Changes of CAMP and cGMP Levels of Rat Antral and Fundic Gastric Mucosa in Different Ulcer Models GABOR A. BALINT First
AND VINCE VAR& School,
Box 469, H-6701
As prostacyclin (PG-13, one of the most active cytoprotective (antiulcerogenic) drugs, has a significant effect on the gastric mucosal intracellular second messenger system, i.e., on cyclic nucleotides (CAMP, cGMP) of the antral and fundic gastric mucosa, (1,2) the question is raised, what is the effect of the noxious agents on the CAMP and cGMP levels of gastric mucosa in the course of ulceration? To investigate the problem, the following experiments were done. EXPERIMENTAL
Indomethacin (IND) and stress (STR)-restraint-induced-gastric ulcer models were investigated. (a) ZND ulcer. The animals received 30 mg/kg IND suspension intraperitoneally at the beginning of the experimental period (0 min). The animals were killed and their stomachs removed 60, 120, and 240 min following IND treatment. (b) STR ulcer. The animals were immobilized (restraint) lying on their backs. The animals were killed and their stomachs removed after 8, 16, and 24 hr of immobilization. Stomachs were opened in both experimental series and the changes were evaluated using an ulcer index (UI) as follows (3,4): Every mm2 of lesion Bleeding Perforation
+ 1 point +5 points + 10 points
Seven groups of female Wistar rats (n = lo/group), weighing 200-230 g were used. Prior to the experiment the animals were fasted for 24 hr but allowed water ad libitum. The CAMP and cGMP contents of the gastric antral and fundic mucosa were determined at the lst, 2nd and 4th hours after IND application; in the case of the STR ulcer, CAMP and cGMP were determined at the gth, 16th, and 24th 121 0885-4505187
Copyright 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved.
TABLE 1 Degree of Ulceration in Different Experimental Gastric Ulcer Models of Rat UI Group Control Fundus (oxyntic cell area) IND 60 min 120 min 240 min STR 8 hr 16 hr 24 hr Antrum
X ? SEM
2.9 -c 0.8 6.7 2 1.9 14.4 f 2.7 0.6 6.1 10.2 No
20.1 46.5 100.0
+ 0.2 5.9 k 2.0 59.8 f 2.4 100.0 erosion or ulceration was detected
hours of restraint. (The usual duration of the two experimental gastric ulcer models is 4 and 24 hr, respectively.) Appropriate control groups were included. At the end of the experimental period the animals were killed, and the removed stomachs were opened. The fundic (oxyntic cell area) and antral mucosa was scraped down and homogenized in ice-cold Tris-EDTA buffer, pH 7.5. Emphasis was put on rapid processing and the whole procedure was carried out in an ice bath. The CAMP and cGMP contents of the homogenizate have been determined by radioimmunoassay, according to the manufacturer’s instructions, using TRK432 (Lot 47B) and TRKJOO (Lot 17A) kits of Amersham International (Amersham, U.K.), respectively. The reproducibility and intraassay variation for CAMP and cGMP measurements showed a coefficient of variation less than 12% over the range 0.2-16 pmole/50 ~1 sample of CAMP, and 100 ~1 of cGMP, respectively. These data are in accordance with the manufacturer’s data. The cyclic nucleotide content was expressed as picomoles per milligram of protein. The protein content of the samples has been estimated using Lowry’s method (5). Within each animal group, means & SEM were calculated and statistical evaluation was performed by analysis of variance. Significant differences were assumed when the probability was less than 5%. RESULTS AND DISCUSSION The experimental results are presented in Tables 1 and 2. Under the same conditions and with identical methods, the Fossible late effects (60 and 120 min after application) of 100 pug/kg orally administered (by gastric tube) PG-I2 on cyclic nucleotides of rat gastric mucosa were investigated. The results are presented in Table 3.
