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Ischemia/reperfusion-induced increase in the hepatic level of prostacyclin is mainly mediated by activation of capsaicin-sensitive sensory neurons in rats

NAOAKI HARADA, KENJI OKAJIMA, MITSUHIRO UCHIBA, and TAKESHI KATSURAGI


KUMAMOTO and FUKUOKA, JAPAN

Capsaicin-sensitive sensory neurons are nociceptive neurons that release calcitonin gene-related peptide (CGRP) on activation by various noxious stimuli. CGRP has been shown to increase the endothelial production of prostacyclin, which reduces ischemia/reperfusion (I/R)-induced liver injury. Therefore, if the sensory neurons can be activated by the pathologic process of hepatic I/R, they might help ameliorate I/R-induced liver injury by promoting the endothelial production of prostacyclin, also known as prostaglandin I2. In this study, we examined these possibilities using a rat model of I/R-induced liver injury. Male Wistar rats were subjected to 60-minute hepatic ischemia and subsequent reperfusion. Hepatic levels of 6-keto-prostaglandin F1 (6-keto-PGF1 ), a stable metabolite of prostacyclin, were significantly increased after hepatic I/R, peaking 1 hour after reperfusion. Administration of capsaicin and CGRP significantly enhanced I/R-induced increases in hepatic levels of 6-keto-PGF1 , increased hepatic-tissue blood flow after reperfusion, and inhibited the I/R-induced increase in tissue levels of both tumor necrosis factor- (TNF- ) and myeloperoxidase. Capsazepine, a vanilloid receptor antagonist; CGRP(8-37), a CGRP-receptor antagonist; L-nitro-arginine-methyl-ester (L-NAME), a nonselective inhibitor of nitric oxide (NO) synthase (NOS); and indomethacin, a nonselective inhibitor of cyclooxygenase, inhibited the I/R-induced increases in hepatic tissue levels of 6-keto-PGF1 and decreased hepatic-tissue blood flow after reperfusion. These compounds significantly enhanced the I/R-induced increases in hepatic tissue levels of both TNF- and myeloperoxidase. Although I/R-induced liver injury was significantly reduced by capsaicin and CGRP, it was exacerbated by capsazepine, CGRP(8-37), L-NAME, and indomethacin. Administration of aminoguanidine, a selective inhibitor of the inducible form of NOS, and NS-398, a selective inhibitor of cyclooxygenase-2, demonstrated no effects on the liver injury or the hepatic levels of 6-keto-PGF1 . These findings strongly suggest that the activation of the sensory neurons helps ameliorate I/R-induced liver injury both by increasing hepatic-tissue blood flow and by limiting inflammatory response through the enhancement of endothelial production of prostacyclin. In the sensory neuron-mediated enhancement of endothelial production of prostacyclin, CGRP-induced activation of both endothelial NOS and cyclooxygenase-1 may be critically involved. (J Lab Clin Med 2002;139:218-26)
Abbreviations: ALT alanine aminotransferase; AST aspartate aminotransferase; CGRP calcitonin gene-related peptide; CMC carboxymethyl cellulose; I/R, ischemia/reperfusion; L-NAME L-nitro-arginine-methyl-ester; NO nitric oxide; NOS nitric oxide synthase; TNFtumor necrosis factor-

From the Department of Laboratory Medicine, Kumamoto University School of Medicine, and the Department of Pharmacology, School of Medicine, Fukuoka University. Submitted for publication November 14, 2001; revision submitted November 27, 2001; accepted November 27, 2001. Reprint requests: Kenji Okajima, MD, Department of Laboratory

Medicine, Kumamoto University School of Medicine, Honjo 1-1-1, Kumamoto 860-0811, Japan; e-mail: whynot@kaiju.medic.kumamotou.ac.jp. Copyright 2002 by Mosby, Inc. 0022-2143/2002 $35.00 0 5/1/121856 doi:10.1067/mlc.2002.121856

