Although occasional reflux of gastric contents into

Transcription

1 GASTROENTEROLOGY 1996;11: Mast Cells Mediate Aid-Indued Augmentation of Opossum Esophageal Blood Flow Via Histamine and Nitri Oxide MICHAEL J. FELDMAN, GERALD P. MORRIS, PARITOSH K. DINDA, and WILLIAM G. PATERSON Gastrointestinal Diseases Researh Unit, Hotel Dieu Hospital and Queen's University, Kingston, Ontario, Canada Bakground & Aims: Inreased esophageal blood flow during reflux episodes may play an important role in muosal resistane to injury, although the mehanism remains unlear. Dereased stainable mast ells and inreased luminal histamine release during aid exposure has been previously doumented. Therefore, the role of mast ells, nerves, histamine, and nitri oxide in mediating inreased blood flow during aid hallenge of the distal esophagus was investigated. Methods: The effets of the mast ell stabilizers disodium romoglyate and doxantrazole, the neurotoxin tetrodotoxin, the histamine H1. reeptor antagonist promethazine, and the NO synthase inhibitor NW-nitro-L-arginine methyl ester were examined by monitoring opossum esophageal histamine release and blood flow during perfusion with 1 mmoljl He!. Results: Luminal aid hallenge signifiantly inreased both histamine release and blood flow (P <.5). Disodium romoglyate, promethazine, and NW-nitro-L-arginine methyl ester attenuated the inrease in blood flow to basal (saline-perfused) levels. Tetrodotoxin did not prevent an aute inrease in blood flow that rapidly returned to baseline, likely from the ensuing hypotension. Conlusions: These findings provide evidene that mast ellderived histamine, ating through an NO-dependent mehanism, plays a entral role in the response of the esophageal miroirulation to luminal aid. Although oasional reflux of gastri ontents into the distal esophagus ours in normal individuals, 1 frequent or prolonged reflux episodes may lead to the development of esophagitis. The apaity of the esophagus to resist the damaging effets of gastri aid depends on a number of defense mehanisms, inluding lower esophageal sphinter funtion, esophageal learane, biarbonate and muous seretion, and intrinsi muosal resistane to injury.2 Inreased muosal blood flow during luminal aid exposure may enhane the ability of the esophageal epithelium to resist damage by maintaining the interstitial aid-base balane. The protetive effets of inreased blood flow are likely the result of enhaned delivery of plasma biarbonate for buffering of hydrogen ions within the muosa, although delivery of oxygen and nutrients and removal of aidi metaboli by-produts may also ontribute to muosal defenses. 3 Although the ontribution of inreased blood flow in muosal defenses has been well doumented in other regions of the gastrointestinal trat, the mehanism and signifiane of blood flow hanges in the esophagus remain unlear. A number of studies have explored the role of sensory neurons in inreasing esophageal blood flow and in muosal protetion. Stimulation of these apsaiin-sensitive nerves has been shown to inrease blood flow 4 and to enhane epithelial resistane to injury.5 However, topial intraluminal lidoaine at onentrations suffiient to blok apsaiin-indued hyperemia had no effet on aidindued inreases in blood flow. 6 More reently, MKie et al. have suggested that alitonin gene-related peptide release in response to luminal sodium deoxyholate inreases esophageal blood flow. 7 Previous investigators in our laboratory have doumented inreased histamine release and dereased stainable mast ell numbers assoiated with the development of esophageal muosal injury following luminal exposure to aid. s We therefore hypothesized that the release of mast ell-derived vasoative mediators ould aount for inreased blood flow in response to luminal aid. The present study was designed to examine the role of esophageal mast ells and enteri nerves in mediating the inreased muosal blood flow found early in the ourse of aid-indued esophageal muosal injury and to investigate possible pathways by whih these effets are mediated. Preliminary data have been reported in abstrat form. 9,lo Materials and Methods Animal Preparation Ethial approval was obtained from the Queen's University Animal Care Committee in aordane with guidelines established by the Canadian Counil on Animal Care. Fasted Abbreviations used in this paper: o-name, IV'"-nitro-o-arginine methyl ester; DOX, doxantrazole; DSCG, disodium romoglyate; L NAME, IV'"-nitro-L-arginine methyl ester; PMZ, promethazine; TTX, tetrodotoxin by the Amerian Gastroenterologial Assoiation /96/$3.

