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Different susceptibility of stress-induced gastric ulcer and the autonomic nervous function in the hereditary hypertensive rats
Journal of the Autonomic Nervous System, 46 (1994) 229-236
229
© 1994 Elsevier Science B.V. All rights reserved 0165-1838/94/$07.00 JANS 01473
Different susceptibility of stress-induced gastric ulcer and the autonomic nervous function in the hereditary hypertensive rats Masahiro Ito
a,,, Kazuko Shichijo a Ichiro Sekine a and Masayori Ozaki b
a Department of Pathology, Atomic Disease Institute, and b Second Department of Pharmacology, Nagasaki University School of Medicine, Nagasaki, Japan (Received 19 April 1993) (Revision received 18 June 1993) (Accepted 21 June 1993)
Key words: Stress-induced ulcer; Spontaneously hypertensive rat; Autonomic nervous system Abstract Susceptibility of stress-induced ulcer by restraint water immersion (RWI) was examined in Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR) and stroke-prone spontaneously hypertensive rats (SHRSP). Ulcer formation was slight in SHR, and very slight in SHRSP. The ulcer index and blood pressure showed a significant inverse correlation (P < 0.001). Acid secretion was lowest in SHRSP, and hypergastrinemia was present in SHRSP. Gastric motility was suppressed during RWI in SHR and SHRSP. The noradrenaline content of the gastric mucosa and muscle layer was significantly greater in these hypertensive strains, and histologically noradrenergic innervation in the mucosa was also denser in SHRSP and SHR. Choline acetyltransferase activity in the stomach was significantly lower in SHR and SHRSP than WKY (P < 0.01). These findings suggest that susceptibility of stress-induced ulcer is inversely correlated with systemic blood pressure and that the alteration of autonomic nervous function contributes to inhibition of stress ulcers by suppressing acid secretion and motility in the hereditary hypertensive rats.
Introduction
Little is known about of the relation between hypertension and gastric ulcer or gastritis. Massarrat et al. [18] found that hypertension was associated with a lower incidence of antral gastritis. Mizuno et al. also reported that the incidence of gastric ulcer is low in patients with untreated hypertension [22]. On the other hand, Sonnenberg reported that gastric ulcers coincided more
* Corresponding author. Department of Pathology, Atomic Disease Institute, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki 852, Japan
SSDI 0 1 6 5 - 1 8 3 8 ( 9 3 ) E 0 0 9 0 - R
frequently with ischemic heart disease related to hypertension [35]. The spontaneously hypertensive rat (SHR) [25] is widely used as a model of essential hypertension; they develop hyperfunctioning of the sympathetic nervous system from the prehypertensive state without any treatment [2,16]. Studies have reported the susceptibility to stress-induced gastric ulcers in SHR, but the results have been conflicting, indicating both a higher [9] and lower [1,28] incidence of stress-induced lesions as compared to controls. We reported previously that stress-induced ulcer formation is significantly milder in SHR than in normotensive Wistar Kyoto rats (WKY), which supported the hypothesis
230 of low susceptibility to stress in gastric lesions [32,33]. In this study, we evaluated the stress-induced ulcers in stroke-prone spontaneously hypertensive rats (SHRSP) [26] and discussed the relationship between hereditary hypertension and stress ulcer susceptibility. The role of the autonomic nervous system in the pathogenesis of stress-induced ulcer was also investigated.
Materials and Methods
The male SHRSP used in this study were raised at the Second Department of Pharmacology, Nagasaki University School of Medicine. SHR and WKY were obtained from Charles River Japan (Atsugi, Japan). The animals were kept in an air-conditioned room (25 _+ 2, 50-60% humidity) illuminated 12 h a day (7:00am-7:00pm) at the Laboratory Animal Center of Biomedical Research, Nagasaki University School of Medicine. They were fed a conventional diet (F2, Funabashi Farm, Chiba, Japan) and water ad libitum until the experiment. Thirty-six-week-old male rats without any symptoms of apoplexy were mainly used in this study. The systolic blood pressure was measured by an indirect tail-cuff method (automatic blood pressure recorder USM-105-R type, Ueda Manufactory, Tokyo) at resting state. The animals (n = 23) were subjected to restraint water immersion (RWI), that is placed in a restraint cage and vertically immersed up to the xyphoid process in water at 23°C after fasting for 17 h as described previously [36]. After immersion for 5 h, the rats were killed and their stomachs fixed in a 4% neutral-buffered paraformaldehyde fixative for 24 h. Gastric mucosal lesions were recognized as linear breaks at the mucosal surface of the glandular stomach, and the extent of the lesions (ulcer index) was expressed as the sum of the area of these mucosal breaks. Rectal temperatures was monitored with a tubular thermometer probe (thermal sensor MGA-219A, Shibaura Electronics Co., Tokyo) and thermometer (MGAI-219S, Shibaura Electronics Co., Tokyo) inserted 3 cm rectally every hour during stress.
