Differences in action of topical and systemic cysteamine on gastric blood flow, gastric acid secretion and gastric ulceration in the rat

J Physiology

OElsevier,

(Park)

(1996) 90, 63-73

Paris

Differences in action of topical and systemic cysteamine on gastric blood flow, gastric acid secretion and gastric ulceration in the rat OME Abdel-Salama, J SzolcsBnyib, G M6zsika* “First

Department

of Medicine

and bDepanmenr

of Pharmacology.

(Received

Medical

14 May 1996; accepted

Universiv

of P&s,

H-7643

Pks.

@sci(:

ut 13. Hungary

10 June 1996)

Summary - The effect of cysteamine on gastric blood flow and on the indomethacin-induced gastric mucosal damage was studied. In anesthetized rats, cysteamine (280 mg/kg) given subcutaneously (SC) decreased gastric blood flow measured by the laser Doppler flowmetry technique. In contrast, cysteamine (160 mg/ml) applied topically to the serosal surface of the stomach evoked a concentration-dependent and long-lasting increase in gastric blood flow. At 60 mg/ml, cysteamine increased blood flow by 166.8 It 26.1% of predrug control value. Pretreatment with indomethacin (20 mg/kg, SC), intravenous (iv) atropine (1 mg/kg), propranolol (1 mg/kg, iv), combined Ht and Hz-blockade or bilateral cervical vagotomy alone or combined with iv guanethidine (8 mg/kg), or pretreatment with the capsaicin analogue resiniferatoxin did not reduce the vasodilator response to cysteamine. The vasodiiator response to topical capsaicin, was not reduced after sc cysteamine (280 mgikg) pretreatment. In conscious pylon&ligated rats, SCcysteamine (100 or 280 mg/kg) given simultaneously with indomethacin inhibited gastric acid output but had variable effects on the indomethacin-induced gastric mucosal damage. Cysteamine (100 or 280 mg/kg) administered sc 4 h prior to indomethacin enhanced gastric injury by sc indomethacin, but did not prevent the gastroprotective action of capsaicin. In contrast, oralJy administered cysteamine (60 mg/ml) reduced gastric injury induced by sc indomethacin plus intragastric HCI. These data provide the first evidence for the effect of cysteamine on gastric microcirculation in the rat and suggest a diit vasodilator effect for topical cysteamine. The microvascular effects of cysteamine are largely responsible for the different effects of this agent on experimental gastric injury gastric microcirculation

I laser Doppler flowmetry

I cysteamine I capsaicin

Introduction Cysteamine is a sulfhydryl reducing agent, which possesses variable effects on the gastroduodenal mucosa. In the duodenum, cysteamine evokes severe duodenal ulceration 24-48 h after its administration in a single dose (Selye and Szabo, 1973; Robert ef al, 1974). In the stomach, pretreatment with cystearnine prevents the chemical-induced gastric injury produced by application of ethanol shortly afterwards (Szabb et al, 1983; Trier et al, 1987) or by itself induces ulcers (Holzer and Sametz, 1986). The agent also possesses important neuroendocrine effects. In the rat, cysteamine depletes somatostatin in different tissues including the duodenum and the glandular stomach (Szabo and Reichlin, 1981; Szabo et al, 1992). In recent years experimental evidence have emphasized the microvasculature as being a target both in injury and in protection of the gastric mucosa Thus, the rnicrocirculation has been revealed to be the target site for several mucosal ulcerogens such as aspirin @maw&i et al, 1990), indomethacin (Rainsford, 1983) or ethanol (Gum et al, 1984) in that vascular endothelial injury is an early event following their application. The microcirculation is also the target for many protective agents, eg cytoprotective prostaglandiis (Guth er al, 19&l), sucralfate (Szabo et al, 199 1). isoproterenol (Howard ef al, 1993), epidennal *Correspondence

and reprints

growth factor (Hui et al, 1993) and capsaicin type agents (Lippe et al, 1989; Holzer et al,1991; Abdel-Salam er al, 1996). Gastric mucosal blood flow is a crucial factor which determines the capacity of the mucosa to remove (Starlinger et al, 1981) or neutralize (Kiviluto et al, 1988) the influxing acid load and consequently determines the development of mucosal damage. In experimental ulcer research, most studies have focused on the effect of cysteamine in relation to the duodenal mucosa. Very few evaluated its effect on the gastric mucosa, while little attention was paid to the microvascular responses to the drug. In the rat duodenum, SC cysteamine has been reported to reduce mucosal blood flow measured by the hydrogen gas clearance technique (Kurebayashi et al, 198.5; Leung and Guth, 1985a). The laser Doppler flowmetry technique permits time-real dynamic monitoring of microvascular responses to drugs (Kiel and Shepherd, 1990). This, in addition to monitoring of arterial blood pressure, provides an excellent approach to the evaluation of microcirculatory events evoked by different agents. The present paper presents the first study performed to evaluate the effect of this potent duodenal ulcerogen on the gastric microvasculature. The effect of cysteamine was further evaluated on gastric mucosal damage evoked by indomethacin. Capsaicin, the pungent principle of hot peppers, is a potent and selective stimulant of mammalian primary afferents with C and A6 thin fibers (Szolcsanyi, 1982,