2 122 FELDMAN ET AL. GASTROENTEROLOGY Vol. 11, No.1 adult opossums (Didelphis virginiana) of either sex weighing kg were anesthetized with pentobarbital sodium (4 mg/kg given intravenously) and maintained with intermittent injetions of pentobarbital sodium (5-1 mg/kg). The animals were intubated and mehanially ventilated (Harvard Rodent Ventilator model 683; Ealing Sientifi ltd., St. laurent, Quebe, Canada) at a rate and tidal volume speified by the Harvard Apparatus Co. (South Natik, MA) for the animals' body weight. The right femoral artery was annulated for the monitoring of blood pressure. The right femoral vein was annulated to maintain venous aess for injetion of drugs and fluid replaement. A laparotomy was performed and the region of the lower esophageal sphinter was annulated in a retrograde fashion using a tapered onnetor (size 12; Belart, Pequannok, NJ) inserted via a gastrostomy and attahed to a 15-m length of Tygon tubing (ID, 3 mm; OD, 5 mm). This was held in plae with a ligature plaed at the gastroesophageal juntion, taking are not to disturb the gastri arteries, and was used for olleting esophageal effluent. A atheter for esophageal luminal perfusion was passed orally and advaned until the atheter tip was loated by palpation at the gastroesophageal juntion. The atheter was withdrawn 7. m and seured with a suture to the animal's lip. A fiber-opti endosopi side probe was also passed in a similar manner and seured 3.5 m above the gastroesophageal juntion to allow laser Doppler monitoring of blood flow in the esophageal wall (probe model PR-436, laser Doppler flowmeter model BPM 2 ; Vasamedis In., St. Paul, MN). The esophagus was perfused with.9% saline (normal saline) prewarmed to 37 C at a rate of 2 mlimin using a peristalti assette pump (Manostat, New York, Ny) for 3 minutes to allow for reovery from the surgial manipulation. Experimental Protool The animals were divided into 11 groups with eah group ontaining at least 5 animals. After the 3-minute stabilization period, normal saline was perfused in all groups for an additional 2 minutes (basal period). The perfusate was then hanged to prewarmed solutions of either normal saline (ontrol group), 1 mmolll HCl made isotoni with NaCl (8 groups), or 1 mmolll histamine in normal saline (2 groups). Perfusion of saline, aid, or histamine solutions was ontinued for 6 minutes (experimental period). Blood flow throughout the experimental perfusion was reorded in units of ml' min g -1 tissue at 1-minute intervals, eah reading refleting the average blood flow over the preeding 5 minutes (flowmeter set for ontinuous averaging). The luminal effluent was also olleted during 1-minute intervals for subsequent determination of histamine release. Thirty minutes before the start of the experimental perfusion period, animals in the saline-perfused ontrol group reeived a sham intravenous injetion of vehile (4 mllkg normal saline), whereas animals in the aid-perfused groups reeived either a sham injetion of vehile or one of the following drugs: 8 mg/kg disodium romoglyate (DSCG), 6 mg/ kg doxantrazole (DOX), 1.5 mg' kg -1 promethazine (PMZ) loading dose followed by 5 mg. kg -1 h -1 ontinuous infusion (we have determined this dose bloks the utaneous erythema, weal and flare response to intradermal injetion of 5 Jll of 1 % histamine), 3 mg/kg NW-nitro-L-arginine methyl ester (L-NAME), and 3 mg/kg NW-nitro-D-arginine methyl ester (D-NAME). The doses of mast ell stabilizers were seleted based on their ability to redue mast ell-mediated gastrointestinal damage in previous studies ll,12 and show few nonspeifi effets,13,14 whereas the dose of L-NAME has been used in our laboratory to inhibit nitri oxide-dependent lower esophageal sphinter relaxation. Animals in the histamineperfused group reeived injetion of either vehile or 3 mg/ kg L-NAME 3 minutes before the start of the experimental perfusion. To evaluate the possible ontribution of nerves in mediating responses to aid perfusion, a separate group of 1 mmolll HCl-perfused animals was pretreated with repeated 5 Jlg/kg boluses of tetrodotoxin (TTX) at 2-minute intervals to a total dose that was titrated to ahieve omplete inhibition of esophageal ontrations evoked by eletrial stimulation of the right ervial vagus (stimulation parameters: amplitude, 8 V; duration, 1 milliseond; frequeny, 1 Hz; train length, 3 seonds) or mehanial pharyngeal stimulation. 