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Capsaicin-sensitive sensory neurons are nociceptive neurons that are activated by a wide variety of noxious physical and chemical stimuli.1 Because ablation of the sensory bers can result in a marked increase in the severity of inammation,2 the sensory neurons have been shown to play a role in the maintenance of tissue integrity by regulating local inammatory response. On activation, the sensory neurons release CGRP, which can increase the endothelial production of prostacyclin, also known as prostaglandin I2.3 Prostacyclin is a wellknown cytoprotective agent that increases tissue blood ow and inhibits leukocyte activation.4 In light of the fact that various noxious stimuli that prompt sensory neurons to release CGRP are capable of inducing tissue damage,5 the CGRP-induced increase in endothelial production of prostacyclin may contribute to attenuate the local inammatory responses, thereby reducing tissue damage. TNF- , a proinammatory cytokine that activates the sensory neurons,6 plays important roles in the development of I/R-induced liver injury.7,8 We previously reported that iloprost, a stable derivative of prostacyclin, reduces I/R-induced liver injury both by increasing hepatic-tissue blood ow and by inhibiting leukocyte activation.9 These observations suggest that activation of capsaicin-sensitive sensory neurons contributes to reduction of I/R-induced liver injury by increasing endothelial production of prostacyclin. We examined this possibility using a rat model of I/R-induced liver injury. Furthermore, recent studies have demonstrated that CGRP increases endothelial production of NO10 and that NO, in turn, activates cyclooxygenase-1, thereby increasing endothelial production of prostaglandin.11 These observations suggest that CGRP increases endothelial production of prostacyclin through NO-mediated activation of cyclooxygenase-1. We examined that possibility as well in this study.
METHODS
Reagents. Capsaicin, capsazepine (an antagonist of capsaicin), L-NAME (a nonselective inhibitor of NOS), aminoguanidine (a selective inhibitor of inducible NOS), and indomethacin (a nonselective inhibitor of cyclooxygenase) were purchased from Sigma Chemical (St. Louis, Mo). NS-398 (a selective inhibitor of cyclooxygenase-2) was a generous gift from Taisho Pharmaceutical (Saitama, Japan). Rat -CGRP and human CGRP(8-37) were purchased from Peptide Institute (Osaka, Japan). All other reagents were of analytical grade. Administration of various agents. Capsazepine and capsaicin were dissolved in 10% Tween 20/10% ethanol (10%) with normal saline solution. We then injected 15 mg/kg of capsazepine subcutaneously 30 minutes before the induction of hepatic ischemia, as described previously.12 Capsaicin (1 mg/kg) was injected intraperitoneally 30 minutes before the

induction of hepatic ischemia to activate the sensory neurons, as described previously.13 L-NAME (5 mg/kg) and aminoguanidine (40 mg/kg) were dissolved in normal saline solution and administered subcutaneously 30 minutes before the induction of hepatic ischemia, as described previously.14,15 CGRP (10 g/kg) and CGRP(8-37) (100 g/kg) were dissolved in sterile distilled water and injected intravenously just before the induction of hepatic ischemia, as described previously.16 Indomethacin (20 mg/kg) was suspended in 5% sodium bicarbonate-buffered saline solution and injected subcutaneously 30 minutes before the induction of hepatic ischemia, as described previously.9 NS-398 (30 mg/kg) was suspended in 0.5% carboxymethyl cellulose aqueous suspension and administered orally 1 hour before the induction of hepatic ischemia, as described previously.17 Solutions were prepared immediately before experiments. Each control animal received the vehicle in question. Animal model of hepatic I/R. Adult male pathogen-free Wistar rats (Nihon SLC, Hamamatsu, Japan), weighing 220 to 280 g, were used in each experiment. Care and handling of the animals were carried out in accordance with National Institutes of Health guidelines. All experimental procedures described below were approved by the Kamamoto University Animal Care and Use Committee. All rats were deprived of food, but not water, for 24 hours before each experiment. The hepatic I/R protocol was performed as described previously.18,19 After the induction of anesthesia with ketamine hydrochloride administered intraperitoneally (100 mg/kg; Parke-Davis, Morris Plains, NJ), the liver was exposed through a midline laparotomy. Silk ligatures were placed around the right and left branches of the portal vein and hepatic artery. Complete ischemia of the median and left hepatic lobes was produced by clamping of the left branches of the portal vein and hepatic artery for 60 minutes. The right hepatic lobe was perfused to prevent intestinal congestion. During the period of hepatic ischemia, the animals abdomen was covered with plastic wrap to prevent dehydration. After 60 minutes of ischemia, the ligatures were removed. To accurately evaluate blood ow in the median and left hepatic lobes after ischemia, we ligated the right branch of the portal vein and the hepatic artery to prevent shunting to the right lobe after reperfusion.18 The wound was closed with 3-0 silk. This procedure directed all portal and hepatic blood ow, except for a small amount to the caudal hepatic lobe, through the lobes of the liver previously made ischemic. Sham-operated animals were similarly prepared, but no ligatures were placed. Instead, blood ow to the right lobe of the liver was occluded. Determination of hepatic 6-keto-PGF1 levels. Because we have previously demonstrated that hepatic 6-keto-PGF1 levels are increased after I/R, peaking after 1 hour of reperfusion,9 we measured hepatic 6-keto-PGF1 levels 1 hour after reperfusion, in accordance with methods described previously.9 One hour after reperfusion, each animal was anesthetized by the intraperitoneal injection of ketamine hydrochloride (100 mg/kg) and exsanguinated via the abdominal aorta. The medial hepatic lobe in which blood ow had been measured was weighed and then immediately homogenized in