64

1990; Holzer, 1991). Introduction of capsaicin into the rat stomach in low concentrations that stimulated capsaicin-sensitive sensory nerves, protected against the chemical-induced gastric mucosal damage via enhancement of the local microcirculation (Szolcsanyi and Barthe, 1981; Lippe et al, 1989; Holzer et al, 1991; Abdel-Salam et al, 1995). Capsaicin evokes the release of neuropeptides (Holzer, 1991), of which CGRP is the most likely one implicated in the vasodilator (Li et al, 1991) and antiulcer (Holzer er al, 1990; Evangelista, 1994; Maggi, 1995) effects of capsaicin. The ulcer-preventive effect of CGRP was reported to be absent in rats depleted of somatostatin by cysteamine pretreatment (Clementi et al, 1993). We therefore examined the hypothesis that the protective effect of capsaicin might be mediated through somatostatin release. Thus, the protective and vasodilator effects of capsaicin were evaluated in rats pretreated with cysteamine at a dose previously reported to deplete somatostatin in different rat tissues including the gastric mucosa (Szabo and Reichlin, 1981; Szabd ef al, 1992).

Materials and methods Animuls

Sprague-Dawley strain rats, of both sexes, 180-230 g body weight, were used throughout the study and housed under standardized conditions for light and temperature. Rats were kept in cages with wide meshed floors to prevent coprophagy. Rats were deprived of food for 24 h before the experiments,

but allowed free access to tap water.

Gastric blood jlow studies The effect of cysteamine on gastric blood flow was studied in

anesthetized tats with the laser Doppler flowmetry technique. Animal preparation

Anesthesia was carried out with thiobutobarbital (Inactin), given ip in a dose of 100 mgikg. A cannula (Y shaped) inserted into the trachea served for facilitation of spontaneous breathing and for occasional aspiration of secretions. Respiratory movements were monitored by a low pressure transducer, connected to one side of the cannula. A polyethylene tube filled with heparinized saline was inserted into the right common carotid artery and connected to a pressure transducer (Statham) for continuous monitoring of systemic arterial blood pressure and heart rate with a physiological chart recorder (Beckman Dynograpgh Type RM). The left internal jugular vein was cannulated for drug administration. Body temperature, maintained at 37°C f 05°C by a heating lamp, was monitored with a rectal thermometer. Rats received 1.5 ml of physiological saline every 1 h through the internal

jugular vein to avoid dehydration during the observations, and in addition hemattit values were obtained before and after the experiment to check for adequate hydration of the animals. Laser Doppler ,flowmrt~

Under anesthesia, the abdomen was opened through a midline incision. the gastrohepatic ligaments were carefully cut. and the stomach was gently exteriorized. Since movements of the stomach and respiration during the experiment could cause changes in the probe position, the stomach was gently placed on a transparent four-legged table-like plastic slide and fixed with fine sutures to metal needles inserted at both sides of the stomach. With this method of fixation, motion artifacts were eliminated, since no jumps occurred in the recordings and respiratory movements caused no synchronous change in the tracings. Blood flow measurements were performed with MBF3D dual channel laser Doppler flowmeter (Moor Instruments Inc. UK). The two probes (P5b type) supported by standard probe holders were placed perpendicularly to the outer surface of the stomach (AbdelSalam et a/, 1996). Warm saline (37°C) was poured continuously over the stomach to keep the tissues moist. The tips of the probes were just in contact with the serosal surface as checked from time to time with an operating microscope. Stable recordings could be obtained for long periods of time (I h). Atropine sulfate (I mg/kg) was injected iv at the beginning of the experiments to inhibit gastric acid secretion. Intravenous injection of saline in a volume of I ml had no effect on the stability of the laser Doppler signal. Study desigrl