Gastric acid output was measured with pyloric ligation preparations in control rats (n = 23) after a 24 h fast with free intake of drinking water, and the pyrolus was then ligated under ether anesthesia. The stomach was resected 6 hr after pyloric ligation, and gastric juice was collected. Gastric output during RWI was not evaluated because of contamination of intragastric bleeding. The pH values and volume of secreted gastric acid were measured using an autonomic pH titrator (PHM852 Standard pH Meter, Copenhagen). Serum gastrin values were also measured by radioimmunoassay (Dinabots Co., Tokyo) in animals subjected to an overnight fast. Gastric motility during RWI was evaluated by the strain-gauge force transducer (n = 18). The strain gauge (Star Medical Co., Tokyo) was sutured on the lower portion of the gastric body parallel to the circular muscle under ether anesthesia 30 min before RWI. For quantitative analysis of the motility, the motility index (MI) was calculated from the frequency and magnitude of contracted waves over a 10-rain period. The formula is as follows; M I = H x F ( g m x m i n 1), H = Z h i / n (gm), F: w a v e s / m i n (min i) The noradrenaline and dopamine contents in gastric tissue from non-fasting control rats (n = 24), between 28 and 36 weeks, were measured by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The gastric tissues were immediately resected after the rats were killed and were then frozen with liquid N2, and subsequently divided into mucosal and muscle layers in the body and pyrolic areas, respectively. Choline acetyltransferase (CAT) activity of the fundic wall was determined by the method of Kaneda et al. [14], with minor modifications (n = 30). By this method, the specific CAT activity is obtained by measuring the concentration of acetylcholine formed from choline. The enzyme solution was prepared from each gastric tissue by homogenization in 12.5 ml of 25 mM solution phosphate buffer (pH 7.4) containing 20nM ethylhomocholine per g wet weight, followed by centrifugation at 20,000 x g for 60 min at 4°C. The incubation mixture consisted of the following components: 100 ~zl of substrate solution chlo-
231 ride, and 20 m M E D T A - 2 N a in 0.1 M sodium phosphate buffer (pH 7.4) and 100 /zl of the enzyme solution. Incubation was performed at 37°C for 20 min, and the reaction was stopped with 5 0 / z l of 1M perchloric acid in an ice-bath. Ten min later, the reaction mixture was centrifuged at 1600 × g for 10 min at 4°C. Twenty-five /~1 of the supernatant liquid was injected into the H P L C system. For the control experiments, the enzyme solution was boiled at 95°C for 5 min. The gastric mucosa was observed by light microscopy after fixation with 10% neutrized formaldehyde and paraffin-embedding. Noradrenergic nerve fibers innervating the mucosa were demonstrated using glyoxalic acid fluorescence histochemistry as described previously [14].
BP (mmHg) 300
Results The systolic blood pressure was 245 + 16, 183 + 8 and 1 2 5 + 1 0 m m H g ( P < 0 . 0 0 1 vs. each other) in SHRSP, S H R and WKY, respectively, at the age of 36 weeks (Fig. 1). In W K Y prominent ulcerations developed in the gastric mucosa after cold-restraint stress. In S H R the extent of stress ulcerations was significantly reduced, while they were virtually absent in the S H R S P (Fig. 2). The ulcer index was 0 . 9 + 0 . 6 5 , 9 . 2 + 3 . 6 and 3 8 . 1 + 1 3 . 6 m m 2 in SHRSP, S H R and WKY, respectively. The correlation coefficient between blood pressure and ulcer index was - 0 . 8 9 0 ( P < 0.001, n = 23) (Fig. 3), suggesting the existence of a significant reverse correlation between blood pressure and gastric ulcers. The rectal t e m p e r a t u r e after 5 h of water immersion was 24.9 + 0.3, 24.2 + 0.2 and 25.4 + 0.2°C in SHRSP, S H R and WKY, respectively. The average p H value in gastric juice was significantly increased, and acid secretion was significantly reduced in SHRSP, compared with S H R and W K Y (Table I). The serum gastrin
SHRSP SHR W K
Y
~
200
100
Statistics Results are expressed as mean +_ SD or SE. Statistical significance was assessed using the A N O V A test. P < 0.05 was considered statistically significant.
• ca ~
, ' , ' , ' , ' , ' , ' , ' , ' , ' , " , ' , ' , " , ' , ' , ' 6 8 10 12 14 16 18 20 22 24 2 6 28 30 32 3 4 36
age (weeks) Fig. 1. Systolic blood pressure in WKY, SHR and SHRSP. SHR and SHRSP showed progressive hypertension since 6 weeks of age. In SHRSP the systolic pressure exceeded over 200 mmHg from young age.
levels in S H R S P and S H R were significantly higher than that in W K Y (Table II). These findings indicate the poor functioning of acid secretion in the hypertensive strains, especially in SHRSP. Histologically, all of the gastric lesions were UI-1 ulcers, but the lesions in W K Y reached the deep layer of the propria mucosa while
Fig. 2. Gastric mucosal lesion from WKY, SHR and SHRSP after water immersion stress. In SHR the extent of stress ulcerations was significantlyreduced, while they were virtually absent in SHRSP.
232
UI
TABLE l
mm 2
Acid secretion
80
60
o
WKY
=
SHR
•
SHRSP
o
40 "
20
o ~
Y=-0.321X+74.3 r=-0.891n=23
o e~°
(sig. p<0.001 )
o
e
WKY SHR SHRSP
(n=9) (n=6) (n=8)
Volume ( m l / 6 h)
pH
acid secretion (/,Eq/h)
6.5_+2.3 4.5_+1.2" 3.9_+1.8"
1.5±0.1 1.5±0.1 2.5±0.8"#
95.2_+43.5 53.6_+ 5 . 6 " 30.8_+17.4 *#
Mean_+ SD, * P < 0.05 vs. WKY, # P < 0.05 vs. SHR.
o
TABLE II
Serum gastrin
100
150
200
250
mmHg BP Fig. 3. The relation between systolic blood pressure and ulcer index. A significant reverse correlation exists between blood pressure and ulcer indices.