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5 mL of 0.1 mol/L phosphate buffer (pH 7.4) containing 2 mmol/L indomethacin at 5C. The homogenate was rst centrifuged at 2000g for 10 minutes to remove minute amounts of solid debris; then the supernatant was acidied with 1 mol/L HCl. 6-keto-PGF1 was extracted from the supernatant with the use of columns packed with ethylbonded silica gel (Amersham, Buckinghamshire, UK). The columns were prepared by washing with 2 mL of methanol, followed by 2 mL of water. The acidied supernatant was applied to the column, followed by sequential washes with 5 mL of water, 5 mL of 10% ethanol, and 5 mL of hexane. The elution of 6-keto-PGF1 was conducted with 5 mL of methyl formate, after which the solvent was evaporated under a stream of nitrogen gas. The concentration of 6-keto-PGF1 was assayed with a specic enzyme immunoassay kit (Amersham). This enzyme immunoassay was sensitive enough to detect 10 to 1280 pg/mL of 6-keto-PGF1 . The results are expressed as nanograms of 6-keto-PGF1 per gram of tissue. The cross-reactivities of this assay with prostaglandin E2, prostaglandin F2 , thromboxane B2, and arachidonic acid were 2.8%, 1.4%, 0.03%, and 0.01%, respectively, according to the manufacturers data sheet. The maximum level of prostaglandin E2 in the liver of this animal model was about 4 ng/g tissue. The tissue level of prostaglandin E2 was assayed with a specic enzyme immunoassay kit (Amersham) as described previously.20 Measurement of serum liver enzymes. We took blood samples 12 hours after reperfusion to measure the levels of serum ALT and AST as described previously.8 These blood samples were collected into test tubes from the abdominal aorta of each anesthetized animal with the use of a 22-gauge needle. ALT and AST levels were measured by means of standard clinical automated analysis; ndings are expressed as international units per liter. Measurement of hepatic-tissue blood flow. Hepatic-tissue blood ow was measured by means of laser-Doppler owmeter (ALF21N; Advance, Tokyo, Japan) for 3 hours after reperfusion, as described previously.9 After the induction of anesthesia with intraperitoneal ketamine hydrochloride (100 mg/kg), the right jugular vein was cannulated with a PE-10 catheter for continuous infusion of normal saline solution or test drugs. A Doppler owmeter probe was placed on the medial hepatic lobe. Hepatic-tissue blood ow was measured from 30 minutes before ischemia to 3 hours after reperfusion. Findings are expressed as a percentage of the preischemia level. Determination of hepatic levels of TNF- . Because we have previously demonstrated that hepatic TNF- levels are increased after I/R, peaking after 1 hour of reperfusion,21 we measured hepatic TNF- 1 hour after reperfusion in this study, in accordance with methods described previously.21 One hour after reperfusion, each animal was anesthetized with an intraperitoneal injection of ketamine hydrochloride (100 mg/kg) and exsanguinated through the abdominal aorta. The medial hepatic lobe was weighed and then homogenized in 5 mL of 0.1 mol/L phosphate buffer (pH 7.4) containing 0.05% (vol/wt) sodium azide at 5C. The homogenate was rst centrifuged at 2000g for 10 minutes to remove minute