In the first series of experiments, the effect of SC administration of cysteamine in a dose of 280 mg/kg on gastric blood flow was studied. Gastric blood flow was measured for 60 min after injection of cysteamine. In the second experimental series, the effect of topical cysteamine application was studied. Cysteamine was applied locally to the serosal surface of the stomach at different concentrations (I. 6 and 60 mglml) in rats with an intact vagus nerve. The effect of cysteamine at a very low concentration of 0.2 pg/ml was also tested. In other groups, the effect of topical cysteamine in 60 mg/ml was evaluated after bilateral cervical vagotomy alone or combined with iv guanethidine (8 mg/kg). The vagus nerves were carefully separated and cut at the cervical region. The effect of cysteamine was also studied after pretreatment with iv propranolol (I mg/kg) and SC indomethacin (20 mgikg), iv cimetidine ( 100 mg/kg) and iv chloropyramin (25 mg/kg). In further experiments, cysteamine was locally applied after pretreatment with the capsaicin analogue resiniferatoxin in three consecutive doses of I kg/kg. Histamine chloride (1 mglml) or isoprenaline (200 Kg/ml) was given to compare its effect with that of cysteamine. 15-30 ~1 from each solution was applied around each probe site with a micropipette. In the last series of experiments we examined the vasodilator effect of topical capsaicin in 3.2 and 10 pg/ml on

6.5

gastric blood flow in rats pretreated with SCcysteamine (280 mg/kg) 1 h prior to capsaicin application.

I and 2 mm = 3; lesions between 2 and 4 mm = 4: and lesions bigger than 4 mm = 5 (Mbzsik et al, 1982).

Gustric ulcer and secretory studies

Gastric secretop studies

The effect of cysteamine on the indomethacin-induced gastric mucosal injury and on the gastroprotective effect of capsaicin was evaluated in conscious pylorus-ligated rats.

The volume of gstric contents was measured, the acid output was determined by titration with 0.1 N NaOH to pH 7 (using a Radelkis pH automatic titimeter, Budapest, Hungary) and H+ output expressed in pEq/rat/4 h (means ? SEM).

Surgical procedure Pylorus-ligation was performed under brief ether anesthesia, care being taken not to interfere with the blood supply to the stomach and duodenum. Immediately thereafter, gastric mucosal damage was evoked by the SC administration of indomethacin in a single dose of 20 mg/kg. Rats ig received 2 ml of saline. Animals were killed 4 h later by cervical dislocation after being lightly anesthetized with ether. The esophagus was then ligated and stomach excised. Gastric juice was collected in graduated tubes after removal of the esophageal ligature and stomachs were opened along the greater curvature and inspected for the presence of gastric mucosal lesions.

Immediately after pylorus-ligation, cysteamine (10, 100 or 280 mg/kg, SC)was given simultaneously with SCindomethacin (20 mg!kg) and rats orally received 2 ml of saline. Rats were killed 4 h later. In further experiments, the effect of oral administration of cysteamine in 60 mg/ml was evaluated against gabic mucosal damage evoked by SCindomethacin and onl administration of 2 ml of 0.3 N HCI. This combination has been shown in our experiments to evoke severe gastric mucosal damage (unpublished results). After ligating the pylorus, indomethacin was SCgiven and rats orally received 0.3 N HCI (2 ml) with or without cysteamine (60 mg/ml). Rats were killed 4 h later. The effect of orally administered cysteamine in 60 mg/ml on gastric acid secretion was also evaluated in the 4-h pylorus-ligated rat. Cysteamine was ig given in 2 ml saline after pylorus-ligation and rats killed 4 h later. In studies evaluating the effect of cysteamine pretreatment on the indomethacin-induced gastric injury, cysteamine was given in a single SCdose of 100 or 280 mg/kg. The latter dose was reported to deplete tissue somatostatin levels with the maximal decrease being at 4 h after cysteamine administration (Szabc? and Reichlin. 1981; Szab6 et al, 1992). Therefore. 4 h after cysteamine injection, pylorus-ligation was performed, indomethacin (20 mg/kg) was SCadministered and animals orally given capsaicin (0.2-3.2 pg/ml) or the vehicle (con&o1 groups) in 2 ml saline. Rats were killed 4 h later.