15 For these experiments, esophageal ontrations were monitored using a perfused manometri atheter plaed in the distal esophagus. The responses to eletrial and mehanial stimulation were retested following the experimental perfusion to verify the persistene of the neural blokade. To ounter the hypotension indued by TTX, animals reeived 2 mllkg 1% Dextran 4 infused during a period of 1 hour for volume expansion. To ontrol for hypotension aused by TTX, a group of animals was exsanguinated to produe an equivalent derease in blood pressure during aid perfusion. Animals were killed by pentobarbital overdose after the experimental perfusion period. Determination of Luminal Histamine Release Eah of the effluent samples was neutralized to ph 7. with 5N NaOH, and 9.-ml aliquots were removed. The aliquots were deproteinized using 1. ml of 3% trihloroaeti aid, vortexed briefly, and entrifuged for 1 minutes at 4 C at 15, rpm (Sorvall entrifuge model RC2-B, rotor speifiation SS-34). Histamine levels were determined using a modified method desribed by Shore et al. 16 Briefly, 2.6-ml aliquots were alkalinized to ph 12.3 and inubated with 3 mmolll rerystallized o-phthaldialdehyde for 7 minutes at 25 C. The produt of the ondensation reation between histamine and o-phthaldialdehyde was stimulated at 365 nm and emitted fluoresene at 444 nm, whih was read on a fluorometer (Sequoia-Turner model 45; Sequoia-Turner, Mountainview, CA). The readings were ompared with a standard urve onstruted using solutions ontaining known quantities of histamine. Materials Sodium pentobarbital (Somnotol) was obtained from MTC Pharmaeutials (Cambridge, Ontario, Canada). DSCG,

3 January 1996 MAST CELL REGULATION OF ESOPHAGEAL BLOOD FLOW 123 PMZ, L-NAME, D-NAME, TTX, and histamine were obtained from Sigma Chemial Co. (Sr. Louis, MO). DSCG, L-NAME, D-NAME, PMZ, and histamine were dissolved in normal saline to the appropriate onentrations immediately before use. TTX was prepared as a 1-4 molll stok solution in normal saline and stored at 4 C until use. DOX was provided by Burroughs Wellome In. (Researh Triangle Park, NC) and was dissolved to 1 mg/ml in.1 molll NaHC 3 while heating to 4 C. Ten perent Dextran 4 in.9% NaCl (Gentran) was obtained from Baxter Corp. (Toronto, Ontario, Canada). Statistis Calulations were performed on an IBM PC-ompatible omputer using SYSTAT (SYSTAT, Evanston, IL) analytial software. Group mean differenes were evaluated using repeated measures analysis of variane. Unpaired Student's t tests were used to evaluate effets of drug treatments or perfused solutions on basal blood flow alulated from the mean blood flow during 2-minute saline perfusion. All data are shown as means ± SEM. Blood flow values obtained by laser Doppler flowmetry are expressed as perent hange relative to basal values for eah animal. Differenes between groups were onsidered signifiant at P <.5. Results Intraluminal Histamine Release Figure 1 shows the effet of 1 mmoul HCl perfusion of the distal esophagus on luminal histamine learane per 1-minute period during the experiment. The basal histamine release during the initial 2 minutes during whih normal saline was perfused was similar between groups. Histamine release for the normal salineperfused ontrol group did not hange during the entire SO-minute perfusion. Perfusion with 1 mmoul HCl resulted in a statistially signifiant (P <.1) inrease in histamine release into the luminal effluent, whih inreased abruptly from 7. ± 1. ng!ml to a peak of 39.9 ± 1.9 ng!ml 3 minutes after the start of aid perfusion. Pretreatment with DSCG attenuated