsuura et al., 1989) and to induce a potent centrally mediated antisecretory effect that requires the integrity of prostaglandin pathways (Saperas et al., 1991). The present study also demonstrates that i.ci. injection of indomethacin in doses ranging from 50 to 500 # g / r a t inhibits dose dependently PGE 2 generative capacity not only in the brain but also in the gastric mucosa. Indomethacin given i.ci. at the highest dose (500 p~g) was equally effective in suppressing prostaglandin synthesis in both the brain and gastric mucosa. At a lower i.ci. dose (50 p~g), the inhibition of P G E 2 generation was higher in the brain cortex (85%) than in the gastric mucosa (26%). These data provide the first evidence that CSF injection of indomethacin at 500 p~g also depletes P G E 2 synthesis in the stomach. The underlying pathways and mechanisms involved in i.ci. indomethacin action needs to be further investigated. Since indomethacin injected i.v. at similar doses was equally effective in inhibiting prostaglandin synthesis in the gastric mucosa, peripheral inhibition of prostaglandin synthesis by central injection may involve leakage of the compound from the CSF into the circulation as reported for other substances (Passaro et al., 1982; Pekary et al., 1990; Tannenbaum and Patel, 1986). It may also be mediated by central mechanisms. The fact that indomethacin injected at doses higher than 500 /~g produces fulminant death associated with lung edema and hemorrhage that is not mimicked by peripheral injection suggests a specific action of indomethacin on CNS structures. Irrespective of the underlying mechanisms, these results show that injection of indomethacin into the CSF at 400-600 /zg doses previously used to assess the role of brain prostaglandins in relation with gastric function (Puurunen, 1983a, c; Fargeas et al., 1985; Guglietta et al., 1988; Guglietta et al., 1989), inhibits not only prostaglandin synthesis in the brain but also in the stomach. From the dose response curves, it appears that i.ci. injection of indomethacin at 50 ~.g provides a more selective depletion of prostaglandins in the brain and will be a more suitable dose to use. It is well established that peripheral injection of indomethacin induced a dose related inhibition of prostaglandin generation in the gastric mucosa (Bunnett et al., 1983; Okada et al., 1989; present observation). In addition we found that i.v. or i.p. injection of
indomethacin also suppressed prostaglandin generation in the rat brain. In previous studies, i.m. injection of three cyclooxygenase inhibitors including indomethacin were found to inhibit prostaglandin generation induced by convulsant treatments in mice brain (F6rstermann et al., 1982). The alterations of prostaglandin synthesis in the brain following peripheral administration of cyclooxygenase inhibitor has been linked with changes in sleep pattern and convulsant actions of drugs (Naito et al., 1988; F6rstermann et al., 1982). The mechanisms through which peripheral indomethacin alters brain prostaglandin synthesis remain to be clarified since following i.v. injection of labelled indomethacin in rats little accumulation of the compound were found in the brain and intestine (Hucker et al., 1966). In summary, the present data show that PGE 2 and the analog 16,16-dimethyl PGE2, injected i.ci., act in the brain to inhibit baclofen-stimulated gastric acid secretion in urethane-anesthetized rats. CSF injection of indomethacin at 400-500/zg inhibits prostaglandins in both the brain and the stomach whereas the 50 /xg dose appears to be more selective to deplete only brain prostaglandins. There is no conclusive evidence for a tonic inhibitory role of endogenous brain prostaglandins in the regulation of acid secretion.
Acknowledgements This work was supported by the National Institute of Arthritis Metabolism and Digestive Disease, Grants AM 30110 and DK 38198 and the National Institute of Mental Health, Grant MH 00663. Dr. E. Saperas has received a fellowship from the Spanish 'Fondo de Investigaciones Sanitarias de la Seguridad Social', and SKF. The authors thank Mrs. E. Kolve for her excellent technical assistance and Mr. Paul Kirshbaum for helping in the preparation of the manuscript.
References Amir, S. and A. Schiavetto, 1990, Injection of prostaglandin E 2 into the anterior hypothalamic preoptic area activates brown adipose tissue thermogenesis in the rat, Brain Res. 528, 138. Barocelli, E., M. Impicciatore, J. Seaton, R. Conter and G. Kauffman, 1991, Localization of central prostaglandin E z antisecretory effects, Gastroenterology 100, 320. Bauminger, S., V. Zor and H.R. Lindner, 1973, Radioimmunological assay of prostaglandin synthetase activity, Prostaglandins 4, 313. Bu~no, L., M.J. Fargeas, J. Fioramonti and M.P. Primi, 1985, Central control of intestinal motility'by prostaglandins: a mediator of the actions of several peptides in rats and dogs, Gastroenterology 88, 1888. Bunnett, N.W., J.H. Walsh, H.T. Debas, G.L. Kauffman and E.M. Golanska, 1983, Measurement of prostaglandin E 2 in interstitial fluid from the dog stomach after feeding and indomethacin, Gastroenterology 85, 1391. Burch, R.M., W.C. Wise and P.V. Halushka, 1983, Prostaglandin-independent inhibition of calcium transport by nonsteroidal anti-in-