Stomachs were excised, opened along the greater curvature, briefly rinsed with saline and inspected for the presence of gastric mucosal lesions. The number and severity of mucosal lesions were noted. Lesions were scaled as follows: petechial lesions = I: lesions smaller than I mm = 2; lesions between

The following drugs were used: inactin (BYK, Germany), capsaicin, resiniferatoxin. cysteamine HCI (2-mercaptoethylamine), guanethidine sulfate (Sigma, USA), chloropyramin HCI (Suprastin, Egis, Hungary), atropine sulfate (Egis. Hungary), cimetidine (Histodil, Richter, Hungary), indomethacin (Chinoin, Hungary), isoproterenol (Isuprel HCI, Winthrop. New York, USA), histamine chloride (histaminum bihydrochloricum, Peremin, Chinoin, Hungary), propranolol hydrochloride (Inderal, ICI, UK). Stock solutions of capsaicin (IO mg/ml) and resiniferatoxin (0.5 mg/ml) contained 10% ethanol, IO% Tween 80, 80% saline solution. Drugs were freshly dissolved with isotonic NaCl immediately before the experiments to obtain the necessary doses. Cysteamine was dissolved in distilled water to obtain the necessary dilutions.

Data are means f SEM The number of animals used and number of observations in different experiments is indicated in the text in parentheses. The drug-induced changes in the laser Doppler signal were expressed as a percentage change of the average data recorded over at least 3-min periods immediately prior to drug administration. The maximum values of the responses are presented. These maximum values are measurements over a 3-min period. Statistical analysis of the results was performed using Student’s t-test. The MannWhitney’s test was applied for mathematical analysis of nonparametric results (ulcer severity). P values of less than 0.05 were considered significant.

Results

Effect of subcutaneous blood flow

cysteamine

on gastric

Cysteamine injected SC in a dose of 280 mg/kg in four rats produced a 30 f 5.2% decrease in gastric blood flow 1 h later. Blood pressure was unchanged or showed a slight increase after cysteamine injection. Injection of the solvent for cysteamine (distilled water) did not affect the laser Doppler signal or arterial blood pressure.

66

Effect P ow

of topical

cysteamine

on gastric

blood

Cysteamine applied on the serosal surface of the stomach in 1 and 6 mg/ml increased gastric blood flow by 50 * 5.5% (n = four rats, seven observations) and 65.9 + 10.3% (n = four rats, nine observations) of predrug control level respectively. A pronounced and sustained vasodilatation was seen when cysteamine was applied on the stomach surface in 60 mg/ml. At this concentration, cysteamine increased gastric blood flow by 166.8 f 26.1 (n = six rats, 19 observations). The vasodilatory response to cysteamine in 60 mg/ml showed a variable time course (fig lA, B). On average the laser Doppler signal started to rise 39.4 f 8.9 s after application of cysteamine, reached maximum at 5.8 + 1 min, then remained elevated for 16.8 f 1.6 min. Blood pressure was practically unchanged after serosal application of the agent (fig IA, B). The response to lower concentrations was tested by application of the agent in 0.2 pg/ml, which was still effective (fig 1C) At this concentration, cysteamine increased gastric blood flow by 42.1 f 14% (n = three rats, eight observations). These responses lasted between 1.5 and 15 min. Application of the solvent (distilled water) did not affect the laser Doppler signal (fig ID). Further experiments were performed in order to elucidate the pathways mediating the vasodilator response to topical cysteamine in the rat stomach. Thus the effect of topical cysteamine (60 mg/ml) was then evaluated under different conditions. This concentration was chosen since it produced a sustained vasodilation. In all studies employing this agent orally for induction of duodenal ulcers (Selye and Szabd 1973), depleting somatostatin (Szabb and Reichlin, 1981) or for eliciting gastroprotection (Szabo et al, 1983; Trier et al, 1987) much higher concentrations were used (100 mg/ml). After acute cervical vagotomy alone or combined with iv guanethidine (8 mg/kg), cysteamine evoked 209.4 f 57% (n = six rats, 12 observations) and 172.9 f 31.1% (n = six rats, 11 observations) increases in gastric blood flow, respectively. These responses also lasted for several minutes (17.8 f 2.9 and 19.1 + 3.3 min, respectively)(fig 2A, B). After treatment with iv propranolol (1 mg/kg), cysteamine produced a 162 + 43% increase in gastric blood flow (n = six rats, 10 observations). After HI- and Hz-receptor blockade, cysteamine application resulted in 136.3 f 28.3% increase in gastric blood flow (n = five rats, 12 observations). In rats pretreated with SC indomethacin (20 mg/kg), cysteamine increased gastric blood flow by 200.2 + 41.4% (n = 10 rats, 19 observations)(fig 2C).