SHRSP showed a very shallow and confined erosion. Gastric motility was markedly enhanced by RWI around after 3 h of stress in WKY, although
WKY SHR SHRSP
(n = 8) (n=6) (n = 8)
262_+ 65 463+ 8 3 " * 505 _+236 * *
Mean _+SD (pg/ml), * * P < 0.05 vs, WKY.
the rhythmic contractions with low amplitudes were observed in all strains of rats at the beginning of RWI. By contrast SHR and SHRSP sustained the rhythmic and relatively low amplitude contractions (Fig. 4). Mls at the fifth hour of
WKY
SHR
SHRSP
~[ l O g i
i
4h 5t3 Fig. 4. Gastric motility monitored with force transducer after about 5 h of RWl. Remarkably enhanced contraction waves appeared in WKY, while lower amplitude waves occurred in SHR and SHRSP.
233 R W I were significantly smaller in S H R and SHRSP than W K Y ( P < 0.05) (Table liD. The noradrenaline content of gastric tissue was significantly higher in SHRSP and S H R than in W K Y in the mucosal and muscular layers of the pylorus and body (Table IV). However, there was no significant difference between SHRSP and SHR. Dopamine content in the gastric mucosa and proper muscle layer was also significantly higher in SHRSP and S H R than in WKY. C A T activity was significantly lower in the corpus in S H R and SHRSP than WKY ( P < 0.01) (Table V). SHRSP showed a tendency to have lower C A T activity than SHR. Fluorescence noradrenergic fibers were most densely innervated in the fundic mucosa in S H R S P (Fig. 5). These findings indicated that the sympathetic innervation is well
TABLE III Motility index during RWI at 5 h
WKY SHR SHRSP
(n = 6) (n=6) (n = 6)
32.65:11.6 11.65: 3.0"* 12.9 + 4.8 **
Mean + SD (gm/min), * * P < 0.05 vs. WKY. developed in the gastric tissues and functionally suppresses cholinergic activity in the hypertensive strains.
Discussion The pathogenesis of essential hypertension is yet to be clarified, although factors such as ab-
Fig. 5. Catecholamine fluorescence of the gastric mucosa in WKY (a) and SHRSP (b). Fluorescence sympathetic fibers were more densely distributed in the mucosa of SHRSP (b) than in that of WKY (a).
234 T A B L E IV
Noradrenaline and dopamine content of the gastric tissues Tissue
Antrum
(ng/g)
mucosa
muscle
mucosa
muscle
231 382 304
157 221 157
278 425 383
270 392 411
NE
WKY SHR SHRSP
DA
WKY SHR SHRSP
+36 _+84" +93 *
9.3_+ 5.0 18.9_+ 6.5 * 19.7_+ 8.9 *
Body
_+58 +36 _+42
1.8 + 0.2 9 . 2 + 7.7 5.3+_ 2.4 *
+ 69 + 60" _+114 *
7.9_+ 22.5_+ 25.6_+
5.3 6.9 * 15.6 *
_+50 _+91 * -+59 *
4.9_+ 2.2 11.4-+ 6.7 11.5+ 4.8 *
Mean _+ SD, * P < 0.05 vs. WKY. NE, noradrenaline; DA, dopamine; WKY (n = 6); S H R (n = 6); SHRSP (n = 12).
normalities in the central nervous system, calcium metabolism, renin-angiotensin system and adrenoceptors have been postulated. Sympathetic hyperfunction is one of the pathogenetic factors in the onset and maintenance of hypertension in SHR and SHRSP [2,16]. The role of sympathetic hyperactivity in stress ulcer formation is not as clear as that of parasympathetic hyperactivity. It may facilitate causative factors [4,6,31] such as mucosal vasoconstriction and protective factors [24,27], such as inhibition of gastric secretion [10,23] and maintenance of blood flow [5,29]. We reported previously that the gastric lesions induced in SHR were aggravated by treatment with 6-hydroxydopamine, an agent for chemical sympathectomy, following decreases in norepinephrine and dopamine contents [33]. As in SHR, 6-hydroxydopamine treatment deteriorated the stress-induced ulcers in SHRSP mainly due to increase of gastric acidity [34]. In this study, the correlation coefficient between ulcer index and blood pressure was -0.890 (P < 0.001), suggesting that susceptibility of stress ulcer is inversely
TABLE V
Choline acetyltran~ferase activity of the fundic tissues in the hypertensive rats WKY SHR SHRSP
5.40 + 0.24 4.57 + 0.35 * 4.30+ 0.24 *
Mean + SE ( n m o l / m i n per g), * P < 0.01 vs. WKY. WKY (n = 12); SHR (n = 6); SHRSP (n = 12).