flammatory drugs: differential effects of carboxylic acids and picroxicam, J. Pharm. Pharmacol. 227, 84. Chiu, E.K.Y. and J.S. Richardson, 1985, Behavioral and neurochemical aspects of prostaglandins in brain function, Gen. Pharmacol. 16, 163. Cornell, R.P., 1989, Central interleukin 1-elicited hyperinsulinemia is mediated by prostaglandins but not autonomics, Am. J. Physiol. 257, R839. Cseh, G., I.K. Szabo, T. Lang and M. Palkovits, 1978, Distribution of prostaglandins E and F in different regions of rat brain, Brain Res. 3, 293. Fargeas, M.J., J. Fioramonti and L. Bu6no, 1984, Prostaglandin E2: a neuromodulator in the central control of gastrointestinal motility and feeding behavior by calcitonin, Science 225, 1050. Fargeas, M.J., J. Fioramonti and L. Bu6no, 1985, Central action of calcitonin on body temperature and intestinal motility in rats: evidence for different mediations, Reg. Pept. 11, 95. F6rstermann, U., R. Heldt, F. Knappen and G. Herning, 1982, Potential anticonvulsive properties of endogenous prostaglandins formed in mouse brain, Brain Res. 240, 303. Goto, Y. and H.T. Debas, 1983, GABA-mimetic effect on gastric acid secretion. Possible significance in central mechanisms, Dig. Dis. Sci. 28, 56. Goto, Y., J.W. Hollinshead and H.T. Debas, 1984, A new intraoperative test for completeness of vagotomy: the PCP-GABA (betaparachlorophenol-gamma-aminobutyric acid) test, Am. J. Surg. 147, 159. Goto, Y., Y. Tach6, H. Debas and D. Novin, 1985, Gastric and vagus nerve response to GABA agonist baclofen, Life Sci. 36, 2471. Guglietta, A., B.J. Irons and L.H. Lazarus, 1988, Interaction between bombesin and brain prostaglandins in the control of gastric secretion, Ann. N.Y. Acad. Sci. 547, 486. Guglietta, A., R.V. Nardi and L.H. Lazarus, 1989, Indomethacin (i.c.v.) reverses the inhibitory action of peptides on gastric secretion, Eur. J. Pharmacol. 170, 87. Giillner, H-G., 1983, The interactions of prostaglandins with the sympathetic nervous systemza review, J. Auton. Nerv. Syst. 8, 1. Hayaishi, O., 1991, Molecular mechanisms of sleep-wake regulation: roles of prostaglandins D z and Ez, FASEB J. 5, 2575. Horiguchi, S., R. Ueno, M. Hyodo and O. Hayaishi, 1986, Alterations of nociception after intracisternal administration of prostaglandin D e, E2, or F2,~ to conscious mice, Eur. J. Pharmacol. 122, 173. Hucker, H.B., A.G. Zacchei, S.V. Cox, D.A. Brodie and N.H.R. Cantwell, 1966, Studies on the absorption, distribution and excretion of indomethacin in various species, J. Pharmacol. Exp. Ther. 153, 237. Katsuura, G., A. Arimura, K. Koves and P.E. Gottschall, 1990, Involvement of organum vasculosum of lamina terminalis and preoptic area in interleukin-1/3-induced ACTH release, Am. J. Physiol. 258, E163. Katsuura, G., P.E. Gottschall, R.R. Dahl and A. Arimura, 1989, Interleukin-1 beta increases prostaglandin E 2 in the rat astrocyte cultures: modulatory effect of neuropeptides, Endocrinology 124, 3125. Ligumsky, M., E.M. Golanska, D.G. Hansen and G.L. Kauffman Jr., 1983, Aspirin can inhibit gastric mucosal cyclo-oxygenase without causing lesions in rats, Gastroenterology 84, 756. Matsumura, K., Y. Watanabe, H. Onoe and O. Hayaishi, 1990, High density of prostaglandin E 2 binding sites in the anterior wall of the 3rd ventricle: a possible site of its hyperthermic action, Brain Res. 533, 147. Naito, K., H. Osama, R. Ueno, O. Hayaishi, K. Honda and S. lnoue, 1988, Suppression of sleep by prostaglandin synthesis inhibitors in unrestrained rats, Brain Res. 453, 329. Northover, B.J., 1977, Indomethacin, a calcium antagonist, Gen. Pharmacol. 8, 293.
Okada, M., N. Hiromichi, K. Takeuchi and S. Okabe, 1989, Role of prostaglandin deficiency in pathogenetic mechanism of gastric lesions induced by indomethacin in rats, Dig. Dis. Sci. 34, 694. Otorii, T., K. Ohkubo and K. Suzuki, 1985, Central noradrenergic neurons and the hypertensive effects of intracerebroventricularly administered prostaglandin E 2 in anaesthetized rabbits, Prostaglandins 29, 25. Passaro, E. Jr., H. Debas, W. Oldendorf and T. Yamada, 1982, Rapid appearance of intraventricularly administered neuropeptides in the peripheral circulation, Brain Res. 241,335. Pekary, A., R. Stephens, M. Simard, X-P. Pang, V. Smith, J.J. Distephano III and J.M. Hershman, 1990, Release of thyrotropin and prolactin by a thyrotropin-releasing hormone (TRH) precursor, TRH-Gly: conversion to TRH is sufficient for in vivo effects, Neuroendocrinology 52, 618. Puurunen, J., 1983a, Central inhibitory action of prostaglandin E 2 on gastric secretion in the rat, Eur. J. Pharmacol. 91,245. Puurunen, J., 1983b, Inhibition of gastric acid secretion by intracerebroventricularly administered prostaglandin E 2 in anaesthetized rats, Br. J. Pharmacol. 78, 131. Puurunen, J., 1983c, Central effects of prostaglandin E 2 and indomethacin on insulin-stimulated gastric acid secretion in anaesthetized rats, Acta Pharmacol. Toxicol. 52, 318. Robert, A., J.E. Nezamis, C. Lancaster and A.J. Hanchar, 1979, Cytoprotection by prostaglandins in rats. Prevention of gastric necrosis produced by alcohol, HCL, NaOH, hypertonic saline, and thermal injury, Gastroenterology 77, 433. Saperas, E., H. Yang, C. Rivier and Y. TachS, 1990, Central action of recombinant interleukinn-1 to inhibit acid secretion in rats, Gastroenterology 99, 1599. Saperas, E., H. Yang and Y. Tach6, 1991, Interleukin-13 acts in specific hypothalamic nuclei to inhibit gastric acid secretion through prostaglandin pathways in the rat, Gastroenterology 100, A664.
