Verapamil attenuates stress-induced gastric ulceration

JOURNALOFSURGICALRESEARCH js,

424-428(1985)

Verapamil Attenuates

Stress-Induced

Gastric Ulceration’-2

RICHARD B. WAIT, M.D., PH.D., AUSTIN L. LEAHY, M.B., FRCSI, JOHN M. NEE, M.B., FRCSI, AND THOMAS W. POLLOCK, M.D. Department of Surgery, State University of New York, Downstate Medical Center, Brooklyn, New York 11203 Presented at the Annual Meeting of the Association for Academic Surgery, San Antonio, Texas, October 31-November 3, 1984 Since many of the proposed etiologic factors leading to gastric stressulceration involve stimulation of calcium influx, the effect of verapamil, a potent calcium channel blocker, on the gastric mucosa in cold-restrained inbred rats was assessed.Twenty-nine rats received intraperitoneal normal saline (2 ml) while the experimental group (N = 29) received 1 mgjkg verapamil in an equal volume of normal saline intraperitoneally. All animals were then stressedat 4°C for 4 hr and sacrificed. Gastrin and fatty acid levels were measured and blinded ulcer scoring of the gastric mucosa was carried out. Verapamil-treated animals had decreasedfrequency and severity of gastric stressulceration as assessed by ulcer index, ulcer grade, and number of ulcers/animal. In addition, the plasma gastrin levels tended to be lower in the verapamil group. Fatty acid levels were similarly depressedfollowing cold restraint in both groups. Pretreatment with verapamil significantly decreasedgastric ulcerative responseto coldrestraint stress in the rat. This effect of verapamil pretreatment may be secondary to cytoprotection of the gastric mucosa, preservation of gastric mucosal bIood flow, or blockade of calcium-mediated ulcerogenic stimuli. 0 1985 Academic Press, ITIC.

Stress ulceration of the gastric mucosa is a well-described clinical entity; however, the exact mechanisms involved in the formation of the superficial mucosal lesions have not been fully elucidated. Many factors contributing to the formation of stress ulcers have been described. These factors include altered gastric acid secretion or concentration [6, 81, gastric mucosal ischemia [ 14, 15, 171, decreasedmucosal ATP [30], altered vagal and sympathetic tone [ 11, 15, 161, altered prostaglandin production [3, 41, and increased platelet aggregation [2]. Many therapeutic interventions have been attempted with variable success.Because many of the proposed causes of stress ulcer formation ultimately involve the stimulation of calcium influx, and since calcium has been implicated in ’ Verapamil was generously supplied by Knoll Pharmaceutical Company, Whippany, N. J. ‘Animal experiments were conducted according to the principles set forth in Guidefor the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, Department of Health, Education and Welfare, Publication No. (National Institutes of Health) 78-23.

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Q 1985 by Academic of reproduction

MATERIALS

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Inc. reserved.

AND

METHODS

Fifty-eight male Sprague-Dawley rats 260 to 330 g were randomly assigned to one of two experimental groups. Rats were housed four per cage in a room maintained at constant temperature and with light/dark cycles of 12 hr. Rats were maintained on Purina rat chow and water ad libitum for 7 days before the experiments. Food was withheld for 48 hr prior to experimentation; however, the animals were allowed free access to water. Following light ether anesthesia, rats were injected intraperitoneally with either 0.9% saline (2 ml) or verapamil (1 mg/kg) dissolved in an equal volume of 0.9% saline. All rats were then restrained in galvanized steel window screen weighing

424

0022-4804/85 $1SO Copyright

ulcer formation in certain pathologic states [ 10, 181, calcium blockade with verapamil could effectively alter the initiating events and inhibit ulcer formation. This study was designed to test this hypothesis in the commonly used model of cold-restraint ulceration in the rat [7].