correlated with blood pressure. This finding is consistent with previous clinical reports on ulcer susceptibility in patients with hypotension [11,21] and hypertension [18,22]. We introduced the hereditarily hypertensive rats as a model of the hyperfunctioning sympathetic nervous system in view of autonomic nervous abnormality. The high concentration of catecholamine in the gastric mucosa of SHR and SHRSP supports this hypothesis. Morphologically, we demonstrated the dense noradrenergic fibers in the gastric mucosa of SHRSP by the fluorescence technique. However, SHRSP did not show the significant differences in noradrenaline and dopamine content in the gastric tissues to SHR. One of characteristic differences between these strains is an adenal function. The catecholamine content of the adrenal gland is about twofold higher in adult SHRSP than in SHR, and this may be involved in the marked hypertension in SHRSP [17] and possibly exerts an additional influence upon gastric sympathetic nervous system humorally. There exist adrenoceptors in the mucosa and muscle of the stomach that are innervated by sympathetic nerves. Activation of 13-adrenoceptor has been shown to inhibit acid secretion [5] and motility [8]. The concept that the sympathetic nervous system suppresses acetylcholine release to the parietal cells presynaptically is generally accepted. Acid secretion is regulated by various mediators but the presence of adrenoceptors on the parietal cells is a subject of controversy. We reported previously that the number of parietal
235 cells was not reduced in the hypertensive strains but mitochondrial degeneration progressed significantly in the parietal ceils of aged SHRSP, indicating that acid secretion was involved organically as well as functionally in lasting sympathetic facilitation [13]. Hypergastrinemia and G-cell hyperplasia occur in the states of vagotomy, long administration of a proton p u m p inhibitor [37], partial corpectomy [19] and aging. In 36-week-old SHRSP, hypergastrinemia and G-cell hyperplasia might be induced as a result of a feedback mechanism against persistently poor acid secretion [13]. Lowest acidity might greatly contribute to less susceptibility to R W I in S H R S P than in SHR. In view of parasympathetic function, the low C A T activity indicates inhibition of parasympathetic nervous system in the hypertensive strains. By R W I acetylcholine content decreased in W K Y but not in SHR, indicating that acetylcholine release was significantly suppressed in S H R during RWI. Although the noradrenaline content decreased in both S H R and W K Y as a result of RWI, it remained still higher in S H R than in W K Y [33]. Gastric motility has been suggested to be an important factor in the induction of several kinds of experimental ulcer [20], but the influence of gastric hypercontraction on mucosal blood flow is controversial [3,7]. We recently reported that enhancement of gastric contraction and the degree of drop in gastric blood flow during hypothermia was significantly smaller in S H R than in W K Y [12]. Inhibition of gastric motility is considered to be mediated mainly via the '~z adrenoceptor presynaptically, thereby suppressing acetylcholine release to smooth muscles. In S H R and SHRSP, noradrenergic hyperinnervation in the muscle layer might play an important role in the inhibition of motility during RWI. To investigate the relation between genetic inheritance and sympathetic function, we previously used the borderline hypertensive rats (BHR), a cross between S H R and W K Y in the stress experiment [30]. As the result, B H R showed intemediate values of blood pressure, ulcer indices, tissue catecholamine and gastric acidity, suggesting that ulcer susceptibility conforms to a polygenal regulation.
In conclusion, the S H R S P exhibiting marked hypertension was more resistant to stress in gastric ulcer formation than the other normotensive and hypertensive strains, suggesting that blood pressure is inversely correlated with stress-induced ulcer susceptibility. Alterative autonomic nervous function, namely sympathetic facilitation in association with low cholinergic activity, might play an important role in inhibiting gastric ulcer formation by suppressing acid secretion and gastric motility in the hereditary hypertensive rats.
Acknowledgments We would like to express sincere thanks to Dr. D. Murosec for editorial assistance and to Mr. T. Kawada for preparing the photographs.
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236 9 Goldenberg, M.M., Stress-induced gastric lesions in spontaneously hypertensive rats, Life Sci., 19 (1974) 353-360. 10 Grund, E.R., Ree, J.D. and Sanders, D.J., The effect of sympathetic nerve stimulation on acid secretion, regional blood flow and oxygen usage by stomachs of anesthetized cats, J. Physiol., 248 (1975) 639-647. 11 Hartman, H.R. and Brown, G.E., The systolic blood pressure in duodenal and gastric ulcer, Arch. Intern. Med., 44 (1929) 843-855. 12 Ito, M., Shichijo, K. and Sekine, I., Gastric motility and blood flow in spontaneously hypertensive rat (SHR), Gastroenterology, 100 (1991) A90. 13 Ito, M., Shichijo K., Fujimoto, C., Onizuka, S., Sekine, I., Taniyama, K. and Imamura, K., Persistent sympathetic facilitation and parietal cell activation in stroke-prone spontaneously hypertensive rats(SHRSP), Auton. Nerv. Syst., 29 (1992) 276-282. (in Japanese) 14 Kaneda, N., and Nagatu, T., Highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection. J. Chromatogr, 341(1985) 23-30. 15 Kawase, Y., Shikuwa, S., Shichijo, K., Ito, M. and Sekine, I., Adrenergic innervation of the mesenteric arteries in Wistar-Kyoto rats, spontaneously hypertensive rats and rats treated with monosodium glutamate, Auton. Nerv. Syst., 26 (1989) 463-468. 16 Limas, C. and Limas, C.J., Decreased number of betaadrenergic receptors in hypertensive vessels, Biochim. Biophys. Acta, 582 (1979) 533-539. 17 Maemura, S., Niwa, M. and Ozaki, M., Characteristic alterations in adrenal catecholamine contents in SHR, SHRSP, and WKY during development of hypertension and stroke, Jpn. Heart J., 23 (1982) 593-601. 18 Massarrat, S., Paidlik, A., Pittner, P., Schmitz-Moormann, P. and Wurbs, M., The role of certain habits and various diseases in the occurrence of gastritis, Hepatogastroenterology, 30 (1983) 249-253. 19 Mattsson, H., Havu, N., Brautigam, J., Carlsson, K., Lundell, L. and Carlsson, E., Partial gastric corpectomy results in hypergastrinemia and development of gastric enterochromaffinlike-cell carcinoids in the rat, Gastroenterology, 100 (1991) 311-319. 20 Mersereau, W.A. and Hinchey, E.J., Hypothermia-induced gastric hypercontractility in the genesis of the restraint ulcer, Can. J. Surg., 24 (1981) 622-625. 21 Mizuno, T., Easy ulcerative tendency of the stomach in hypotensive patients, Yokohama Med. Bull., 38 (1987) 87-97. 22 Mizuno, T. and Shindo, K., Acute gastric ulceration due to excessive reduction of blood pressure in hypertensive patients, Jpn. J. Med., 28 (1989) 269-271.