Sautereau, D. and C. Roz6, 1988, Central effects of prostaglandins on the digestive tract, Gastroenterol. Clin. Biol. 12, 222. Sirko, S., I. Bishai and F. Coceani, 1989, Prostaglandin formation in the hypothalamus in vivo: effect of pyrogens, Am. J. Physiol. 256, R616. Staumont, G., J. Fioramonti, J. Frexinos and L. Bu~no, 1990, Oral prostaglandin E analogues induce intestinal migration motor complexes after a meal in dogs. Evidence for a central mechanism, Gastroenterology 98, 888. Tach6, Y., 1987, Central regulation of gastric acid secretion, in: Physiology of the Gastrointestinal Tract, Vol. 2, eds. L.R. Johnson, J. Christensen, M. Jackson, E.D. Jacobson and J.H. Walsh (Raven Press, New York) p. 911. TachS, Y., Y. Goto, D. Hamel, A. Pekary and D. Novin, 1985, Mechanisms underlying intracisternal TRH-induced stimulation of gastric acid secretion in rats, Reg. Pept. 13, 21. Tannenbaum, G.S. and Y.C. Patel, 1986, On the fate of centrally administered somatostatin in the rat: massive hypersomatostatinemia resulting from leakage into the peripheral circulation has effects on growth hormone secretion and glucoregulation. Endocrinology 118, 2137. Whittle, B.J.R., G.A. Higgs, K.E. Eakins, A. Moncada and J.R. Vance, 1980, Selective inhibition of prostaglandin production in inflammatory exudates and gastric mucosa, Nature 284, 271. Yang, H., H. Wong, G. Rosenquist, J.H. Walsh and Y. TachS, 1990, Somatostatin monoclonal antibody immunoneutralization increases gastrin and gastric acid secretion in urethane-anesthetized rats, Gastroenterology 99, 659. Yang, H., H. Wong, J.H. Walsh and Y. Tach6, 1989, Effect of gastrin monoclonal antibody 28.2 on acid response to chemical vagal stimulation in rats, Life Sci. 45, 2413.
1
© 1991 Elsevier Science Publishers B.V. All rights reserved 0014-2999/91/$03.50
EJP 52171
Role of central prostaglandin E 2 in the regulation of gastric acid secretion in the rat Esteban Saperas, Gordon Kauffman ~ and Yvette Tach6 Center for Ulcer Research and Education, VA Wadsworth Medical Center, Department of Medicine and Brain Research Institute, UCLA, Los Angeles, CA, U.S.A. and l Division of General Surgery, The Milton Hershey Medical Center, The Pennsyh,ania State Unit,ersity, Hershey, PA, U.S.A.
Received 10 September 1991, accepted 17 September 1991
The central action of prostaglandin E 2 (PGE2) on gastric acid secretion was investigated in rats by comparing the effects of intracisternal (i.ci.) and i.v. administration of PGE 2 and the influence of i.ci. injection of indomethacin on acid secretion and PGE 2 generation in the brain and stomach. I.ci. injections of PGE 2 (1-10 /xg) or the stable analog, 16,16-dimethyl PGE2, (0.01-0.1 /xg) induced a dose dependent inhibition of baclofen-stimulated gastric acid secretion by 0-82% and by 7-87% respectively. I.v. infusion of PGE 2 also induced a dose related inhibition of baclofen-stimulated acid secretion, but 10 fold higher doses were required. I.ci. or i.v. injection of indomethacin in doses ranging from 50 to 500 /xg/rat, produced a similar dose dependent inhibition of the PGE 2 generation in both the gastric mucosa and brain cortex measured 1 h post injection. I.ci. injection of indomethacin (500 p.g) increased within 10 min acid secretion with a peak response at 20-30 rain; 60-120 rain post injection, when prostaglandin synthesis was inhibited by 90%, basal and baclofen-stimulated acid output were not altered. These results further establish that PGE 2 acts in the brain to inhibit vagally stimulated gastric acid secretion in rats, and do not support a tonic inhibitory influence of endogenous brain PGE 2 in the regulation of gastric acid secretion. In addition, these data showed that indomethacin injected i.ci. at 500/xg does not induce a selective inhibition of prostaglandin synthesis in the brain.
Prostaglandin E2; Indomethacin; Gastric acid secretion; Vagus; Baclofen
1. I n t r o d u c t i o n
Prostaglandin E 2 ( P G E 2) receptors are widely distributed in the central nervous system (CNS) (Cseh et al., 1978; M a t s u m u r a et al., 1990). Growing n e u r o p h a r macological evidence suggests that prostaglandins act in the brain to influence homeostasis and a u t o n o m i c functions. P G E 2 injected into the cerebrospinal fluid (CSF) or into specific brain sites elicits behavioral changes (Chiu and Richardson, 1985), analgesia (Horiguchi et al., 1986), fever (Amir and Schiavetto, 1990), wakefulness (Hayaishi, 1991), hyperinsulinemia (Cornell, 1989), A C T H release (Katsuura et al., 1990; Cornell, 1989), sympathetic activation (Gfillner, 1983), and increases blood pressure and heart rate (Otorii et al., 1985). Prostaglandins, along with peptides, also exert C N S actions that influence gastrointestinal secretory and m o t o r functions (Puurunen, 1983a; BuEno et al., 1985; Tach6, 1987; Sautereau and Roz6, 1988; S t a u m o n t et al., 1990). In particular, one g r o u p of investigators has established that P G E 2 acts in the
Correspondence to: Y. TachS, VA Wadsworth Medical Center, Building 115, Room 203, Wilshire and Sawtelle BI, Los Angeles, CA 90073, U.S.A. Tel. 1.213.206 5489, fax 1.213.824 6752.
brain to inhibit vagally stimulated acid secretion in rats (Puurunen, 1983a, b, c). W h e t h e r e n d o g e n o u s P G E 2 in the brain exerts a tonic inhibition on gastric secretion is still to be established. In the present study we further investigated the influence of central prostaglandins in the regulation of gastric acid secretion in the rat. Comparison of the dose-related inhibition of vagally stimulated acid secretion in response to CSF injection or i.v. infusion of P G E 2 or the stable P G E 2 analog, 16,16-dimethyl P G E 2, was performed. In addition, the influence of i.ci. injection of the prostaglandin synthesis inhibitor, indomethacin, on basal and stimulated acid secretion was m o n i t o r e d over time along with its inhibitory effect on e n d o g e n o u s prostaglandin generation in the brain and stomach.
2. M a t e r i a l s a n d m e t h o d s 2.1. A n i m a l s
Male S p r a g u e - D a w l e y rats, weighing 2 0 0 - 2 8 0 g (Simonsen Laboratories, Gilroy, California) were fasted with free access to water 24 h before the experiment.
