Interaction of substance P and leukotriene C4 in ethanol-induced mucosal injury of rat stomach

Regulatory Pepndes, 46 (1993) 241-243

241

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Interaction of substance P and leukotriene C 4 in ethanol-induced mucosal injury of rat stomach M. Katori, T. Ohno and K. N i s h i y a m a Department of Pharmacology. Kttasato Umversay School of Me&cme, Kanagawa(Japan) Key words: Leukotriene

C4; Gastnc

mucosal injury; Capsaacin; Spantide; Substance P release

Introduction Intragastric administration of ethanol in concentrations of higher than 30% to rats resulted in mucosal injury of the stomach, which showed dark red in colour. This initial change was attributed to blood 'congestion' of the mucosal blood flow, not to bleeding [1]. Bleeding was observed 30 min later. The injured area of the gastric mucosa was reduced by pretreatment of rats with a 5-hpoxygenase inhibitor [1 ]. Exposure of the rat gastric mucosa to 30 ~o ethanol caused generation of leukotriene (LT) C4 in the gastric wall [2]. Immunohistochemical study with polyclonal antibody revealed that stained cells were scattered in the gastric mucosa and seemed to be the mucosal type mast cells. The connective tissue type mast cells in the submucosa of the stomach were not stained [ 3 ]. Furthermore, pretreatment of the stomach with capsaicin (0.16 and 1.6 mM) intragastncally or spantlde (0.1 mg/kg, i.v.) or calcitonln-gene related peptide (CGRP, 10 #M, intragastrically)reduced the Injured area induced by 30% ethanol, Correspondenceto M Katon, Department of Pharmacology,Katasato University School of Mechcme, Katasato 1-15-1, Sagamahaxa, Kanagawa 228, Japan_

whereas substance P (10 and 100 nmol/kg, i.v.) increased the injured area. It was also reported that substance P reduced degranulation of the connective tissue type mast cells to release histamine [3] The present study was performed by observation of the submucosal microclrculatlon to test the possibility that substance P may contribute to the mucosal injury directly or through release of LTC 4 from the mucosal type mast cells.

Materials and Methods SD strain male specific pathogen free rats were anesthetized by urethane (1.25 g/kg, i.p.). After laparotomy, the greater curvature was cut longitudinally The dorsal side of the glandular stomach was fixed on the mucosal side in the plastic chamber and perfused with warmed Tyrode solution at 37 ° C. Partial removal of the smooth muscle allowed us to observe the submucosal microcirculatlon with translllurmnation (Fig. 1). In the separate experiments, the stomach was cannulated from the esophageal and duodenal sides. Physiological saline, capsaicin (1.6 mM in 5~o gum arabic), or 30 or 50~o ethanol was administered to

242

S

surface. T h e caplUarles j o i n to the collecting v e n u l e s on the m u c o s a l surface a n d the latter r u n d o w n and j o i n to the s u b m u c o s a l m i c r o c l r c u l a t l o n . W h e n the s u b m u c o s a l m i c r o c i r c u l a t l o n w a s o b s e r v e d f r o m the

serosa

serosal side u n d e r the m i c r o s c o p e , the collecting v e n u l e s c a n be seen as the c r o s s section

Effect of Spantide (3 nmol/kg/min) Time Course After 50%EtOH (Arterioles) 5O0 Spanllden=4 ....e,,,, Vehiclen=3

400

E -6

300

c 20O "6 o-e

tO0

0 -10

0

10

mucosa Fig 1 Illustration of the observation method of the submucosal mlcrocIrculatlon under the light microscope Partial removal of the smooth muscle and attachment of a plastic window allowed to observe the submucosal mxcrocirculation with transdlummation The vascular structure of the mucosal mlcroclrculat]on ]s also Illustrated_

the gastric l u m e n t h r o u g h the e s o p h a g e a l c a n n u l a a n d 10 m i n later t h e y w e r e c o l l e c t e d t h r o u g h the d u o d e n a l c a n n u l a and the s u b s t a n c e P level m the gastric fluid w a s m e a s u r e d

20

30

40

Time (ram)

by E L I S A

(Cayman

C h e m t c a l Co., A n n A r b o r , M I , U S A ) .