23 Moraes, M.F., Nyhus, L.M., Kalananis, N.G., Bombeck, C.T. and Gupta, T.K.D., Role of the sympathetic nervous system in peptic ulcer production in rats, Surgery, 83 (1978) 194-199. 24 Nobrega, J.N. and Wiener, N.I., Effects of catecholamine agonist and antagonist drugs on acute stomach ulceration induced by medial hypothalamic lesions in rats, Pharmacol. Biochem. Behav., 19 (1983) 831-838. 25 Okamoto, K. and Aoki, K., Development of a strain of spontaneously hypertensive rats, Jpn. Circ. J., 27 (1963) 282-293. 26 Okamoto, K., Yamori, Y. and Nagaoka, A., Establishment of the stroke-prone spontaneously hypertensive rat (SHR), Circ. Res. 34 and 35 (Suppl. I) (1974) 143-153. 27 Orlando, R.C., Hernandez, D.E., Prange, A.J. and Nemeroff, C.B., Role of the autonomic nervous system in the cytoprotective effect of neurotensin against gastric stress ulcers in rats, Psychoneuroendocrinology, 10 (1985) 149157. 28 Pare, W.P. and Schimmel, G.T., Stress ulcer in normotensive and spontaneously hypertensive rats, Physiol. Behav., 36 (1986) 699-705. 29 Reed, J.D., Sanders, D.J. and Thorpe, V., The effect of splanchnic nerve stimulation and mucosal blood flow in the anesthetized cat, J. Physiol., 214 (1971) 1-13. 30 Sekine, i., Ito, M., Shichijo, K., Kawase, Y., Shukuwa, S., Hsu, C.T. and Imamura, K., Pathophysiological study of stress ulcer using borderline hypertensive rat, Ther. Res., 11 (1990) 1104-1107. 31 Sethbhadki, S., Roth, LEA. and Pfeiffer, C.H., Gastric mucosal ulceration after epinephrine. A study of the etiologic mechanisms, Am. J. Dig. Dis., 15 (1970) 1055-1065. 32 Shichijo, K., Sekine, I., Nishimori, I. and Ozaki, M., Experimental stress ulcer and gastric catecholamine contents in spontaneously hypertensive rats, Gastroenterol. Jap., 21 (1986) 567-572. 33 Shichijo, K., Ito, M. and Sekine, I., The mechanism of low susceptibility to stress in gastric lesions of spontaneously hypertensive rats, Life Sci., 49 (1991) 2023-2029. 34 Shichijo, K., Ito, M., Taniyama, K. and Sekine I., The role of sympathetic neurons for low susceptibility to stress in gastric lesions, Gastroenterology, 104 (1993) A190(abst). 35 Sonnenberg, A., Concordant occurrence of gastric and hypertensive disease, Gastroenterology, 95 (1988) 42-48. 36 Takagi, K. and Okabe, S., The effects of the production and recovery processes of the stress ulcer, Jap. J. Pharmacol., 18 (1968) 9-18. 37 Tielemans, Y., Proliferation of enterochromaffinlike cells in omeprazole-treated hypergastrinemic rats, Gastroenterology, 96 (1989) 723-729.
229
© 1994 Elsevier Science B.V. All rights reserved 0165-1838/94/$07.00 JANS 01473
Different susceptibility of stress-induced gastric ulcer and the autonomic nervous function in the hereditary hypertensive rats Masahiro Ito
a,,, Kazuko Shichijo a Ichiro Sekine a and Masayori Ozaki b
a Department of Pathology, Atomic Disease Institute, and b Second Department of Pharmacology, Nagasaki University School of Medicine, Nagasaki, Japan (Received 19 April 1993) (Revision received 18 June 1993) (Accepted 21 June 1993)
Key words: Stress-induced ulcer; Spontaneously hypertensive rat; Autonomic nervous system Abstract Susceptibility of stress-induced ulcer by restraint water immersion (RWI) was examined in Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR) and stroke-prone spontaneously hypertensive rats (SHRSP). Ulcer formation was slight in SHR, and very slight in SHRSP. The ulcer index and blood pressure showed a significant inverse correlation (P < 0.001). Acid secretion was lowest in SHRSP, and hypergastrinemia was present in SHRSP. Gastric motility was suppressed during RWI in SHR and SHRSP. The noradrenaline content of the gastric mucosa and muscle layer was significantly greater in these hypertensive strains, and histologically noradrenergic innervation in the mucosa was also denser in SHRSP and SHR. Choline acetyltransferase activity in the stomach was significantly lower in SHR and SHRSP than WKY (P < 0.01). These findings suggest that susceptibility of stress-induced ulcer is inversely correlated with systemic blood pressure and that the alteration of autonomic nervous function contributes to inhibition of stress ulcers by suppressing acid secretion and motility in the hereditary hypertensive rats.
Introduction
Little is known about of the relation between hypertension and gastric ulcer or gastritis. Massarrat et al. [18] found that hypertension was associated with a lower incidence of antral gastritis. Mizuno et al. also reported that the incidence of gastric ulcer is low in patients with untreated hypertension [22]. On the other hand, Sonnenberg reported that gastric ulcers coincided more
* Corresponding author. Department of Pathology, Atomic Disease Institute, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki 852, Japan
SSDI 0 1 6 5 - 1 8 3 8 ( 9 3 ) E 0 0 9 0 - R
frequently with ischemic heart disease related to hypertension [35]. The spontaneously hypertensive rat (SHR) [25] is widely used as a model of essential hypertension; they develop hyperfunctioning of the sympathetic nervous system from the prehypertensive state without any treatment [2,16]. Studies have reported the susceptibility to stress-induced gastric ulcers in SHR, but the results have been conflicting, indicating both a higher [9] and lower [1,28] incidence of stress-induced lesions as compared to controls. We reported previously that stress-induced ulcer formation is significantly milder in SHR than in normotensive Wistar Kyoto rats (WKY), which supported the hypothesis
230 of low susceptibility to stress in gastric lesions [32,33]. In this study, we evaluated the stress-induced ulcers in stroke-prone spontaneously hypertensive rats (SHRSP) [26] and discussed the relationship between hereditary hypertension and stress ulcer susceptibility. The role of the autonomic nervous system in the pathogenesis of stress-induced ulcer was also investigated.