amounts of solid debris. The supernatant was assayed with a Rat TNF enzyme-linked immunosorbent assay(Amersham). This assay is sensitive enough to detect 31 to 2500 pg/mL TNF- . Results are expressed as picograms of TNF- per gram of tissue. Determination of hepatic myeloperoxidase activity. Because we have previously demonstrated that hepatic myeloperoxidase activity, which reects the tissue accumulation of neutrophils in the liver, was increased after I/R, peaking 6 hours after reperfusion,9 we measured hepatic myeloperoxidase activity 6 hours after reperfusion, in accordance with a previously described method.9 After 6 hours of reperfusion, the livers were quickly removed and the accumulation of leukocytes assessed through measurement of myeloperoxidase activity. In brief, the livers were weighed and suspended in 6 mL of 50 mmol/L phosphate buffer (pH 6.0) containing 1% hexadecyltrimethylammonium bromide. The samples were homogenized, after which the homogenate was sonicated, freeze-thawed, and centrifuged (4500g for 15 minutes at 4C). Myeloperoxidase activity in the supernatant (0.1 mL) was determined after the addition of 0.6 mL phosphate buffer (pH 6.0) containing 0.167 mg/mL o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. The change in absorbance at 460 nm over 10 minutes was measured in a spectrophotometer (DU-54; Beckman, Irvine, CA). One unit of myeloperoxidase activity was dened as the amount of enzyme needed to reduce 1 mol peroxide/min. Results are expressed as units of myeloperoxidase activity per gram of tissue. Five separate groups of animals (n 40, each group) were used to assess hepatic 6-keto-PGF1 levels, liver-enzyme levels, tissue blood ow, hepatic TNF- levels, and hepatic myeloperoxidase activity. This procedure avoided problems associated with excessive blood loss resulting from blood sampling or associated with uid loss caused by blood-ow measurement. Statistical analysis. Data are expressed as mean SD. We compared the results with the use of analysis of variance followed by Scheffes post hoc test or with an unpaired Student t test. We considered P values less than 0.05 statistically signicant. RESULTS
Effects of capsazepine, CGRP(8-37), L-NAME, aminoguanidine, indomethacin, and NS-398 on I/R-induced increases in hepatic-tissue levels of 6-keto-PGF1 after reperfusion. Hepatic-tissue levels of 6-keto-PGF1 in-

creased after hepatic I/R, peaking 1 hour after reperfusion.9 Capsaicin (1 mg/kg, intraperitoneally) and CGRP (10 g/kg, intravenously) signicantly enhanced I/Rinduced increases in hepatic-tissue levels of 6-ketoPGF1 (Fig 1, A and C). Capsazepine (15 mg/kg, subcutaneously) and CGRP(8-37) (100 g/kg, intravenously) signicantly inhibited I/R-induced increases in hepatic-tissue levels of 6-keto-PGF1 1 hour after reperfusion (Fig 1, B and C). L-NAME (5 mg/kg, subcutaneously) and indomethacin (20 mg/kg, subcu-

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Fig 1. Effects of (A) capsaicin, (B) capsazepine, (C) CGRP(8-37) and CGRP, (D) NOS inhibitors L-NAME and aminoguanidine, and cyclooxygenase inhibitors (E) indomethacin and (F) NS-398 on I/R-induced increase in hepatic-tissue levels of 6-keto-PGF1 1 hour after reperfusion. Animals were injected intraperitoneally with capsaicin (1 mg/kg) 30 minutes before the induction of hepatic ischemia. Animals were injected subcutaneously with capsazepine (CPZ, 15 mg/kg), L-NAME (5 mg/kg), aminoguanidine (AG, 40 mg/kg), or indomethacin (IM, 20 mg/kg) 30 minutes before the induction of hepatic ischemia. Animals were injected intravenously with CGRP (10 g/kg) and CGRP(8-37) (100 g/kg) immediately before the induction of ischemia. NS-398 (30 mg/kg) was administered orally 1 hour before the induction of ischemia. Each control animal received the vehicle, as described in the Methods section. Each bar represents the mean SD. *P .01 vs I/R vehicle; NS, not signicantly different vs I/R vehicle.