To investigate whether the vasodilator response to cysteamine could be mediated through a release of neuropeptides from sensory nerve endings, cysteamine was applied on the stomach in rats pretreated with resiniferatoxin in three consecutive doses of 1 pg&g with a 20-min interval. Resiniferatoxin is a potent capsaicin analogue, capable of exciting and desensitizing capsaicin-sensitive neurons (Maggi et al, 1990; Szolcsanyi et al, 1990; Szallasi and Blumberg, 1992). This was confirmed by the finding that iv injection or topical application of resiniferatoxin or capsaicin thereafter and for 3-4 h later (the end of experiments) failed to evoke any changes in blood flow or in arterial blood pressure, thus indicating that resiniferatoxin in the doses used above produced long-lasting desensitization of sensory receptors and neuropeptide depletion. Intravenous resiniferatoxin in 1 l.q$g, evoked a 176.8 + 23.8% increase in gastric blood flow that lasted between 9 and 15 min (11.7 + 0.8 min) (n = six rats, six observations). Subsequent injection of the agent produced minimal or no vasodilatation, while the third dose had no effect on the laser Doppler signal. Cysteamine applied 20 or 60 min after the last dose of resiniferatoxin evoked a 161.6 f 49% (n = six rats, 12 observations) and 133.9 f 48% (n = six rats, eight observations) increases in gastric blood flow, respectively. Vasodilatation was slow in onset (fig 2D). The duration of these responses lasted between 3 and 27 min (14.1 f 1.7 min), with one response lasting only 30 s. Application of the solvent for cysteamine (distilled water) in all experiments had no effect on the laser Doppler signal. Histamine (1 mg/ml) applied on the serosal surface of the stomach for comparison evoked 174.9 * 46.5% which lasted between 10 and 28 min (16.9 f 2.4 min) (n = live rats, nine observations) (fig 3). Isoprenaline (200 l.tg/ml) evoked 122.7 + 15.8% increase in gastric blood flow that lasted from 7-55 min (20 f 2.8 min) (n = 14 rats, 25 observations).

Effect of capsaicin cysteamine-pretreated

on gastric rats

blood flow

in

Cysteamine administered SC in 280 mg/kg 1 h prior to capsaicin, did not prevent the vasodilator effect of topical capsaicin application. Capsaicin applied on the serosa of the stomach in 3.2 or 10 pg/ml evoked 53 f 7% (n = four rats, six observations) and 119 f 17.3% (n = eight rats, nine observations) increases in gastric blood flow, respectively.

67

B 1000

GBF A”

0

1

T

r

200 ASP

mw 100

Ii 1 ~Jr~~~~~rtir~~~~~~irr~ t

0

Cysteamine

TS

I

I 4

min

Fig 1. Experimental tracing of laser Doppler flowmetry from four different rats showing gastric blood flow (GBF) in arbitrary units (AU) and arterial blood pressure (ABP; mmHg). A, B. Following topical application of cysteamine (60 mg/ml) on the serosal surface of the stomach in two rats with intact vagus nerve. C. After topical application of cysteamine (0.2 pg/ml) on the serosal surface of the stomach. D. After topical application of distilled water on the serosal surface of the stomach. In figures C and D, two laser probes were applied simultaneously on the serosal surface of the stomach.

68

Fig 2. Experimental tracing of laser Doppler flowmetry from four different rats, showing gastric blood flow (GBF) in arbitrary units (AU), arterial blood pressure (ABP; mmHg) following topical application of cysteamine (60 mg/ml) on the serosal surface of the stomach. A. After bilateral cervical vagotomy. B. After bilateral cervical vagotomy combined with guanethidine treatment (8 mgikg iv). C. After pretreatment with indomethacin (20 mg/kg SC). D. After pretreatment with the capsaicin analogue resiniferatoxin (in three consecutive doses of 1 pgkg iv) to deplete neuropeptides from sensory nerve endings. In figures A and B, two laser probes were applied simultaneously on the serosal surface of the stomach.

69

GBF AU

GBP AU

0 I 200 ABP mm 100

1

r

Histamine

TS

Fig 3. Experimental tracing of laser Doppler flowmetry showing gastric blood flow (GBF) in arbitrary units (AU), arterial blood pressure (ABP; tnmHg) following topical application of histamine (I mg/ml) on the serosal surface of the stomach in a rat with intact vagus nerve. Two laser probes were applied simultaneously on the serosal surface of the stomach.