Materials and Methods
The male SHRSP used in this study were raised at the Second Department of Pharmacology, Nagasaki University School of Medicine. SHR and WKY were obtained from Charles River Japan (Atsugi, Japan). The animals were kept in an air-conditioned room (25 _+ 2, 50-60% humidity) illuminated 12 h a day (7:00am-7:00pm) at the Laboratory Animal Center of Biomedical Research, Nagasaki University School of Medicine. They were fed a conventional diet (F2, Funabashi Farm, Chiba, Japan) and water ad libitum until the experiment. Thirty-six-week-old male rats without any symptoms of apoplexy were mainly used in this study. The systolic blood pressure was measured by an indirect tail-cuff method (automatic blood pressure recorder USM-105-R type, Ueda Manufactory, Tokyo) at resting state. The animals (n = 23) were subjected to restraint water immersion (RWI), that is placed in a restraint cage and vertically immersed up to the xyphoid process in water at 23°C after fasting for 17 h as described previously [36]. After immersion for 5 h, the rats were killed and their stomachs fixed in a 4% neutral-buffered paraformaldehyde fixative for 24 h. Gastric mucosal lesions were recognized as linear breaks at the mucosal surface of the glandular stomach, and the extent of the lesions (ulcer index) was expressed as the sum of the area of these mucosal breaks. Rectal temperatures was monitored with a tubular thermometer probe (thermal sensor MGA-219A, Shibaura Electronics Co., Tokyo) and thermometer (MGAI-219S, Shibaura Electronics Co., Tokyo) inserted 3 cm rectally every hour during stress.
Gastric acid output was measured with pyloric ligation preparations in control rats (n = 23) after a 24 h fast with free intake of drinking water, and the pyrolus was then ligated under ether anesthesia. The stomach was resected 6 hr after pyloric ligation, and gastric juice was collected. Gastric output during RWI was not evaluated because of contamination of intragastric bleeding. The pH values and volume of secreted gastric acid were measured using an autonomic pH titrator (PHM852 Standard pH Meter, Copenhagen). Serum gastrin values were also measured by radioimmunoassay (Dinabots Co., Tokyo) in animals subjected to an overnight fast. Gastric motility during RWI was evaluated by the strain-gauge force transducer (n = 18). The strain gauge (Star Medical Co., Tokyo) was sutured on the lower portion of the gastric body parallel to the circular muscle under ether anesthesia 30 min before RWI. For quantitative analysis of the motility, the motility index (MI) was calculated from the frequency and magnitude of contracted waves over a 10-rain period. The formula is as follows; M I = H x F ( g m x m i n 1), H = Z h i / n (gm), F: w a v e s / m i n (min i) The noradrenaline and dopamine contents in gastric tissue from non-fasting control rats (n = 24), between 28 and 36 weeks, were measured by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The gastric tissues were immediately resected after the rats were killed and were then frozen with liquid N2, and subsequently divided into mucosal and muscle layers in the body and pyrolic areas, respectively. Choline acetyltransferase (CAT) activity of the fundic wall was determined by the method of Kaneda et al. [14], with minor modifications (n = 30). By this method, the specific CAT activity is obtained by measuring the concentration of acetylcholine formed from choline. The enzyme solution was prepared from each gastric tissue by homogenization in 12.5 ml of 25 mM solution phosphate buffer (pH 7.4) containing 20nM ethylhomocholine per g wet weight, followed by centrifugation at 20,000 x g for 60 min at 4°C. The incubation mixture consisted of the following components: 100 ~zl of substrate solution chlo-
231 ride, and 20 m M E D T A - 2 N a in 0.1 M sodium phosphate buffer (pH 7.4) and 100 /zl of the enzyme solution. Incubation was performed at 37°C for 20 min, and the reaction was stopped with 5 0 / z l of 1M perchloric acid in an ice-bath. Ten min later, the reaction mixture was centrifuged at 1600 × g for 10 min at 4°C. Twenty-five /~1 of the supernatant liquid was injected into the H P L C system. For the control experiments, the enzyme solution was boiled at 95°C for 5 min. The gastric mucosa was observed by light microscopy after fixation with 10% neutrized formaldehyde and paraffin-embedding. Noradrenergic nerve fibers innervating the mucosa were demonstrated using glyoxalic acid fluorescence histochemistry as described previously [14].