taneously) signicantly inhibited I/R-induced increases in hepatic-tissue levels of 6-keto-PGF1 (Fig 1, D and E), but neither aminoguanidine (40 mg/kg, subcutaneously) nor NS-398 (30 mg/kg, orally) showed any effects (Fig 1, D and F).
Effects of capsazepine, CGRP(8-37), L-NAME, aminoguanidine, indomethacin, and NS-398 on hepatic-tissue blood flow after reperfusion. Although capsaicin (1 mg/

Effects of capsaicin, capsazepine, CGRP(8-37), L-NAME, aminoguanidine, indomethacin, and NS-398 on I/R-induced increases in hepatic-tissue levels of TNF- and myeloperoxidase. Although capsaicin (1 mg/kg, intra-

kg, intraperitoneally) and CGRP (10 g/kg, intravenously) markedly increased hepatic-tissue blood ow after reperfusion (Fig 2, A and C), capsazepine (15 mg/kg, subcutaneously) and CGRP(8-37) (100 g/kg, intravenously) signicantly decreased hepatic-tissue blood ow after reperfusion compared with our ndings in controls (Fig 2, B and C). L-NAME (5 mg/kg, subcutaneously) and indomethacin (20 mg/kg, subcutaneously) signicantly decreased hepatic-tissue blood ow after reperfusion (Fig 2, D and E), but neither aminoguanidine (40 mg/kg, subcutaneously) nor NS398 (30 mg/kg, orally) affected hepatic-tissue blood ow (Fig 2, D and F).

peritoneally) and CGRP (10 g/kg, intravenously) signicantly inhibited I/R-induced increases in hepatictissue levels of both TNF- and myeloperoxidase (Fig 3, A, B, E, and F), capsazepine (15 mg/kg, subcutaneously) and CGRP(8-37) (100 g/kg, intravenously) enhanced those increases (Fig 3, C D, E, and F). L-NAME (5 mg/kg, subcutaneously) and indomethacin (20 mg/ kg, subcutaneously) enhanced the I/R-induced increase in hepatic-tissue levels of both TNF- and myeloperoxidase (Fig 3, G, H, I, and J), whereas neither aminoguanidine (40 mg/kg, subcutaneously) nor NS-398 (30 mg/kg, orally) showed any effects (Fig 3, G, H, K, and L).
Effects of capsaicin, capsazepine, CGRP(8-37), L-NAME, aminoguanidine, indomethacin, and NS-398 on I/R-induced increases in serum levels of transaminases after reperfusion. Serum levels of transaminases were in-

creased after hepatic I/R, peaking 12 hours after reper-

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Fig 2. Effects of (A) capsaicin, (B) capsazepine, (C) CGRP(8-37) and CGRP, (D) NOS inhibitors L-NAME and aminoguanidine, and cyclooxygenase inhibitors (E) indomethacin and (F) NS-398) on I/R-induced changes in hepatic-tissue blood ow. Animals were injected intraperitoneally with capsaicin (1 mg/kg) 30 minutes before the induction of hepatic ischemia. Animals were injected subcutaneously with capsazepine (15 mg/kg), L-NAME (5 mg/kg), aminoguanidine (40 mg/kg), or indomethacin (20 mg/kg) 30 minutes before the induction of ischemia. Animals were injected intravenously with CGRP (10 g/kg) or CGRP(8-37) (100 g/kg) immediately before the induction of ischemia. NS-398 (30 mg/kg) was administered orally 1 hour before the induction of ischemia. Each control animal received the vehicle, as described in the Methods section. Each value represents the mean SD derived from 5 animals. A, open circle I/R vehicle; closed circle I/R capsaicin. B, open circle I/R vehicle; closed circle I/R capsazepine. C, open circle I/R vehicle; closed circle I/R CGRP; open box I/R CGRP(8-37). D, open circle I/R vehicle; closed circle I/R L-NAME; closed triangle I/R aminoguanidine. E, open circle I/R vehicle; closed circle I/R indomethacin. F, open circle I/R vehicle; closed circle I/R NS-398. *P .01 vs I/R vehicle.