The effect of cysteamine on the indomethacin-induced gastric injury cupsaicin-induced gastroprotection Simultaneous

cysteamine

and the

administration

Subcutaneous cysteamine. Cysteamine in a 10 mg/kg dose did not affect gastric acid output or indomethacin-induced gastric lesions, while in a 100 mg/kg dose it markedly inhibited gastric acid output and the indomethacin-induced gastric lesions in the 4-h ligated rat. Cysteamine at 280 mg/kg inhibited gastric acid output, but failed to reduce the indomethacin-induced injury (table I). Intragastric cysteamine. Oral administration of 2 ml of 0.3 N HCl to rats treated with SC indomethacin evoked severe gastric mucosal damage with the number and severity of gastric lesions being 29.7 f 4 and 75.5 If: 9, respectively (n = 5). Cysteamine administered into the stomach simultaneously at a concentration of 60 mg/ml with HCl and SC indomethacin reduced gastric mucosal damage caused by indomethacin plus HCl from the above control values to 21 f 3.4 and 47 f 6, respectively (n = 5, P < 0.05 for both). Cysteamine introduced into the stomach in the same concentration

in 2 ml saline significantly increased gastric acid output in the 4-h pylorus-ligated rats. Gastric acid output was 1468.8 f 152 w control value of 797.2 @,q/rat/4 h (P < 0.001). Cysteamine pretreatment. Rats pretreated with cysteamine ( 100 or 280 mg/kg SC). a specific depletor of tissue somatostatin (Szabo and Reichlin, 198 1; Szabo et al. 1992). exhibited more severe gastric mucosal damage in response to SCindomethacin than their corresponding control group. The changes caused by cysteamine were dose-dependent and were not associated with increased gastric acid secretion. On the contrary, gastric acid output was significantly reduced in cysteamine-pretreated rats compared with the control group (table II). Capsaicin administered ig in 0.2 and 0.8 pg/ml did not reduce the indomethacin-induced damage in cysteamine-pretreated rats, although the incidence of lesions was reduced by 60% with 0.8 pg/ml capsaicin in those pretreated with 100 mg/kg cysteamine. A higher degree of mucosal damage was seen after indomethacin in rats pretreated with cysteamine in 280 mg/kg. Ulcers were also noted on the non-glandular forestomach in addition to the glandular stomach. The severity of lesions was significantly reduced by

70

Table I. Effect of simultaneous pylorus-ligated rat.

administration

of cysteamine on the indomethacin-induced

Lesions/rat/4 Treatment

ND control + Cysteamine + Cysteamine + Cysteamine

( IO mg/kg) (100 mgkg) (280 mg/kg)

h

I1

Number

Severit)

11 8

3.7 3.7 0.4 2.8

5.1 5.0 0.6 3.7

8 10

f f f f

gastric mucosal injury in the 4-h

0.6 1.6NS 0.3*** 0.9NS

Volume (ml/rat/4 h)

f I.1 * 1.9NS + 0.5** IL l.ONS

9.1 7.9 3.3 4.3

+ f * k

0.5 0.8NS 0.2*** 0.5***

Acid output (FEq/rat/4 h) 1001 826.1 151.7 326.0

+ f f +

85.8 134NS 26.0*** 85.0**

Values are means f SEM. n = number of animals in each group. Abbreviations: IND, indomethacin. Cysteamine was administered in a single SC dose of 10, 100 or 280 mgikg immediately after pylorus-ligation and simultaneously with SC indmethacin (20 m&g). Rats were killed 4 h after indomethacin. Statistical significance of the difference between the solvent-treated indomethacin control group and the cysteamine-treated groups was denoted by **P < 0.01; ***P < 0.001: NS, not significant; Student’s f-test (Mann-Whitney’s test for ulcer severity).

Table II. Effect of cysteamine pretreatment on the gastroprotective gastric mucosal injury in the 4-h pylorus-ligated rat. Lesions/rat/4 Treatment

n

IND control 8 + Cysteamine ( 100 mg/kg, sc) 11 + Capsaicin 0.2 Fg/ml 6 + Capsaicin 0.8 pg/ml 12 IND + cysteamine (280 mg/kg, SC) 16 + Capsaicin 0.8 yg/ml 8 + Capsaicin 1.6 Fglml 11 + Capsaicin 3.2 ug/ml 8

Number

2.9 5.5 6 3.6 17.6 11.2 8.9

f f f f f f f

0.6 1.3” 2.0NS 1.4NS 3.1d 2.5NS 2.2NS

7.2 + 1.4*

effect of capsaicin

against the indomethacin-induced

h Severity

4.2 10.5 1I .6 5.0 36.7 20.5 16.8

f 0.8 f 2.0b f 2.7NS f 1.9NS f 7d zk 4.8NS + 4.3*

11.8 f 2.7”