BP (mmHg) 300
Results The systolic blood pressure was 245 + 16, 183 + 8 and 1 2 5 + 1 0 m m H g ( P < 0 . 0 0 1 vs. each other) in SHRSP, S H R and WKY, respectively, at the age of 36 weeks (Fig. 1). In W K Y prominent ulcerations developed in the gastric mucosa after cold-restraint stress. In S H R the extent of stress ulcerations was significantly reduced, while they were virtually absent in the S H R S P (Fig. 2). The ulcer index was 0 . 9 + 0 . 6 5 , 9 . 2 + 3 . 6 and 3 8 . 1 + 1 3 . 6 m m 2 in SHRSP, S H R and WKY, respectively. The correlation coefficient between blood pressure and ulcer index was - 0 . 8 9 0 ( P < 0.001, n = 23) (Fig. 3), suggesting the existence of a significant reverse correlation between blood pressure and gastric ulcers. The rectal t e m p e r a t u r e after 5 h of water immersion was 24.9 + 0.3, 24.2 + 0.2 and 25.4 + 0.2°C in SHRSP, S H R and WKY, respectively. The average p H value in gastric juice was significantly increased, and acid secretion was significantly reduced in SHRSP, compared with S H R and W K Y (Table I). The serum gastrin
SHRSP SHR W K
Y
~
200
100
Statistics Results are expressed as mean +_ SD or SE. Statistical significance was assessed using the A N O V A test. P < 0.05 was considered statistically significant.
• ca ~
, ' , ' , ' , ' , ' , ' , ' , ' , ' , " , ' , ' , " , ' , ' , ' 6 8 10 12 14 16 18 20 22 24 2 6 28 30 32 3 4 36
age (weeks) Fig. 1. Systolic blood pressure in WKY, SHR and SHRSP. SHR and SHRSP showed progressive hypertension since 6 weeks of age. In SHRSP the systolic pressure exceeded over 200 mmHg from young age.
levels in S H R S P and S H R were significantly higher than that in W K Y (Table II). These findings indicate the poor functioning of acid secretion in the hypertensive strains, especially in SHRSP. Histologically, all of the gastric lesions were UI-1 ulcers, but the lesions in W K Y reached the deep layer of the propria mucosa while
Fig. 2. Gastric mucosal lesion from WKY, SHR and SHRSP after water immersion stress. In SHR the extent of stress ulcerations was significantlyreduced, while they were virtually absent in SHRSP.
232
UI
TABLE l
mm 2
Acid secretion
80
60
o
WKY
=
SHR
•
SHRSP
o
40 "
20
o ~
Y=-0.321X+74.3 r=-0.891n=23
o e~°
(sig. p<0.001 )
o
e
WKY SHR SHRSP
(n=9) (n=6) (n=8)
Volume ( m l / 6 h)
pH
acid secretion (/,Eq/h)
6.5_+2.3 4.5_+1.2" 3.9_+1.8"
1.5±0.1 1.5±0.1 2.5±0.8"#
95.2_+43.5 53.6_+ 5 . 6 " 30.8_+17.4 *#
Mean_+ SD, * P < 0.05 vs. WKY, # P < 0.05 vs. SHR.
o
TABLE II
Serum gastrin
100
150
200
250
mmHg BP Fig. 3. The relation between systolic blood pressure and ulcer index. A significant reverse correlation exists between blood pressure and ulcer indices.
SHRSP showed a very shallow and confined erosion. Gastric motility was markedly enhanced by RWI around after 3 h of stress in WKY, although
WKY SHR SHRSP
(n = 8) (n=6) (n = 8)
262_+ 65 463+ 8 3 " * 505 _+236 * *
Mean _+SD (pg/ml), * * P < 0.05 vs, WKY.
the rhythmic contractions with low amplitudes were observed in all strains of rats at the beginning of RWI. By contrast SHR and SHRSP sustained the rhythmic and relatively low amplitude contractions (Fig. 4). Mls at the fifth hour of
WKY
SHR
SHRSP
~[ l O g i
i
4h 5t3 Fig. 4. Gastric motility monitored with force transducer after about 5 h of RWl. Remarkably enhanced contraction waves appeared in WKY, while lower amplitude waves occurred in SHR and SHRSP.
233 R W I were significantly smaller in S H R and SHRSP than W K Y ( P < 0.05) (Table liD. The noradrenaline content of gastric tissue was significantly higher in SHRSP and S H R than in W K Y in the mucosal and muscular layers of the pylorus and body (Table IV). However, there was no significant difference between SHRSP and SHR. Dopamine content in the gastric mucosa and proper muscle layer was also significantly higher in SHRSP and S H R than in WKY. C A T activity was significantly lower in the corpus in S H R and SHRSP than WKY ( P < 0.01) (Table V). SHRSP showed a tendency to have lower C A T activity than SHR. Fluorescence noradrenergic fibers were most densely innervated in the fundic mucosa in S H R S P (Fig. 5). These findings indicated that the sympathetic innervation is well
TABLE III Motility index during RWI at 5 h
WKY SHR SHRSP
(n = 6) (n=6) (n = 6)
32.65:11.6 11.65: 3.0"* 12.9 + 4.8 **
Mean + SD (gm/min), * * P < 0.05 vs. WKY. developed in the gastric tissues and functionally suppresses cholinergic activity in the hypertensive strains.
Discussion The pathogenesis of essential hypertension is yet to be clarified, although factors such as ab-
Fig. 5. Catecholamine fluorescence of the gastric mucosa in WKY (a) and SHRSP (b). Fluorescence sympathetic fibers were more densely distributed in the mucosa of SHRSP (b) than in that of WKY (a).