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WAIT ET AL.: VERAPAMIL ATTENUATES STRESSULCERS

according to the methodof Bassoet al. [4]. velopedstressulcerswhichwereconfinedto The restrained rats were placed in a cold the glandular portion of the stomach.Results room (4°C) for 4 hr. of the microscopic analysis of the stomachs

Aftercoldrestraint, theanimals weresac- at autopsyaregivenin Table1.Therewere rificed by decapitation. Blood was collected in chilled tubes for serum gas&in assay [20] and measurement of fatty acid levels. At autopsy the severity of gastric erosions was evaluated in a blinded fashion using a dissecting microscope. After the stomach was opened along the greater curve, the ulcers were counted and measured. The product of the absolute number of ulcers and the total length of the ulcers was designated as the ulcer index. A severity grade was also assigned to each stomach, graded by the following criteria: Grade 0, no lesions; Grade I, punctate ulcers (1 to 4 mm); Grade II, linear ulcers >4 mm; and Grade III, linear ulcers with frank hemorrhage and gastritis. Mucosal erythema alone, which occurred frequently, received a score of 0. All blood samples for fatty acid analysis were collected in EDTA and centrifuged immediately. Plasma lipids were extracted by the method of Folch [ 131. After addition of 500 mg of heneicosanoic acid (2 1:0) as an internal standard, methyl esters of the fatty acids were prepared using boron trifluoride-methanol f33]. Plasma fatty acids were assayedby gas-liquid chromatography [27] (Perkin-Elmer, Sigma 3, Norwalk, Conn.). Individual fatty acid peaks were identified by comparison with commercially available fatty acids (Sigma Chemical) and quantitated by reference to the known concentration of heneicosanoiate internal standard, a fatty acid which does not appear in rat blood [26]. Plasma fatty acid levels are expressed in milligrams per milliliter as the mean + SEM. Statistical analysis was performed using the unpaired Student’s t test between groups. Distribution of ulcers by score was assessed using the x2 test. RESULTS

Eighty-nine percent of the control group and 79% of the verapamil-treated group de-

significantly fewer ulcers per animal in the verapamil-treated group as compared to the control group. Moreover, the ulcerations were less severe in the treated group as evidenced by the difference in the ulcer indices. In the treated group, 86% of the animals had no ulcers or mild (Grade I) ulceration, whereas only 52% of the control group fell into this favorable category. The distribution of severity grades was different between groups, with the control group having more frequent and severe lesions than the verapamil-treated group (P < 0.05 by x2) (Fig. 1). Analysis of serum gastrin levels revealed lower values in rats treated with verapamil as compared with controls; however, this difference approached but did not attain statistical significance (P < 0.08). Serum gastrin levels in control rats averaged 27 + 7.7 fm/ ml and in treated animals averaged 22 f 7 fm/ml. Results of fatty acid analysis are given in Table 2. We have previously noted that cold restraint significantly decreasestotal fatty acid, and particularly linoleic acid, to levels below values found in fasted control rats. The serum levels of all fatty acids measured were lower than normal, but virtually identical in the two groups of cold-restrained animals. DISCUSSION

This study utilized a cold-restraint model for stressulceration which is dependent upon TABLE INCIDENCE

I

AND SEVENTY

OF ULCERATION

NO. ulcers/ animal

Length (mm)

Control [29] SEM

9.91 I .54

Verapamil SEM

6.34 1.21

Group

P value

[29]

<0.04

Ulcer index

Ulcer grade

12.38 2.35

208.9 I 51.93

1.66 0.18

6.02 1.27

78.40 28.76

0.97 0.12

<0.04


<0.02

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JOURNALOF SURGICALRESEARCH: VOL.38,NO. 5, MAY 1985