2.2. Drugs and treatment P G E 2 and 16,16-dimethyl PGE 2 (Upjohn Co., Kalamazoo, Michigan)were dissolved in absolute ethanol (10 mg ml-~) and stored at - 2 0 ° C. The stock solution was diluted in sterile 0.9% saline immediately prior to i.ci. injection (10 /xl) or i.v. infusion (0.5 ml/h). /3-(pChlorophenyl)-¢-aminobutyric acid (baclofen, Lioresal; Geigy Pharmaceutical, Summit, New Jersey) was dissolved in saline and injected s.c. in 1 ml volume. Indomethacin (Sigma Chemical, St. Louis, Missouri) was dissolved in 1% sodium bicarbonate and injected i.ci. (10/xl), i.v. (1 ml kg -~) or i.p. (1 ml).
2.3. Measurement of gastric acid secretion Gastric acid secretion was measured as previously reported (Tach6 et al., 1985). In rats under urethane anesthesia (1.25 g / k g i.p.), the pylorus was ligated and a double-lumen cannula was acutely implanted in the forestomach. The inlet and outlet tubes of the gastric cannula were continuously perfused with a perislatic pump at flow rate of 3.5 m l / m i n with normal saline adjusted to pH 5.5. Gastric acid output was recorded every 2 min by titration of the gastric perfusate to a pH 5.5 with 0.02 N NaOH, using an automatic titrator with a zero suppression adaptor (TOA Electronics, Tokyo, Japan). In one group of experiments, gastric acid secretion was measured every 10 min by flushing the gastric lumen with two 5 ml boli of 0.15 M NaC1 and 5 ml of air at the end of each 10 min period. Acid output was determined by titration of the flushed perfusate with 0.1 M N a O H to pH 7.0 on an automatic titrator (Radiometer, Copenhagen).
2.4. Experimental design 2. 4.1. Effect of PGE 2 administration Basal gastric acid secretion was determined every 2 min for 20 min then baclofen was injected s.c. (4 m g / k g ) and 80 min later, vehicle or various doses of P G E 2 or 16,16-dimethyl P G E 2 were injected i.ci. or infused i.v. over a 30 rain period. Incremental doses of P G E 2 (1, 3 and 10 p~g) or the P G E 2 analog (0.01, 0.03 and 0.1 /xg) were injected i.ci. at 30 min intervals. I.v. infusion of P G E 2 was maintained throughout the experiment and doses (10, 30 and 1 0 0 / x g / r a t per 30 min) were increased every 30 rain. 2. 4. 2. Effects of indomethacin administration Basal acid secretion was measured for 30 rain before and 100 min after i.ci. injection of vehicle or indomethacin (500/xg) using the 10 min flush technique. In another experiment, indomethacin (500 p.g) was injected i.ci. and 1 h later basal acid secretion was measured every 2 min for 30 min, then baclofen was
injected s.c. (4 mg/kg) and acid secretion was measured for the following 90 min period.
2.5. Measurement of PGE 2 generation Under light ether anesthesia the animals were injected i.ci., i.v. through the jugular vein or i.p. with vehicle or indomethacin. Conscious rats were killed 1 h later by CO 2 inhalation and the corpus mucosa was stripped and brain tissue specimens were collected, weighed and processed for ex vivo P G E 2 generation as previously d'escribed (Whittle et al., 1980). The tissue was chopped with scissors for 45 s in microfuge plastic tubes containing 1 ml of phosphate buffer (100 raM, pH 7.4) and centrifuged in a fixed-speed bench centrifuge at 15000 rpm for 30 s. After discarding the supernatant, the tissue was suspended in 1 ml of phosphate buffer and mixed by vortex for 1 min at room temperature. Then, 10/xg (10/xl) of indomethacin was added to each sample to inhibit any further formation of prostaglandins. The samples were centrifuged for 1 min and the supernatants were stored at - 2 0 °C until assayed. P G E 2 was determined by a modification of a radioimmunoassay technique previously described (Bauminger, Zor and Lindner, 1973) using [3H]PGE2 as tracer, specific P G E 2 antiserum, and standard. The assay medium was 0.1 M phosphate buffer saline (0.9%) with gelatin (0.1%), pH 7.4. The radioimmunoassay was performed as previously published (Ligumsky et al., 1983). The generative capacity is expressed as nanograms of PGE 2 per gram wet weight of tissue per min (ng g J min-l).
2.6. Statistical analysis The results are presented as means _+ S.E.M. Statistical probabilities were calculated by Student's t-test or one-way analysis of variance followed by multiple comparisons with Dunnett's t-test.
3. Results
3.1. Effect of i.ci. injection or i.v. infusion of PGE 2 on baclofen-stimulated gastric acid secretion Baclofen (4 m g / k g s.c.) significantly stimulated the low basal acid secretion (0.2_+ 0.1 /xmol/2 rain) in urethane-anesthetized rats. A plateau response was observed within 58 + 3 min and maintained at this level for 90 min in rats injected i.ci. with vehicle (1% ethanol in 0.9% saline) every 30 min starting 80 min post baclofen injection (fig. 1). I.ci. injections of 16,16-dimethyl P G E 2 at 0.01, 0.03 and 0.1 p,g every 30 min, dose dependently inhibited baclofen-stimulated acid secretion by 7, 41 and 87% respectively. Thirty minutes
16,16 d m P G E 2 Dose (ng/rat)
A
Intravenous 30
10
Intraclsternal
I00 20,
'E ,o
i
~ 15,
g0-
Z
~
!..... ~?.. -y
20-
o Ix¢ nIJJ Z UJ (3
-o
10,
5'
LI,Ie4 o
•
20
•
0
20
40
80
60 TIME
100
120
140
160
(3
-
18'0
(min)
0
Fig. 1. Dose-related inhibition of baclofen-stimulated gastric acid secretion by i.ci. injection of 16,16-dimethyl PGE 2 in urethaneanesthetized rats. After 20 min basal period, baclofen was injected s.c. and 80 min later vehicle (©) or the PGE 2 analog (e) was injected i.ci. at various doses every 30 min. Each point represents the mean + S.E.M. of four rats. There is a significant (P < 0.05) dose-related inhibition of acid secretion by 16,16-dimethyl PGE 2 compared with vehicle-treated rats.
after i.ci. injection of 16,16-dimethyl PGE 2 at 0.1 /xg, acid secretion was completely inhibited (fig. 1). Likewise, PGE 2 injected i.ci. every 30 rain at step dose increments (1, 3 and 10 /xg), dose dependently inhibited baclofen-stimulated gastric acid output by 0, 45 and 82% respectively (fig. 2). The gastric inhibitory effect was observed within 3.7_+ 0.5 rain after i.ci. injection of PGE 2 or 16,16-dimethyl PGE 2. I.v. infusion of PGE 2 in doses ranging from 10 to 100 /xg/rat per 30 min every 30 min inhibited dose dependently baclofen-stimulated gastric acid output by 17-86% in urethane-anesthetized rats (fig. 2).