fusion.8 Although capsaicin (1 mg/kg, intraperitoneally) and CGRP (10 g/kg, intravenously) markedly inhibited I/R-induced increases in serum levels of transaminases (Fig 4, A, B, E, and F), capsazepine (15 mg/kg, subcutaneously) and CGRP(8-37) (100 g/kg, intravenously) signicantly enhanced those increases (Fig 4, C, D, E, and F). L-NAME (5 mg/kg, subcutaneously) and indomethacin (20 mg/kg, subcutaneously) signicantly enhanced I/R-induced increases in serum levels of transaminases (Fig 4, G, H, I, and J), whereas neither aminoguanidine (40 mg/kg, subcutaneously) nor NS-398 (30 mg/kg, orally) showed any effects (Fig 4, G, H, K, and L).
DISCUSSION

In this study, both capsaicin and CGRP had signicantly enhanced I/R-induced increases in hepatic-tissue levels of 6-keto-PGF1 1 hour after reperfusion, and both capsazepine, a vanilloid receptor antagonist, and CGRP(8-37), a CGRP-receptor antagonist, had significantly inhibited them, suggesting that CGRP released on the activation of capsaicin-sensitive sensory neurons is involved in the I/R-induced increase in hepatic tissue level of 6-keto-PGF1 . These in vivo observations are consistent with previous in vitro ndings showing that

CGRP increased the release of prostacyclin from cultured human umbilical vein endothelial cells.3 Although both capsazepine and CGRP(8-37) signicantly inhibited the I/R-induced increase in hepatic-tissue level of 6-keto-PGF1 , the inhibitory effect of capsazepine appeared more predominant than that of CGRP(8-37) (Fig 1). Because capsazepine inhibits acetylcholine receptors22 and acetylcholine is involved in the endothelial production of prostacyclin,23 capsazepine may also inhibit the acetylcholine-induced endothelial production of prostacyclin, accounting for the apparent difference between effects of capsazepine and CGRP(8-37) on the hepatic-tissue levels of 6-ketoPGF1 in this study. CGRP has been shown to induce vasorelaxation by increasing the endothelial production of NO.10 Clancy et al11 demonstrated that NO activated cyclooxygenase-1 selectively, thereby increasing the endothelial production of prostaglandin in vitro. Consistent with this nding is our observation that the I/R-induced increase in the hepatic level of 6-keto-PGF1 was inhibited by indomethacin and by L-NAME, a nonselective inhibitor of NOS, but not by NS-398, a selective inhibitor of cyclooxygenase-2. Because aminoguanidine, a selective inhibitor of inducible NOS, did not

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Fig 3. Effects of capsaicin (A and B), capsazepine (C and D), CGRP(8-37) and CGRP (E and F), NOS inhibitors L-NAME and aminoguanidine (G and H) and cyclooxygenase inhibitors indomethacin and NS-398 (I, J, K, and L) on I/R-induced increases in hepatic-tissue levels of myeloperoxidase and TNF- . Animals were injected intraperitoneally with capsaicin (1 mg/kg) 30 minutes before the induction of hepatic ischemia. Animals were injected subcutaneously with capsazepine (CPZ, 15 mg/kg), L-NAME (5 mg/kg), aminoguanidine (AG, 40 mg/kg), or indomethacin (IM, 20 mg/kg) 30 minutes before the induction of ischemia. Animals were injected intravenously with CGRP (10 g/kg) or CGRP(8-37) (100 g/kg) immediately before the induction of ischemia. NS-398 (30 mg/kg) was administered orally 1 hour before the induction of ischemia. Each control animal received the vehicle, as described in the Methods section. Each bar represents the mean SD. *P .01 vs I/R vehicle; NS, not signicantly different vs I/R vehicle.

affect the I/R-induced increase in hepatic levels of 6-keto-PGF1 , the NO produced by endothelial NOS may activate cyclooxygenase-1, thereby increasing the endothelial production of prostacyclin in this animal model. Hajjar et al24 showed that S-nitrosylation of cysteine residue of cyclooxygenase-1 by NO may be important in the activation of cyclooxygenase-1.

Prostacyclin, a well-known cytoprotective agent, is synthesized in endothelial cells.25 In light of the fact that prostacyclin has a vasodilatory action, the CGRPinduced increase in endothelial production of prostacyclin in the liver may increase hepatic-tissue blood ow. Consistent with this hypothesis is the observation made in our study.