Results and Discussion As seen in Fig 1, the arterioles m the s u b m u c o s a l microc~rculation o f the s t o m a c h , after p e n e t r a t i o n o f the l a m i n a m u s c u l a n s m u c o s a , b r a n c h o f f t o the c a p fllaries in the m u c o s a a n d run t o w a r d the m u c o s a l

Time Course After 50%EtOH

(Coll.venules)

140 ~)

120

'~

loo

_ == ~

so

"6

40

*

~

60

20 0 -10

m EtOH 0

~ Spanhden=7 ....6... V e h i c l e n=S 1°

2'0

30

40

Time (ram)

Fig 2 Effect of the close arterial Infusion of spantlde on changes of the diameters of the microvessels after exposure of the mucosa to 50~ ethanol. The upper and lower panels indicate changes of the diameters of the submucosal arterioles and of the collecting venules, respectively The dotted hnes indicate time course of the changes of the vascular diameters after exposure of the mucosa to 50 ~o ethanol dunng lnfuston of the vehicle (physlologacal saline), whereas the sohd lines show those dunng the artenal infusion of spant]de (3 nmol/kg/min) Mean + S_E

243 Application of 50~'o ethanol to the mucosal side caused the marked dilatation of the arterioles and marked constriction of the collecting venules, as seen by disappearance of the cross section. This may well explain the congestion in the gastric mucosa by ethanol Fig 2 shows time course of changes of microvessels in the gastric microclrculation after application of 5 0 ~ ethanol on the mucosal side. Ethanol application resulted in dilatation of the arterioles in the submucosa and long lasting constriction of the collecting venules with transient dilatation. Infusion of spantide (3 nmol/kg/mln) from the close gastric artery did not significantly inhibit the artenolar dilatation. This result is in agreement with observation of no dilatation of the arterioles after application of substance P to the submucosal microvessels. Spantlde slightly prolonged the dilating phase of the constriction of the collecting venules, indicating that substance P may contribute to the constriction partly, but the major part of the constrictlon was attributable to LTC4, since the strong constriction of the collecting venules was markedly inhibited by topical application of a peptide leukotrlene antagonist, ONO-1078 (10 #M) Administration of substance P on the microvessels in the submucosal rmcroctrculation induced constriction of the collecting venules, but this constriction was not inhibited by ONO-1078, indicating that substance P did not induce the venular constriction through release of LTC 4. In separate experiments, the substance P level in the gastric fluid was measured The level (mean + S E , ng/stomach/mm) was significantly in-

creased after 50 ~o ethanol (31.7 + 8.8, n = 3, P < 0.01) or capsalcln (24.8 + 10.2, n = 4, P < 0 . 0 5 ) for 10 min, comparing with that in physiological saline (0 9 + 0.1, n=5). In summary, exposure of the gastnc mucosa to 30-50~o ethanol induced the congestion of the mucosal blood flow by constriction of the collecting venules, together with release of substance P and LTC 4 in the gastric mucosa. LTC 4 may be released from the mucosal type mast cells, but the present results indicated that substance P may not release LTC 4 by degranulation of the mast cells. Also the direct constriction of the collecting venules by substance P was not supported, since only the earlier dilating phase of the constriction of the collecting venules was prolonged by spantide. LTC 4 may be the major factor to constrict the collecting venules. The further study with new selective antagonists against substance P is required for the confirmation of the observation

References 1 Katon, M, Nishlyama, K, Ueno, A. and Suzuki, Y, Possible role of endogenousprostaglandlns agmnst ethanol injury m rat stomach, J Clm Gastroenterol, 12 (Suppl. 1) (1990) $25-$31_ 2 Katon, M, Nlsh~yama,K, Ueno, A and Ohno, T, An Important role of leukotnene C4 m mlcroclrculatlon dunng ethanolreduced gastric mucosal injury m rats In Samuelsson, Bet al (Eds), Advancesin Prostaglandms,Thromboxane,Leukotnene Research, Vol 21, Raven Press, New York, 1990, pp 771776 3 Lowman,M A, Benyon,C R and Church, M_K, Characterlzatlon of neuropeptlde-mduced histamine release from human dispersed skin mast cells, Br J Pharmacol, 95 (1988) 121-130