50
100
500
INDOMETHACIN
0 DOSE
50
100
500
(~tg/rat)
Fig. 3. Inhibition of PGE 2 generation in the brain cortex by i.v. or i.ci. injection of indomethacin. Rats fasted for 24 h were injected i.v. or i.ci. under light ether anesthesia with vehicle or indomethacin (50, 100 or 500 /xg/rat). Animals were killed I h later and brain cortex collected for PGE 2 generation. Each column represents the mean + S.E.M. of four rats. * P < 0.01 compared with vehicle-treated group.
3.2. Effect of i.ci. and i.u. injection of indomethacin on PGE2-generative capacity in L,arious tissues
I.v. or i.ci. injection of indomethacin (50-500 /zg) significantly suppressed PGE2-generative capacity in both the brain cortex and gastric mucosa as assessed 1 h after indomethacin injection (figs. 3 and 4). The inhibition of PGE 2 syntheses in the gastric mucosa in response to i.ci. or i.v. injection of indomethacin was dose-related (fig. 4). The percentages of inhibition produced by indomethacin in doses ranging from 50 to 500 /xg/rat were similar in the cortex after the i.ci. or i.v. route of administration (fig. 3). At the 500 /xg dose, indomethacin injected i.ci. inhibited PGE 2 generation 300
120,
Intraclaternal
A
E
10o.
o E
eo-
l--
60.
O. k..~ 0
40-
a <
0
1
i; 3
Intravenous
Intravenous
o
10
PGE 2
0 DOSE
0
30
100
50
a0o
Intraclstemal
500
0
so
100
5oo
INDOMEI'HACINDOSE 0ag/rat)
(gg/rat)
Fig. 2. Dose-related inhibition of baclofen-stimulated gastric acid secretion by i.ci. injection or i.v. infusion of PGE 2 in urethaneanesthetized rats. After 20 min basal period, baclofen (4 m g / k g s.c.) was injected and 80 min later vehicle or PGE 2 was injected i.ci. every 30 min or infused i.v. for 30 min at various doses. Each column represents the mean + S.E.M. of four rats; * P < 0.05 and * * P < 0.01 compared with vehicle-treated group (©).
Fig. 4. Dose-related inhibition of PGE 2 generation in the gastric mucosa induced by i.v. or i.ci. injection of indomethacin. Under light ether anesthesia rats fasted for 24 h received a single i.v. or i.ci. injection of vehicle (1% sodium bicarbonate) or indomethacin (50, 100 or 500 ~ g / r a t ) . Fundic mucosa was stripped and collected for measurement of prostaglandin generation 1 h later. Each column represents the m e a n + S.E.M. of four rats. ~ P < 0.05 and * P < 0.01 compared with vehicle-treated group.
I•
4o,
~
[]
TABLE 1
Control Indomethacin
Effect of indomethacin pretreatment on basal and baclofen-stimulated gastric acid secretion in urethane-anesthetized rats.
3o.
Treatment ;~
F-
2o.
w Z ul
Vehicle Indomethacin
10.
~' Rats fasted for 24 h were anesthetized with urethane and double lumen was implanted in the forestomach, lndomethacin (500 p.g) or vehicle was injected i.ci. and stomach was constantly perfused. One hour after the injection, gastric acid output was measured for 30 min under basal conditions and for 90 min following baclofen injection (4 m g / k g s.c.), b Results are expressed as means_+S.E.M, of four rats in each group; acid output is expressed as 30 min periods before and 60 min after baclofen injection, c p < 0.05 compared with respective basal values.
Z <
Cerebral Cortex
Brain Stem
Hypothalamus Area
Cerebral Cortex
Brain Stem
Hypothalamus Area
Fig. 5. Effect of i.p. (left) or i.ci. (right) injection of indomethacin on PGE z generation in several brain areas. Rats were injected with vehicle or indomethacin i.p. (10 mg/kg), or i.ci. (500 Ixg/rat). Animals were killed 1 h later and cerebral cortex, brain stem and hypothalamus area were collected for prostaglandin generation. Each column represents the mean _+S.E.M. of four rats. * P < 0.01 compared to control groups.
by 96% in the gastric mucosa and by 80% in the brain cortex and when injected i.v. by 89 and 72% respectively. However, indomethacin injected i.ci. at a lower dose (50 /xg) induced a higher inhibitory effect in the brain (85%) than in the gastric mucosa (26%) (figs. 3 and 4). The inhibition of P G E 2 synthesis in several brain areas including the cerebral cortex, brain stem and hypothalamus was similar after i.p. (10 mg/kg) or i.ci. (500 txg/rat) injection of indomethacin (fig. 5).
3.3. Effect of i. ci. injection of indomethacin on basal and baclofen-stimulated gastric acid secretion I.ci. injection of indomethacin (500 /zg) increased gastric acid output (fig. 6). The onset was observed within 10 rain and a peak response with values three fold over basal occurred 20-30 min post injection;
o
V II-
g
"
-~o
"
~
"
2b
TIME
(mln)
"
~i~
"
6b
"
~
"
16o
Fig. 6. Effect of i.ci. injection of indomethacin (500 p~g) on basal acid output in urethane-anesthetized rats. Each point represents the mean _+S.E.M. of six rats. In the indomethacin-treated group, the acid output for the 60 min period was significantly increased (P < 0.01) compared with saline-treated group. Vehicle ©; indomethacin I.