TABLE 2 Changes of Cyclic Nucleotide Levels of Rat Gastric Mucosa in Different Experimental Ulcer Models Group Antrum Control IND 60 min 120 min 240 min STR 8 hr 16 hr 24 hr
9.50 2 0.71
pmole/mg protein 1.62 f 0.12
1.39 r 0.72*** 3.90 + 1.14* 15.24 t 3.06*
0.81 -c 0.10** 0.93 2 0.21* 1.26 k 0.32
1.7 4.2 12.1
17.01 2 2.23** 0.53 k 0.14*** 6.24 k 2.13
0.54 5 0.05*** 0.46 ? 0.04*** 1.14 k 0.32
31.5 1.2 5.5
Fundus (oxyntic cell area) Control 5.61 2 0.41 IND 60 min 1.94 2 0.23*** 120 min 3.92 k 0.44* 240 min 3.18 2 0.32***,” STR 8 hr 5.10 2 0.39” 16 hr 4.08 2 0.51*,“” 24 hr 6.63 2 1.24
0.72 2 0.11
0.51 r 0.11 0.53 f 0.12” 0.46 ” 0.08*-
3.8 7.4 6.9
0.21 k 0.04***.” 0.54 t 0.10 0.42 k 0.09”
24.3 7.6 15.8
Note. Data are expressed as X + SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, vs control. a P < 0.05, OrnP < 0.001, vs antrum.
According to the literature, PG-I, is a potent stimulator of adenylate cyclase, resulting in enhanced CAMP levels in different tissues (6,7). PG-I* activates adenylate cyclase in human fundic mucosa at concentrations greater than 1 PM. PG-I*, after 120 min of administration, shows a significant CAMP-elevating effect in both parts (antrum and fundus) of the gastric mucosa. This is in complete TABLE 3 The Late Effect of Prostacyclin on Cyclic Nucleotide Content of Rat Gastric Mucosa Group
pmole/mg protein Antrum Control 5 PG-I, 60 min 5 PG-I, 120 min 5 Fundus (oxyntic cell area) Control 5 PG-I, 60 min 5 PCS, 120 min 5
8.86 ? 0.58 4.11 2 1.02* 13.56 +- 3.39*
1.64 -+ 0.34 1.98 ? 0.35 1.69 2 0.35
5.4 2.1 8.0
5.23 ‘- 0.40 4.76 2 0.97 8.36 + 1.73*
0.72 2 0.15 0.35 f 0.07* 0.38 f 0.09*
7.3 13.6 21.8
Note. Data are expressed as X + SEM. * = P < 0.05 vs control.
agreement with data in the literature but at the same time it calls attention to our previous results (1,2), i.e., during the initial periods PC-I2 significantly decreases the CAMP content, up to the 60th min. This CAMP-decreasing effect of PG-I2 is not a contradiction of the present data, because the dose applied (100 pg/kg, po) is less than 0.3 FM, moreover, our (previous) experimental period was only 60 min. In the IND ulcer model both components of the intracellular second messenger system significantly decrease, together with the cAMP/cGMP ratio. At the end of the experimental period (240 min) the antral CAMP level shows a marked elevation, causing a sharp rise in the cAMP/cGMP ratio. The fundic values remain low. In STR-induced gastric ulceration the antral CAMP level is significantly elevated, at the same time cGMP decreases dramatically; this is the cause of the very high cAMP/cGMP ratio. At the end of the experimental period (24 hr) all values were within the normal range. In the fundic mucosa the CAMP level remains near the normal level but the cGMP content is always below the normal value; therefore, at the beginning of our experimental period (8th hour), as at the end of it (24th hour), the cAMP/cGMP ratio is very high. It is worth mentioning that the control values determined at different times showed no changes; moreover, additional 24-hr fasting also has no effect on UI, CAMP, and cGMP (unpublished observations). PG-IZ elevates the cAMP/cGMP ratio. This elevation was especially marked in the fundic area. As PG-I, is one of the most potent antiulcerogenic (cytoprotective) drugs, we conclude that a rise in the cAMP/cGMP ratio is one of the indicators of the antiulcerogenic (reparative? metabolic?) processes. SUMMARY
In both ulcer models investigated, except the 240-min IND and 8-hr STR CAMP values in the antrum, the cyclic nucleotides showed a significant decrease in the gastric mucosa, which is most probably a sign of cellular exhaustion. REFERENCES 1. 2. 3. 4. 5.
Bdint, Balint, Ntir&di, Balint, Lowry,
G. A., and Varr6, V., Agents Actions 17, 46 (1985). G. A., and Varr6, V., Agenls Actions 18, 329 (1986). J., Balint, G. A., C&i, S., and Varr6, V., Agents Acfions 15, 449 (1984). G. A., Karksony, Gizella, and Varr6, V., Agents Actions 16, 404 (1985). 0. H., Rdsenbrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem.
6. Gorman, R. R., Bunting, S., and Miller, 0. V., Prostaglandins 13, ‘177 (1977). 7. SolI, A. H., and Whittle, B. J. R., Prostaglundins 21, 353 (1981).