234 T A B L E IV
Noradrenaline and dopamine content of the gastric tissues Tissue
Antrum
(ng/g)
mucosa
muscle
mucosa
muscle
231 382 304
157 221 157
278 425 383
270 392 411
NE
WKY SHR SHRSP
DA
WKY SHR SHRSP
+36 _+84" +93 *
9.3_+ 5.0 18.9_+ 6.5 * 19.7_+ 8.9 *
Body
_+58 +36 _+42
1.8 + 0.2 9 . 2 + 7.7 5.3+_ 2.4 *
+ 69 + 60" _+114 *
7.9_+ 22.5_+ 25.6_+
5.3 6.9 * 15.6 *
_+50 _+91 * -+59 *
4.9_+ 2.2 11.4-+ 6.7 11.5+ 4.8 *
Mean _+ SD, * P < 0.05 vs. WKY. NE, noradrenaline; DA, dopamine; WKY (n = 6); S H R (n = 6); SHRSP (n = 12).
normalities in the central nervous system, calcium metabolism, renin-angiotensin system and adrenoceptors have been postulated. Sympathetic hyperfunction is one of the pathogenetic factors in the onset and maintenance of hypertension in SHR and SHRSP [2,16]. The role of sympathetic hyperactivity in stress ulcer formation is not as clear as that of parasympathetic hyperactivity. It may facilitate causative factors [4,6,31] such as mucosal vasoconstriction and protective factors [24,27], such as inhibition of gastric secretion [10,23] and maintenance of blood flow [5,29]. We reported previously that the gastric lesions induced in SHR were aggravated by treatment with 6-hydroxydopamine, an agent for chemical sympathectomy, following decreases in norepinephrine and dopamine contents [33]. As in SHR, 6-hydroxydopamine treatment deteriorated the stress-induced ulcers in SHRSP mainly due to increase of gastric acidity [34]. In this study, the correlation coefficient between ulcer index and blood pressure was -0.890 (P < 0.001), suggesting that susceptibility of stress ulcer is inversely
TABLE V
Choline acetyltran~ferase activity of the fundic tissues in the hypertensive rats WKY SHR SHRSP
5.40 + 0.24 4.57 + 0.35 * 4.30+ 0.24 *
Mean + SE ( n m o l / m i n per g), * P < 0.01 vs. WKY. WKY (n = 12); SHR (n = 6); SHRSP (n = 12).
correlated with blood pressure. This finding is consistent with previous clinical reports on ulcer susceptibility in patients with hypotension [11,21] and hypertension [18,22]. We introduced the hereditarily hypertensive rats as a model of the hyperfunctioning sympathetic nervous system in view of autonomic nervous abnormality. The high concentration of catecholamine in the gastric mucosa of SHR and SHRSP supports this hypothesis. Morphologically, we demonstrated the dense noradrenergic fibers in the gastric mucosa of SHRSP by the fluorescence technique. However, SHRSP did not show the significant differences in noradrenaline and dopamine content in the gastric tissues to SHR. One of characteristic differences between these strains is an adenal function. The catecholamine content of the adrenal gland is about twofold higher in adult SHRSP than in SHR, and this may be involved in the marked hypertension in SHRSP [17] and possibly exerts an additional influence upon gastric sympathetic nervous system humorally. There exist adrenoceptors in the mucosa and muscle of the stomach that are innervated by sympathetic nerves. Activation of 13-adrenoceptor has been shown to inhibit acid secretion [5] and motility [8]. The concept that the sympathetic nervous system suppresses acetylcholine release to the parietal cells presynaptically is generally accepted. Acid secretion is regulated by various mediators but the presence of adrenoceptors on the parietal cells is a subject of controversy. We reported previously that the number of parietal
235 cells was not reduced in the hypertensive strains but mitochondrial degeneration progressed significantly in the parietal ceils of aged SHRSP, indicating that acid secretion was involved organically as well as functionally in lasting sympathetic facilitation [13]. Hypergastrinemia and G-cell hyperplasia occur in the states of vagotomy, long administration of a proton p u m p inhibitor [37], partial corpectomy [19] and aging. In 36-week-old SHRSP, hypergastrinemia and G-cell hyperplasia might be induced as a result of a feedback mechanism against persistently poor acid secretion [13]. Lowest acidity might greatly contribute to less susceptibility to R W I in S H R S P than in SHR. In view of parasympathetic function, the low C A T activity indicates inhibition of parasympathetic nervous system in the hypertensive strains. By R W I acetylcholine content decreased in W K Y but not in SHR, indicating that acetylcholine release was significantly suppressed in S H R during RWI. Although the noradrenaline content decreased in both S H R and W K Y as a result of RWI, it remained still higher in S H R than in W K Y [33]. Gastric motility has been suggested to be an important factor in the induction of several kinds of experimental ulcer [20], but the influence of gastric hypercontraction on mucosal blood flow is controversial [3,7]. We recently reported that enhancement of gastric contraction and the degree of drop in gastric blood flow during hypothermia was significantly smaller in S H R than in W K Y [12]. Inhibition of gastric motility is considered to be mediated mainly via the '~z adrenoceptor presynaptically, thereby suppressing acetylcholine release to smooth muscles. In S H R and SHRSP, noradrenergic hyperinnervation in the muscle layer might play an important role in the inhibition of motility during RWI. To investigate the relation between genetic inheritance and sympathetic function, we previously used the borderline hypertensive rats (BHR), a cross between S H R and W K Y in the stress experiment [30]. As the result, B H R showed intemediate values of blood pressure, ulcer indices, tissue catecholamine and gastric acidity, suggesting that ulcer susceptibility conforms to a polygenal regulation.
In conclusion, the S H R S P exhibiting marked hypertension was more resistant to stress in gastric ulcer formation than the other normotensive and hypertensive strains, suggesting that blood pressure is inversely correlated with stress-induced ulcer susceptibility. Alterative autonomic nervous function, namely sympathetic facilitation in association with low cholinergic activity, might play an important role in inhibiting gastric ulcer formation by suppressing acid secretion and gastric motility in the hereditary hypertensive rats.
Acknowledgments We would like to express sincere thanks to Dr. D. Murosec for editorial assistance and to Mr. T. Kawada for preparing the photographs.
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