Gastric acid output 0 z m o l / 3 / / m i n ) b Basal
Baclofen
14+2 16 + 2
78_+ 4 ~ 88 + 11 ~
thereafter acid secretion gradually decreased to reach basal level at 60 min (fig. 6). I.ci. injection of indomethacin (500/xg) 1 h before the experiment, modified neither basal nor baclofen-stimulated acid output (table 1). Indomethacin injected i.ci. at higher doses (750-1000 /xg/rat) induced acute severe vascular injury in the lung and lethality within 10 rain. Gross anatomic examination revealed numerous punctate and mainly confluent hemorrhagic spots throughout the lungs.
4.
Discussion
Gastric acid secretion is low in rats anesthetized with urethane. We reported that urethane anesthesiainduced low gastric acid secretion results from the enhanced expression and release of somatostatin in the stomach (Yang et al., 1990). Electrophysiological, surgical and pharmacological studies have established that peripheral injection of the GABA B receptor agonist, baclofen, stimulates gastric acid secretion through vagal pathways (Goto et al., 1984, 1985; Goto and Debas, 1983). In the present study, s.c. injection of baclofen in urethane-anesthetized rats induced within 1 h a plateau stimulation of gastric acid secretion which was maintained throughout the 2 h experimental period as reported in previous studies (Goto et al., 1985; Goto and Debas, 1983; Yang et al., 1989). PGE 2 and 16,16-dimethyl PGE2, injected i.ci. caused a dose-related inhibition of baclofen stimulated acid secretion in urethane-anesthetized rats. The minimal effective dose of 16,16-dimethyl PGE 2 was 0.03 /xg and 100 fold lower compared to that of PGE 2. This shows the potency of the stable PGE z analog to inhibit gastric secretion as demonstrated in other experimental conditions (Robert et al., 1979). I.v. infusion of P G E 2 also inhibited baclofen-stimulated gastric acid secretion. However, ten fold higher i.v. doses were required to produce a
significant effect compared to CSF injection. These results indicate that P G E 2 or P G E 2 analog injected i.ci. acts in the brain to inhibit vagally stimulated acid secretion and not in the periphery through leakage into the circulation. These findings further support Puurunen's previous report (Puurunen, 1983b) showing that PGE 2 injected i.c.v, dose dependently inhibited gastric acid secretion induced by chemical (insulin) or electrical vagal stimulation through a CNS-mediated action. Injection of P G E 2 into the lateral ventricle was also reported to inhibit acetylcholine- and pentagastrin-stimulated acid secretion (Puurunen, 1983b; Barocelli et al., 1991). Specific brain sites of P G E 2 to inhibit acid secretion stimulated by pentagastrin or pylorus ligation have been recently localized in the central amygdala, ventromedial hypothalamic nucleus, nucleus accumbens and preoptic area (Barocelli et al., 1991; Saperas et al., 1991). Although previous and present pharmacological studies provide consistent results on the central inhibitory effect of P G E 2 on stimulated acid secretion, it is still unclear whether endogenous prostaglandins play a physiological role in the central regulation of gastric acid secretion. Earlier studies indicate that i.c.v, injection of the prostaglandin synthetase inhibitor, indomethacin (400-500 p~g) significantly increased basal gastric acid secretion in 1 h pylorus-ligated conscious rats and insulin-stimulated gastric acid output in urethane-anesthetized rats (Puurunen, 1983a, c). These data were interpreted as suggesting a tonic inhibitory influence of central prostaglandins in the regulation of acid secretion (Puurunen, 1983a, c). By contrast, i.ci. injection of indomethacin (400 ~g) was reported to not modify basal gastric acid secretion measured 2 h after pylorus ligation in conscious rats (Guglietta et al., 1989). The present kinetic study indicates that i.ci. injection of indomethacin (500 /~g) stimulates gastric acid output. The response started within 10 min, with a peak occurring at 20-30 min, and thereafter values returned to basal levels. However, at 60-120 min post indomethacin injection, neither basal, nor baclofenstimulated gastric acid secretion were modified. The stimulation of gastric acid secretion immediately following i.ci. injection of indomethacin added to the vanishing of the secretory response 1 or 2 h later at a time when brain prostaglandin synthesis is inhibited, do not favor a tonic inhibitory influence of brain P G E 2 on gastric acid secretion. Indomethacin is not only known to inhibit prostaglandin synthesis but also calcium uptake (Burch et al., 1983; Northover, 1977). To what extent the initial secretory response to central injection of indomethacin may reflect alteration of calcium flux rather than prostaglandin synthesis inhibition remains to be further investigated. Likewise, the immediate increase in postprandial intestinal spike activity induced by CSF (cerebrospinal fluid) injection of
indomethacin (500 p,g) was shown to be unrelated to alterations of brain prostaglandin synthesis (Fargeas et al., 1985). Although there is no convincing evidence that brain P G E 2 exerts a tonic inhibition on gastric acid secretion in rats under basal conditions, brain P G E 2 may however be involved in pathophysiological conditions associated with enhanced release. For instance, interleukin-1/3 are known to increase P G E 2 production in several tissues including the brain (Sirko et al., 1989; Katsuura et al., 1989) and to induce a potent centrally mediated antisecretory effect that requires the integrity of prostaglandin pathways (Saperas et al., 1991). The present study also demonstrates that i.ci. injection of indomethacin in doses ranging from 50 to 500 # g / r a t inhibits dose dependently PGE 2 generative capacity not only in the brain but also in the gastric mucosa. Indomethacin given i.ci. at the highest dose (500 p~g) was equally effective in suppressing prostaglandin synthesis in both the brain and gastric mucosa. At a lower