Volume (ml/rat/4 h) 10.1 8.1 9.8 7.6 8.0 7.4 8.5

+ 0.3 f 0.9b rf: l.3NS f 0.8NS + 0.5’ f 0.6NS f 0.6NS

8.6 f 0.8NS

Acid output (pEq/rat/4 h) 1152.2 837.9 1087.6 723.2 854.6 819.1 834.2

+ f + + f. + +

62.6 137b 184.6NS II 1.6NS 85.6b 130NS 90NS

975.2 + 164NS

Values are means + SEM. n = number of animals in each group. Abbreviations: IND, indomethacin. Cysteamine was administered in a single SC dose of 100 or 280 mg/kg, 4 h prior to pylorus-ligation and SC indomethacin (20 mg/kg). Capsaicin or its solvent was introduced into the stomach in 2 ml volumes of saline simultaneously with indomethacin administration. Rats were killed 4 h after indomethacin. Statistical analysis between the IND control group and the IND + cysteamine-treated group is denoted by: “not significant: ‘P C 0.05; ‘P < 0.01; dP < 0.001. Statistical significance of the difference between the IND + cysteamine-treated control group and the capsaicin-treated groups is denoted by *P < 0.05; NS, not significant; Student’s f-test (Mann-Whitney’s test for ulcer severity).

1.6 l.tg/ml capsaicin (P < O.OS), while both the number and the severity were significantly reduced by 3.2 pg/ml capsaicin (P < 0.05 for both) (table II).

Discussion The present paper is the first study reporting on the effect of cysteamine on the gastric microvasculature in the rat. The laser Doppler flowmetry technique is capable of providing a continuous, localized measurement of tissue perfusion and thus provides a useful tool for

recording dynamic events in the microcirculation (Kiel and Shepherd, 1990). The technique is particularly suitable for evaluating the effects of drugs on the microcirculation under self-control conditions. Calibration of the laser Doppler flowmetry in absolute units of blood flow may not be necessary inasmuch as significant hypotheses can be tested by determining relative or directional changes in blood flow (Leung, 1992). Data obtained by this technique linearly correlated with those obtained with other currently established techniques for measuring blood flow such as electromagnetic flowmeter and hydrogen gas clearance techniques (Kiel and Shepherd, 1990; Leung, 1992).

71

Subcutaneous administration of cysteamine reduced gastric blood flow in accordance with the effects reported in the duodenum using the same route of administration (Kurebayashi et aE, 1985; Leung and Guth, 1985a). Topical application of cysteamine on the gastric serosa, however, evoked a pronounced and long-lasting enhancement of the microcirculation. This vasodilator effect can be demonstrated even when a very low concentration (0.2 mg/ml) of the agent was applied. The vasodilator response to topical cysteamine is not a sympathetic or vagal reflex since it was not prevented by cervical vagotomy or by vagotomy combined with sympathetic neuron blockade with guanethidine. Pretreatment with indomethacin at a dose which is known to block prostaglandin synthesis (Rainsford, 1983) did not prevent the vasodilator effect of topical cysteamine, suggesting that it is not mediated through prostaglandins. In the rat, histamine increases gastric mucosal blood flow via stimulation of HI- and possibly Hz-receptors (Guth and Leung, 1987). Further, cysteamine increases histamine-forming capacity of the rat’s gastric mucosa (Boesby et al, 1983). Accordingly, the effect of cysteamine was evaluated after HI and Hz-receptor blockade. Data, however, do not suggest that the effect of topical cysteamine is mediated via histamine release. It has been reported that cysteamine (900 mg/kg, po) resulted in selective depletion of CGRP in the rat stomach and duodenum. This effect of cysteamine was absent in capsaicin-desensitized rats, suggesting that the reduction in CGRP derives from a capsaicin-sensitive pool (Evangelista et al, 1992). We therefore, examined the hypothesis that the observed vasodilator effect of topical cysteamine might be mediated through the release of CGRP from capsaicin-sensitive sensory nerves. Thus, the vasodilator response to cysteamine was examined in rats pretreated with the capsaicin analogue resiniferatoxin (Maggi et al, 1990; Szolcsanyi et al, 1990; Szallasi and Blumberg, 1992). Resiniferatoxin in desensitizing doses did not prevent the vasodilator response to topically applied cysteamine, suggesting that the latter effect does not involve neuropeptide release of capsaicin-sensitive sensory nerve endings. Results suggest that the vasodilator effects of local cysteamine most likely to result from direct action of the drug on the vascular smooth muscles. Findings in the present study indicate that the route of administration determines not only the effect of cysteamine on gastric blood flow but also on gastric acid secretion. Systemic administration of the agent reduces gastric blood flow and produces an inhibition of gastric acid output that is most marked in the first 4 h following its administration. Topical application of cysteamine on the stomach enhances gastric blood flow, while its oral administration produces a marked in-

