Bile Nucleotides Exaggerate Ischemia–Reperfusion-Induced Epithelial Injury via P2Y, Not P2X Purinoceptor in Rat Jejunum

Bile Nucleotides Exaggerate Ischemia–Reperfusion-Induced Epithelial Injury via P2Y, Not P2X Purinoceptor in Rat Jejunum

Misa Mizumori

, Yasutada Akiba

, Soichiro Miura

, Hidekazu Suzuki

, Hiroshi Nagata

, and Hiromasa Ishii

Key words. P2 receptor, Bile acid, Ischemia–reperfusion, Jejunal epithelial injury, Intracellular pH

Introduction

Extracellular nucleotides have important roles to mediate and regulate the physiological functions such as neurotransmission and secretion [1,2]. Bile contains enough concentration of nucleotides to activate purinoceptors [3], such as P2X and P2Y adenosine triphosphate (ATP) receptor expressed in the lumen of epithelial cells [2]. However, the effects of luminal nucleotides on the intestinal epithelial functions are unknown. We have demonstrated that luminal taurocholic acid (TCA) acidifies epithelial cells and enhances ischemia–reperfusion (I/R)-induced epithelial injury in rat jejunum [4]. The present study was undertaken to examine the effects of luminal nucleotides with or without TCA on intracellular pH (pH

) and cell injury induced by I/R in rat jejunal epithelia.

115

1

Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan

2

CURE/UCLA & BBRI, Building 114, Suites 217, WLA VAMC, 11301 Wilshire Boulevard, Los Angeles, CA 90073, USA

3

Second Department of Internal Medicine, National Defense Medical College, 3-2 Namiki,

Tokorozawa-shi, Saitama 359-8513, Japan

Materials and Methods

Male Wistar rats (250–300 g) were used. Under urethane anesthesia (1.25 g/kg i.p.), the abdomen was opened and the posterior wall of the proximal jejunum 2 cm distal to the ligament of Treitz was exposed. Using in vivo fluorescent microscopy, pH

was measured with BCECF loaded into the epithelial cells, and epithelial injury was assessed by propidium iodide (PI) staining in vivo in situ as previously described for rat duodenum [5]. After the stabiliza- tion and time was set as t = 0, the jejunal mucosa was topically superfused through the mucosally-placed chamber with Krebs buffer (pH 7.4) for 5 min, followed by Krebs superfusion with or without TCA (20 mM), P2X agonist ab-methylene-ATP (abMeATP, 0.1 mM), P2X antagonist pyridoxal phosphate- 6 -azo (benzene-2,4-disulfonic acid) (PPADS, 0.1 mM), P2Y agonist 2- methylthio-ATP (2MeSATP, 0.1 mM), P2X/P2Y antagonist suramin (0.1 mM), or P2Y2 and P2Y4 agonist UTP (0.1 mM) (from t = 5 min to t = 35 min).

Ischemia–reperfusion was performed by a 5-min occlusion of the anterior mesenteric artery (ischemia, from t = 5 to t = 10) followed by the release of the occlusion (reperfusion, from t = 10 to t = 35).

Results

ab-Methylene-ATP and 2MeSATP acidified the epithelial cells, whereas PPADS and suramin alkalinized cells. Each chemical alone had no effect on PI-positive cell number. pH

was transiently decreased by 5 min I/R without cell injury. Under this “mild” I/R, 2MeSATP enhanced cell injury, whereas others did not. Taurocholic acid alone acidified cells without injury, whereas TCA under mild I/R further acidified cells and induced cell injury. 2- Methylthio-ATP exaggerated and suramin abolished TCA+I/R-induced injury. Furthermore, UTP enhanced TCA+I/R-induced injury by a suramin- inhibitable mechanism.

Discussion

We demonstrated that the cellular acidification by luminal TCA, luminal

P2 agonists, and mild ischemia potentiates triggering the epithelial injury

in rat jejunum. Furthermore, P2Y, not P2X receptor, exaggerates the TCA

toxicity and I/R-induced epithelial injury. Suramin, a nonspecific P2 receptor

antagonist, alkalinized cells, consistent with the observation in the bronchial

epithelium [6], and 2MeSATP, a P2Y receptor agonist, enhanced, but suramin

inhibited, the TCA-induced acidification, suggesting that P2Y receptor is

116 M. Mizumori et al.

spontaneously activated and regulates the TCA absorption under physiolog- ical condition presumably with bicarbonate secretion, since P2Y is involved in the bicarbonate secretion [7]. Furthermore, 2MeSATP exaggerated, but suramin reduced or abolished, the injurious effects of TCA+I/R and UTP, suggesting that P2Y (P2Y2 or P2Y4) activation is involved in the epithelial injury in the ischemic intestine. These findings may account for the chain injurious reaction of epithelial injury during the ischemia due to the leaked extracellular ATP. P2Y antagonist may be useful to reduce the epithelial injury in the ischemic intestine, e.g., surgical operation and small intestine transportation.

References

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3 . Chari RS, Schutz SM, Haebig JE, et al (1996) Adenosine nucleotides in bile. Am J Physiol 270 :G246–G252

4 . Mizumori M, Akiba Y, Miura S, et al (2002) Taurocholate, not tauroursodeoxycholate enhances ischemia-reperfusion-induced epithelial injury in rat jejunum (abstract). Gas- troenterology 122:M1151

5 . Akiba Y, Furukawa O, Guth PH, et al (2001) Cellular bicarbonate protects rat duodenal mucosa from acid-induced injury. J Clin Invest 108:1807–1816

6 . Urbach V, Helix N, Renaudon B, et al (2002) Cellular mechanisms for apical ATP effects on intracellular pH in human bronchial epithelium. J Physiol 543:13–21

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