i.ci. dose (50 p~g), the inhibition of P G E 2 generation was higher in the brain cortex (85%) than in the gastric mucosa (26%). These data provide the first evidence that CSF injection of indomethacin at 500 p~g also depletes P G E 2 synthesis in the stomach. The underlying pathways and mechanisms involved in i.ci. indomethacin action needs to be further investigated. Since indomethacin injected i.v. at similar doses was equally effective in inhibiting prostaglandin synthesis in the gastric mucosa, peripheral inhibition of prostaglandin synthesis by central injection may involve leakage of the compound from the CSF into the circulation as reported for other substances (Passaro et al., 1982; Pekary et al., 1990; Tannenbaum and Patel, 1986). It may also be mediated by central mechanisms. The fact that indomethacin injected at doses higher than 500 /~g produces fulminant death associated with lung edema and hemorrhage that is not mimicked by peripheral injection suggests a specific action of indomethacin on CNS structures. Irrespective of the underlying mechanisms, these results show that injection of indomethacin into the CSF at 400-600 /zg doses previously used to assess the role of brain prostaglandins in relation with gastric function (Puurunen, 1983a, c; Fargeas et al., 1985; Guglietta et al., 1988; Guglietta et al., 1989), inhibits not only prostaglandin synthesis in the brain but also in the stomach. From the dose response curves, it appears that i.ci. injection of indomethacin at 50 ~.g provides a more selective depletion of prostaglandins in the brain and will be a more suitable dose to use. It is well established that peripheral injection of indomethacin induced a dose related inhibition of prostaglandin generation in the gastric mucosa (Bunnett et al., 1983; Okada et al., 1989; present observation). In addition we found that i.v. or i.p. injection of
indomethacin also suppressed prostaglandin generation in the rat brain. In previous studies, i.m. injection of three cyclooxygenase inhibitors including indomethacin were found to inhibit prostaglandin generation induced by convulsant treatments in mice brain (F6rstermann et al., 1982). The alterations of prostaglandin synthesis in the brain following peripheral administration of cyclooxygenase inhibitor has been linked with changes in sleep pattern and convulsant actions of drugs (Naito et al., 1988; F6rstermann et al., 1982). The mechanisms through which peripheral indomethacin alters brain prostaglandin synthesis remain to be clarified since following i.v. injection of labelled indomethacin in rats little accumulation of the compound were found in the brain and intestine (Hucker et al., 1966). In summary, the present data show that PGE 2 and the analog 16,16-dimethyl PGE2, injected i.ci., act in the brain to inhibit baclofen-stimulated gastric acid secretion in urethane-anesthetized rats. CSF injection of indomethacin at 400-500/zg inhibits prostaglandins in both the brain and the stomach whereas the 50 /xg dose appears to be more selective to deplete only brain prostaglandins. There is no conclusive evidence for a tonic inhibitory role of endogenous brain prostaglandins in the regulation of acid secretion.
Acknowledgements This work was supported by the National Institute of Arthritis Metabolism and Digestive Disease, Grants AM 30110 and DK 38198 and the National Institute of Mental Health, Grant MH 00663. Dr. E. Saperas has received a fellowship from the Spanish 'Fondo de Investigaciones Sanitarias de la Seguridad Social', and SKF. The authors thank Mrs. E. Kolve for her excellent technical assistance and Mr. Paul Kirshbaum for helping in the preparation of the manuscript.
References Amir, S. and A. Schiavetto, 1990, Injection of prostaglandin E 2 into the anterior hypothalamic preoptic area activates brown adipose tissue thermogenesis in the rat, Brain Res. 528, 138. Barocelli, E., M. Impicciatore, J. Seaton, R. Conter and G. Kauffman, 1991, Localization of central prostaglandin E z antisecretory effects, Gastroenterology 100, 320. Bauminger, S., V. Zor and H.R. Lindner, 1973, Radioimmunological assay of prostaglandin synthetase activity, Prostaglandins 4, 313. Bu~no, L., M.J. Fargeas, J. Fioramonti and M.P. Primi, 1985, Central control of intestinal motility'by prostaglandins: a mediator of the actions of several peptides in rats and dogs, Gastroenterology 88, 1888. Bunnett, N.W., J.H. Walsh, H.T. Debas, G.L. Kauffman and E.M. Golanska, 1983, Measurement of prostaglandin E 2 in interstitial fluid from the dog stomach after feeding and indomethacin, Gastroenterology 85, 1391. Burch, R.M., W.C. Wise and P.V. Halushka, 1983, Prostaglandin-independent inhibition of calcium transport by nonsteroidal anti-in-
flammatory drugs: differential effects of carboxylic acids and picroxicam, J. Pharm. Pharmacol. 227, 84. Chiu, E.K.Y. and J.S. Richardson, 1985, Behavioral and neurochemical aspects of prostaglandins in brain function, Gen. Pharmacol. 16, 163. Cornell, R.P., 1989, Central interleukin 1-elicited hyperinsulinemia is mediated by prostaglandins but not autonomics, Am. J. Physiol. 257, R839. Cseh, G., I.K. Szabo, T. Lang and M. Palkovits, 1978, Distribution of prostaglandins E and F in different regions of rat brain, Brain Res. 3, 293. Fargeas, M.J., J. Fioramonti and L. Bu6no, 1984, Prostaglandin E2: a neuromodulator in the central control of gastrointestinal motility and feeding behavior by calcitonin, Science 225, 1050. Fargeas, M.J., J. Fioramonti and L. Bu6no, 1985, Central action of calcitonin on body temperature and intestinal motility in rats: evidence for different mediations, Reg. Pept. 11, 95. F6rstermann, U., R. Heldt, F. Knappen and G. Herning, 1982, Potential anticonvulsive properties of endogenous prostaglandins formed in mouse brain, Brain Res. 240, 303. Goto, Y. and H.T. Debas, 1983, GABA-mimetic effect on gastric acid secretion. Possible significance in central mechanisms, Dig. Dis. Sci. 28, 56. Goto, Y., J.W. Hollinshead and H.T. Debas, 1984, A new intraoperative test for completeness of vagotomy: the PCP-GABA (betaparachlorophenol-gamma-aminobutyric acid) test, Am. J. Surg. 147, 159. Goto, Y., Y. Tach6, H. Debas and D. Novin, 1985, Gastric and vagus nerve response to GABA agonist baclofen, Life Sci. 36, 2471. Guglietta, A., B.J. Irons and L.H. Lazarus, 1988, Interaction between bombesin and brain prostaglandins in the control of gastric secretion, Ann. N.Y. Acad. Sci. 547, 486. Guglietta, A., R.V. Nardi and L.H. Lazarus, 1989, Indomethacin (i.c.v.) reverses the inhibitory action of peptides on gastric secretion, Eur. J. Pharmacol. 