the aidindued inrease in histamine release to a maximum of 16.3 ± 4. ng!ml at 4 minutes after the start of aid perfusion. This level was not signifiantly different from normal saline-perfused ontrols. DOX pretreatment also attenuated the aid-indued inrease in histamine release, but the histamine release remained signifiantly greater (P <.5) than in normal saline-perfused ontrols. Pretreatment with TTX did not affet the aid-indued inrease in luminal histamine learane (Figure 2). L NAME redued histamine release to 2.2 ± s.6 ng! ml after 3 minutes of aid perfusion, whih was not signifiantly elevated ompared with normal saline-perfused animals. PMZ did not blok the inrease in luminal histamine learane during aid perfusion (P <.5,,, E 5 Ol 4 '-" Q) E +' III I Aid perfusion E r trll ** 3 J'I'-: 2,-t-,i-'; (DDX) 1 V-V-V"" Figure 1. Effet of mast ell stabilizers on luminal release of histamine into the opossum distal esophagus during 1 mmoljl HCI perfusion. The esophagus was ontinuously perfused in vivo with saline to determine basal levels of histamine release followed by 6 minutes of perfusion of either aid or saline (ontrols only) starting at time O. Aid perfusion resulted in highly signifiant inreases in histamine release ompared with saline-perfused ontrols. DSCG attenuated the aid-indued histamine release to levels not signifiantly different from ontrols. Histamine release with aid + DOX was attenuated but remained signifiantly elevated from ontrols., Control; e, aid; \7, aid + DSCG; T, aid + DOX. *Signifiant differene from ontrol group, P <.5; **Signifiant differene from ontrol group, P<.1. ompared with ontrols), whih reahed 42.3 ± 2S.6 ng! ml 4 minutes after the start of aid perfusion. Beause PMZ ontains a ylial moiety similar to that found in histamine, we tested for the possibility that PMZ might interfere with the histamine assay. We found that fluorometri histamine assay of standard histamine samples ontaining PMZ was unaffeted by the presene of this drug. Esophageal Blood Flow Basal blood flow in the normal saline-perfused ontrol animals was 55.9 ± 9.S ml- min -1-1 g-i tissue and delined slightly during the experimental perfusion to 44.6 ± 5.3 ml- min-i-ioo g-i after 4 minutes. In ontrast, after a 2-minute lateny, perfusion with 1 mmoul HCl resulted in a sustained and statistially signifiant inrease in esophageal blood flow from 5.6 ± S.5 ml- min -1-1 g -1 to a peak of 95.2 ± 17. ml- min-i-ioo g-r, an average inrease of 123% ± 64% more than basal levels after 4 minutes of aid perfusion (Figure 3). Blood flow values measured using

4 1.24 FELDMAN ET AL. GASTROENTEROLOGY Vol. 1.1., No. 1. the laser Doppler flowmeter are presented as a perentage hange in blood flow from basal blood flow during the initial 2-minute saline perfusion. Basal blood flow ompared with ontrols was unhanged by either aid or saline perfusion, hypotension, and all drug treatments exept DOX, whih redued it signifiantly to 24.4 ± 5.2 ml min-i. 1 g-i. Pretreatment with both DSCG and DOX attenuated the inrease in blood flow when aid was introdued into the esophageal lumen, suh that levels that did not hange signifiantly from baseline. TTX did not prevent an early aid-indued inrease in blood flow (Figure 4), but this response was not sustained. Blood flow in the TTX group reahed 92.5% ± 48.6% above basal levels after 3 minutes and delined to near-basal levels of 11.7% ± 12.7% after 5 minutes of aid perfusion. The inrease in blood flow in this group remained signifiantly elevated with respet to baseline values (P <.5). Exsanguination-indued hypotension ompletely bloked the blood flow inrease duting aid perfusion. Aid-indued inreases in blood flow were also ompletely abolished by pretreatment with PMZ or i-name (Fig ute 5). Duting aid perfusion, mean blood flow in the PMZ-treated group arually delined to 21.1 % ± 13.4% below basal levels, whereas blood flow in the i E 5 Aid perfusion.. "- \..(TTX) '-/ 4.