m 0

Ulcer Grade

Verapamil Control

calcium increases feline gastric acid secretion [5]. The importance of gastric acid secretion in stress ulcers remains controversial. In restraint models, Menguy [29] demonstrated a decrease in total acid production, whereas Bonfils et al. [6] found an increase in acid production. Brodie and colleagues [8] found a decreasein volume but an increase in acid concentration. Jeppson and associates [2 l] have clearly shown that blockade of Hl and H2 receptors with cimetidine alone or with chlorpheniramine decreased the incidence and severity of stressulcers in restrained rats. Verapamil may act to inhibit gastric acid secretion through two primary mechanisms. First, verapamil has been shown to inhibit histamine-stimulated acid secretion in a dosedependent fashion, and has also been demonstrated to limit the secretory response to gastrin [23]. Kasbekar [22] has presented evidence in isolated bullfrog gastric mucosa that cellular calcium may play a direct role in the production of H+ by mediating the effect of raising intracellular cyclic AMP. Second, lowering intracellular calcium may inhibit the secretion of gas&in [ 121 and the release of histamine [25], both potent secretagogues. Our study demonstrates a trend toward lower gastrin levels in verapamiltreated animals. Changes in gastric mucosal blood flow have also been implicated in the genesis of stress ulcers. Local mucosal ischemia has clearly been found to be an important predisposing factor in ulcerogenesis [9, 14, 15, 171.In addition to being a direct vasodilator, verapamil has also been shown to inhibit the

,IIll

FIG. 1. Following 4 hr of cold restraint verapamiltreated animals had lesssevere (Grades 0 and I) mucosal lesions. More control (saline-treated) animals developed significant mucosal ulceration and hemorrhage (Grades 11and III).

duration of immobilization, duration of fasting, and temperature. In this study 48 hr of fasting followed by 4 hr of cold restraint resulted in an 89% incidence of gastric ulceration in the control group. Treatment with the calcium blocker verapamil decreasedthe incidence of ulceration to 79%, and the severity of the ulcerations was significantly less. Although the benefits of treatment with verapamil at the dose used in this experiment appear to be modest in comparison to treatment with Hl and H2 blockers [21], the results do demonstrate a significant beneficial effect of verapamil and suggestthat Ca2+flux during stressin this model probably mediates one or more of the causative mechanisms. In support of this concept is the evidence that hypercalcemia in patients with hyperparathyroidism is associatedwith an increased incidence of peptic ulcer disease [ 10, 181 as well as the observation that infusion of

TABLE 2 Emc-rs OF COLD RESTRAINT ON FATN ACID LEVELS (mg/ml) Palmitic

Steak

Oleic

Linoleic

Arachidonic

Control [29] SEM

0.26 0.01

0.17 0.01

0.14 0.01

0.28 0.02

0.43 0.03

1.28 0.07

Verapamil [29] SEM

0.26 0.01

0.17 0.01

0.14 0.01

0.29 0.01

0.43 0.02

1.29 0.06

Total

WAIT ET AL.: VERAPAMILATTENUATESSTRESSULCERS

vasoconstrictor response to norepinephrine [32] and angiotensin II [ 191.Goldman and Rosoff [ 151and Mersereauand Hinchley [ 3 1] have suggestedthat intense gastric muscular contraction with extrinsic compression of the intramural vesselscontributes to the formation of the linear ulcers seen in stress. Verapamil is effective in inhibiting smooth muscle contraction in the esophagus [34] and small intestine, and through this mechanism may prevent gastric muscular activity and subsequent focal ischemia. Local ischemia may be aggravated by platelet aggregation and thrombus formation [2]. Kodama et al. [24] have shown that pretreatment with heparin is effective in preventing stress ulcers in the cold-restraint model. Verapamil is a potent inhibitor of platelet aggregation [ 1, 281. Mehta and colleagues [28] have demonstrated that verapamil inhibits platelet aggregation induced by ADP, arachidonic acid, and epinephrine, and thus part of the beneficial effect seen in the present study may be mediated through this mechanism. In conclusion, we have shown that pretreatment of rats with verapamil significantly diminishes the severity of the gastric ulcerative response to cold restraint. This beneficial effect is associated with a trend toward decreasedserum gastrin levels and no change in fatty acid levels. The beneficial effects of verapamil may be related to its ability to block calcium-mediated ulcerogenic stimuli. REFERENCES 1. Addonizio, B. P., Fisher, C. A., Strauss, J. F., III, and Edmunds, Jr., L. H. Inhibition of human platelet function by verapamil. Thromb. Res. 28: 545, 1982. 2. Balibrea, J. L., Merchant, J., Gomez, J., et a!. Restraint-induced stress ulcer. II. Biochemical and ultrastructural studies of gastric mucosa. World J. Surg. 4: 91, 1980.

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