170, 87. Giillner, H-G., 1983, The interactions of prostaglandins with the sympathetic nervous systemza review, J. Auton. Nerv. Syst. 8, 1. Hayaishi, O., 1991, Molecular mechanisms of sleep-wake regulation: roles of prostaglandins D z and Ez, FASEB J. 5, 2575. Horiguchi, S., R. Ueno, M. Hyodo and O. Hayaishi, 1986, Alterations of nociception after intracisternal administration of prostaglandin D e, E2, or F2,~ to conscious mice, Eur. J. Pharmacol. 122, 173. Hucker, H.B., A.G. Zacchei, S.V. Cox, D.A. Brodie and N.H.R. Cantwell, 1966, Studies on the absorption, distribution and excretion of indomethacin in various species, J. Pharmacol. Exp. Ther. 153, 237. Katsuura, G., A. Arimura, K. Koves and P.E. Gottschall, 1990, Involvement of organum vasculosum of lamina terminalis and preoptic area in interleukin-1/3-induced ACTH release, Am. J. Physiol. 258, E163. Katsuura, G., P.E. Gottschall, R.R. Dahl and A. Arimura, 1989, Interleukin-1 beta increases prostaglandin E 2 in the rat astrocyte cultures: modulatory effect of neuropeptides, Endocrinology 124, 3125. Ligumsky, M., E.M. Golanska, D.G. Hansen and G.L. Kauffman Jr., 1983, Aspirin can inhibit gastric mucosal cyclo-oxygenase without causing lesions in rats, Gastroenterology 84, 756. Matsumura, K., Y. Watanabe, H. Onoe and O. Hayaishi, 1990, High density of prostaglandin E 2 binding sites in the anterior wall of the 3rd ventricle: a possible site of its hyperthermic action, Brain Res. 533, 147. Naito, K., H. Osama, R. Ueno, O. Hayaishi, K. Honda and S. lnoue, 1988, Suppression of sleep by prostaglandin synthesis inhibitors in unrestrained rats, Brain Res. 453, 329. Northover, B.J., 1977, Indomethacin, a calcium antagonist, Gen. Pharmacol. 8, 293.
Okada, M., N. Hiromichi, K. Takeuchi and S. Okabe, 1989, Role of prostaglandin deficiency in pathogenetic mechanism of gastric lesions induced by indomethacin in rats, Dig. Dis. Sci. 34, 694. Otorii, T., K. Ohkubo and K. Suzuki, 1985, Central noradrenergic neurons and the hypertensive effects of intracerebroventricularly administered prostaglandin E 2 in anaesthetized rabbits, Prostaglandins 29, 25. Passaro, E. Jr., H. Debas, W. Oldendorf and T. Yamada, 1982, Rapid appearance of intraventricularly administered neuropeptides in the peripheral circulation, Brain Res. 241,335. Pekary, A., R. Stephens, M. Simard, X-P. Pang, V. Smith, J.J. Distephano III and J.M. Hershman, 1990, Release of thyrotropin and prolactin by a thyrotropin-releasing hormone (TRH) precursor, TRH-Gly: conversion to TRH is sufficient for in vivo effects, Neuroendocrinology 52, 618. Puurunen, J., 1983a, Central inhibitory action of prostaglandin E 2 on gastric secretion in the rat, Eur. J. Pharmacol. 91,245. Puurunen, J., 1983b, Inhibition of gastric acid secretion by intracerebroventricularly administered prostaglandin E 2 in anaesthetized rats, Br. J. Pharmacol. 78, 131. Puurunen, J., 1983c, Central effects of prostaglandin E 2 and indomethacin on insulin-stimulated gastric acid secretion in anaesthetized rats, Acta Pharmacol. Toxicol. 52, 318. Robert, A., J.E. Nezamis, C. Lancaster and A.J. Hanchar, 1979, Cytoprotection by prostaglandins in rats. Prevention of gastric necrosis produced by alcohol, HCL, NaOH, hypertonic saline, and thermal injury, Gastroenterology 77, 433. Saperas, E., H. Yang, C. Rivier and Y. TachS, 1990, Central action of recombinant interleukinn-1 to inhibit acid secretion in rats, Gastroenterology 99, 1599. Saperas, E., H. Yang and Y. Tach6, 1991, Interleukin-13 acts in specific hypothalamic nuclei to inhibit gastric acid secretion through prostaglandin pathways in the rat, Gastroenterology 100, A664.
Sautereau, D. and C. Roz6, 1988, Central effects of prostaglandins on the digestive tract, Gastroenterol. Clin. Biol. 12, 222. Sirko, S., I. Bishai and F. Coceani, 1989, Prostaglandin formation in the hypothalamus in vivo: effect of pyrogens, Am. J. Physiol. 256, R616. Staumont, G., J. Fioramonti, J. Frexinos and L. Bu~no, 1990, Oral prostaglandin E analogues induce intestinal migration motor complexes after a meal in dogs. Evidence for a central mechanism, Gastroenterology 98, 888. Tach6, Y., 1987, Central regulation of gastric acid secretion, in: Physiology of the Gastrointestinal Tract, Vol. 2, eds. L.R. Johnson, J. Christensen, M. Jackson, E.D. Jacobson and J.H. Walsh (Raven Press, New York) p. 911. TachS, Y., Y. Goto, D. Hamel, A. Pekary and D. Novin, 1985, Mechanisms underlying intracisternal TRH-induced stimulation of gastric acid secretion in rats, Reg. Pept. 13, 21. Tannenbaum, G.S. and Y.C. Patel, 1986, On the fate of centrally administered somatostatin in the rat: massive hypersomatostatinemia resulting from leakage into the peripheral circulation has effects on growth hormone secretion and glucoregulation. Endocrinology 118, 2137. Whittle, B.J.R., G.A. Higgs, K.E. Eakins, A. Moncada and J.R. Vance, 1980, Selective inhibition of prostaglandin production in inflammatory exudates and gastric mucosa, Nature 284, 271. Yang, H., H. Wong, G. Rosenquist, J.H. Walsh and Y. TachS, 1990, Somatostatin monoclonal antibody immunoneutralization increases gastrin and gastric acid secretion in urethane-anesthetized rats, Gastroenterology 99, 659. Yang, H., H. Wong, J.H. Walsh and Y. Tach6, 1989, Effect of gastrin monoclonal antibody 28.2 on acid response to chemical vagal stimulation in rats, Life Sci. 45, 2413.