(PMZ) Q) E +' CJJ I Figure 2. Effet of TTX, PMZ, and L-NAME on esophageal luminal histamine release. Animals were pretreated with TTX at a dose titrated to eliminate vagally mediated esophageal ontrations. Perfusion with 1 mmoljl HCI for 6 minutes resulted in signifiant inreases in luminal histamine release in sham-injeted, PMZ-, and TTX-treated animals. L-NAME attenuated histamine release suh that it did not inrease signifiantly over ontrols., Control;., aid + TTX; 17, aid + L-NAME; T, aid + PMZ. *p <.5; **p <.1. NAME-pretreated group delined slightly to ± 15.3% below basal levels. The effet of i-name was shown to be stereospeifi beause esophageal blood flow in animals perfused with aid and pretreated with the enantiomer D-NAME remained inreased over baseline values. Exogenous 1 mmolll histamine perfusion was apable of eliiting a signifiant inrease in blood flow in the distal esophagus, whih reahed 281 % ± 119% above basal levels within 3 minutes (Figure 6). The effet of histamine was ompletely bloked by i-name, whih prevented blood flow from hanging signifiantly from baseline. Blood Pressure 2,... CLl 15 \.. (J '-/ 1 5 ll. " CD II! Aid perfusion T Figure 3. Effet of mast ell stabilizers on esophageal blood flow in the opossum distal esophagus during 1 mmoljl HCI perfusion. The esophagus was perfused in vivo with saline to determine basal blood flow followed by 6 minutes of either aid or saline (ontrols only) starting at time O. Aid perfusion resulted in a signifiant inrease in blood flow over basal levels, whereas blood flow did not inrease signifiantly above baseline in animals pretreated with the mast ell stabilizing agents DSCG and DOX., Control;., aid; 17, aid + DSCG; T, aid + DOX. *p <.5. Mean arterial blood pressure alulated for eah lo-minute period during the basal and experimental periods remained onstant at 14 ± 1 mm Hg in ontrol animals. DSCG, DOX, PMZ, D-NAME, and luminal aid or histamine perfusions had no effet on blood pressure. i-name pretreatment resulted in an aute inrease in mean arterial blood pressure to 144 ± 5 mm Hg that gradually rerurned to normal levels by the start of the protool. TTX pretreatment aused prolonged hypo ten- ;-

5 January 1996 MAST CELL REGULATION OF ESOPHAGEAL BLOOD FLOW IE.. Aid perfusion... u 1 15 C.!: U CI'! '"---' 1 * 5 u... "U - rn Figure 4. Effet of nx and systemi hypotension on esophageal blood flow in the opossum distal esophagus during 1 mmol/l HCI perfusion. Aid perfusion of the nx-pretreated group resulted in an aute inrease in blood flow that was signifiantly elevated with respet to basal levels. Hypotension after exsanguination bloked the aid-indued inrease in blood flow., Control;., aid; V, aid + nx (mean arterial blood pressure, 73 ± 3 mm Hg); T, aid + hypotension (mean arterial blood pressure, 65 ± 1 mm Hg). *p < u 15 1.!: U CI'! '"---' 5 u... "U rn 2 "'" Aid perfusion 1 * -5-2 o Figure 5. Effet of histamine Hi-reeptor antagonist or NO synthase inhibitor on esophageal blood flow in the opossum distal esophagus during 1 mmol/l HCI perfusion. Hi-reeptor blokade with PMZ bloked the aid-indued inrease of blood flow above basal levels. L-NAME (but not the enantiomer D-NAME) also ompletely bloked this response., Control;., aid; V, aid + PMZ; T, aid + L-NAME;, aid + D-NAME. *p < *... u 1 C 4 3.!: u CI'! '"---' 2 G:: "U 1 rn Histamine perfusion "'"... "W** Figure 6. Effet of NO synthase inhibition on esophageal blood flow in the opossum distal esophagus during 1 mmol/l histamine perfusion. Exogenous histamine perfusion for 6 minutes resulted in highly signifiant inreases in blood flow over basal levels, an effet that was ompletely bloked by L-NAME., Control;., histamine; V, histamine + L-NAME. **p <.1. sion, whih was only partly reversed by dextran infusion. Although mean blood pressure during the experimental perfusion in this group (73 ± 3 mm Hg) remained lower than in ontrols, the differene was not statistially signifiant (P =.11). Exsanguination resulted in a mean blood pressure of 65 ± 1 mm Hg during the SO-minute protool, whih was signifiantly