crease in gastric acid secretion in the 4-h pylorus-ligated rat. The opposite effects of cysteamine on the gastric microcirculation and on gastric acid secretion provide an explanation for both the ulcerogenic and gastro-protective effects previously reported or presented in this study following its systemic or intragastric administration, respectively. Inhibition of gastric acid secretion by cysteamine might play a role in the protective effect initially seen with sc cysteamine in 100 mg/kg in the early hours after its administration with indomethacin. It is possible that, with the higher dose of cysteamine, a reduction in gastric blood flow accounted for the loss of protection by SC cysteamine. Changes in gastric acid secretion do not play a role in the aggravation of lesions seen when cysteamine was given 4 h prior to indomethacin. Gastric acid output was even still lower in the cysteamine-treated groups than in control group. Studies by Ritchie (1975) and Whittle (1977) have shown that a reduction in mucosal blood flow in the presence of increased rate of H’backdiffusion following exposure to a mild barrier breaker results in severe gastric mucosal injury. Although we did not measure gastric blood flow for more than 1 h following cysteamine injection, Kurebayashi et ul (1985) using the hydrogen gas clearance technique reported a decrease in duodenal blood flow starting within 1 h and lasting for 10 h following a SC cysteamine dose of 300 mg/kg. Indomethacin, in addition, has been shown to inhibit prostaglandin synthesis (Rainsford, 1983), reduce gastric mucosal blood flow (Whittle, 1977) and to cause vascular endothelial damage (Rainsford, 1983; Nygard et al, 1995) under different experimental conditions. Thus it is possible that the combined microcirculatory derangement evoked by SCindomethacin and cysteamine in the presence of gastric acid accounted for the effect of cysteamine pretreatment on the indomethacin-induced gastric injury. A reduction in gastric blood flow thus could be an important factor in the late aggravation of lesions by SC cysteamine. Cysteamine depletes somatostatin in different tissues including the rat duodenum and glandular stomach and thus can be used to study the effect of local tissue deficiency of somatostation (Szabo and Reichlin, 1981; Szabo et al, 1992). The reduction in tissue levels of somatostatin starts within 1 h after its SC or oral administration and reaches maximum at 4 h and remains for several hours. Therefore, it is possible that enhancement of the indomethacin-induced gastric mucosal damage seen in the present study could be mediated at least in a part through blocking somatostatin release. The peptide exerts potent antiulcer effects (Die1 and Szabo, 1986) and this suggests a protective role for endogenous somatostatin.

72

Studies indicated a linkage between capsaicin, CGRP and somatostatin in the rat stomach. Cap&in is a potent stimulator of both CGRP and somatostatin release. The latter effect is mediated by CGRP in vivo and in vitro (Dunning and Taborsky, 1987; Inui et al, 1991; Ren et al, 1993; Sandvik et al, 1993). Further, data suggested the involvement of somatostatin in the gastro-protective effect of exogenously administered CGRP (Clementi et al, 1993). The latter is the most likely candidate for the antiulcer and vasodilator properties of capsaicin (Holzer et al, 1990; Evangelista, 1994; Maggi, 1995). Therefore, in the present study, pretreatment with cysteamine was used as a tool to investigate the possible role of somatostatin-depletion in the gastroprotective effects of caps&in. The present study shows that the vasodilator and anti-ulcer action of capsaicin was not prevented in cysteamine-treated rats. This indicates that somatostatin is not involved in the antiulcer effect of capsaicin. Indeed, it is unlikely for somatostatin to mediate the gastroprotective effect of capsaicin since: i) enhancement of the microcirculation contributes largely to the anti-ulcer action of capsaicin (Lippe et al, 1989; Holzer et al, 1991; Maggi, 1995); and ii) somatostatin does not increase gastric mucosal blood flow (Leung and Guth, 1985b) and even, somatostatin inhibited the intestinal vasodilatory effect of capsaicin in dogs (Rozsa et al, 1984). In conclusion, the present study demonstrated that cysteamine possesses different effects on gastric blood flow and gastric acid secretion. The blood flow data correlated well with the effect of this agent on the indomethacin-induced gastric mucosal damage. Cysteamine thus modulates gastric mucosal response to noxious injury in different ways depending on its route and time of administration in relation to damaging agents. The antiulcer effects of cap&in do not involve somatostatin.

Acknowledgments This study was supported by grants of the Hungarian National Research Fund (OTKA no T 020098 & T 016945) and the Ministry of Health and Welfare (ETT-03 660/93 & ETT-T 563).

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