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Fig 4. Effects of capsaicin (A and B), capsazepine (C and D), CGRP and CGRP(8-37) (E and F), NOS inhibitors L-NAME and aminoguanidine (AG) (G and H), and cyclooxygenase inhibitors indomethacin (IM) and NS-398 (I, J, K, and L) on I/R-induced increases in serum levels of transaminases 12 hours after reperfusion. Animals were injected intraperitoneally with capsaicin (1 mg/kg) 30 minutes before the induction of hepatic ischemia. Animals were injected subcutaneously with capsazepine (CPZ, 15 mg/kg), L-NAME (5 mg/kg), aminoguanidine (AG, 40 mg/kg), or indomethacin (IM, 20 mg/kg) 30 minutes before the induction of ischemia. Animals were injected intravenously with CGRP (10 g/kg) or CGRP(8-37) (100 g/kg) immediately before the induction of ischemia. NS-398 (30 mg/kg) was administered orally 1 hour before the induction of ischemia. Serum levels of transaminases were determined after 12 hours of reperfusion. Each control animal received the vehicle, as described in the Methods section. Each bar represents the mean SD. P .01 vs sham. *P .01 vs I/R vehicle. NS, not signicantly different vs I/R vehicle.

Activated leukocytes were suggested to play a pivotal role in I/R-induced liver injury by releasing inammatory mediators that are capable of damaging endothelial cells, leading to microcirculatory disturbances.26-28 Prostacyclin has been shown to inhibit monocytic production of TNF.29 TNF- activates both endothelial cells and neutrophils, thereby increasing the accumulation of neutrophils at the endothelial-cell surface, where activated neutrophils

damage the endothelial cell by releasing the inammatory mediators.30 This hypothesis is supported by our previous ndings showing that iloprost, a stable derivative of prostacyclin, reduces I/R-induced liver injury by inhibiting I/R-induced increases in hepatic-tissue levels of cytokineinduced neutrophil chemoattractant, a family of human interleukin-8, and myeloperoxidase.9 These observations strongly suggest that in this study, the CGRP-mediated

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increase in endothelial production of prostacyclin helped reduce liver injury by increasing hepatic-tissue blood ow and inhibiting leukocyte activation. However, the increase in endothelial production of prostacyclin induced by activation of capsaicin-sensitive sensory neurons may not be adequate to reduce I/R-induced liver injury; serum levels of transaminases were increased 12 hours after reperfusion. CGRP, administered exogenously, enhanced I/Rinduced increases in hepatic-tissue levels of 6-ketoPGF1 , thereby increasing hepatic-tissue blood ow, inhibiting local inammatory responses, and reducing inammation of the liver, supporting the hypothesis described above. CGRP is known to be released from both capsaicinsensitive sensory neurons and insensitive sensory neurons.31 However, the effects of capsazepine on I/Rinduced changes in hepatic-tissue levels of both TNFand myeloperoxidase, as well as serum levels of transaminases, were similar to those of CGRP(8-37). These observations suggest that CGRP released from capsaicin-sensitive sensory neurons plays a central role in reduction of liver injury. Because CGRP itself has been shown to inhibit nuclear factor B activation, thereby inhibiting TNFproduction,32,33 it is possible that CGRP is also involved in inhibition of TNF- production. However, this hypothesis appears far-fetched, because inhibition of prostaglandin synthesis by indomethacin enhanced I/R-induced increases in hepatic-tissue levels of TNFand myeloperoxidase and exacerbated liver injury. The major part of anti-inammatory responses induced by activation of the sensory neurons may be explained by the actions of prostacyclin. Capsaicin-sensitive sensory neurons are activated by physical and chemical stimuli, including heat,34 protons,35 histamine,36 and interleukin-1 .36 Increases in tissue levels of these substances could excite nociceptors and evoke pain sensation. In light of the possibility that such noxious substances that activate the sensory neurons induce tissue damage, CGRP released from the nerve ends of the sensory neurons in response to such stimuli may play a role in the attenuation of that tissue damage. These observations strongly suggest that capsaicin-sensitive sensory neurons play a role not only in the sensory nervous system but in the cytoprotective system by increasing the endothelial production of prostacyclin.
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