lower than in ontrols (P <.1). Disussion These data show that in the opossum, the inrease in esophageal blood flow observed during luminal exposure to isotoni 1 mmol/l HCl is preeded by luminal histamine release and is attenuated by mast ell stabilizing agents, histamine H[ blokade, or NO synthase inhibition. These findings suggest that mast ells playa role in mediating reative blood flow inreases in the distal esophagus after aid hallenge. These mast ell-dependent hemodynami hanges are likely mediated by histamine release beause blokade of histamine H[ reeptors using PMZ ompletely abolished the aid-indued blood flow inrease. We have determined the tissue histamine ontent of the normal opossum esophagus to be 44.6 ± 1.6 g/g wet tissue (data not shown), a theoretial onentration of.4 mmolll. Beause histamine released from mast ells is likely to remain in the interstitial

6 126 FELDMAN ET AL. GASTROENTEROLOGY Vol. 11, No.1 ompartment, loal tissue histamine onentration may be even higher than 1 mmolll. Thus, the 39.9 ± 1.9 ng/ml histamine measured in the effluent represents only the small proportion of the tissue histamine that diffused aross the epithelium. NO has been shown by Masini et al. to attenuate histamine release from isolated rat peritoneal mast ells,17 whereas we found that NO synthase inhibition attenuated aid-indued esophageal histamine release. The reason for the disrepany between the present results and those of Masini et al. is unertain bur ould be related to indiret effets of L-NAME in vivo. In any event, L NAME ompletely abolished the hyperemi response to both luminal aid and 1 mmolll histamine perfusion, suggesting that NO is the final endogenous mediator responsible for inreasing esophageal blood flow in response to histamine release. The use of laser Doppler flowmetry allowed noninvasive reording of esophageal blood flow in this study. Laser Doppler flowmetry was shown to be highly orrelated with blood flow measurements obtained with radiolabeled mirospheres in pig skin flaps and is onsidered a sensitive measurement of tissue perfusion at rates more than 2 ml min -I. 1 g -I tissue. 18 Blood flow values in these experiments were generally similar to those previously obtained using radiolabeled mirospheres in this model,19 and the tehnique is well suited for protools that all for ontinuous determination of relative hanges in blood flow. The ability of the muosa to respond to noxious luminal stimuli by modulating blood flow is a phenomenon that has been onsistently observed in the gastrointestinal trat and is likely to play an important role in muosal defense against aid injury.2-23 The mehanisms and mediators responsible for modulating blood flow remain unlear, although apsaiin-sensitive sensory afferent neurons, mast ells, and produts of the ylooxygenase pathway have all been impliated in the literature. 5.24,25 The response of the esophageal miroirulation to luminal hallenge with noxious substanes appears similar to that in other parts of the gut. Bass et al. 26 showed signifiant inreases in blood flow, whih were not mediated by prostaglandins, in the rabbit esophageal muosa after exposure to 1 mmolll HCl for 6 minutes. Hollwarth et al. 27 subsequently reported inreased whole wall distal esophageal blood flow in the at after luminal exposure of the esophagus to ph 1.5 HCl, largely due to inreased blood flow in the musularis. The investigators also deteted inreased blood flow in the upper esophageal sphinter region, whih was not diretly exposed to aid, and proposed that this supported a role for neuronal reflexes in this response. However, bak flow of aid into the upper sphinter ould not be exluded, and our previous studies using the mirosphere tehnique in the opossum showed that the aid-indued inrease in blood flow was a loal phenomenon that did not extend to the proxlma. I esop h agus. 28 An inreasing body of evidene points to the ability of hemosensitive afferent nerves to respond to noxious stimuli via antidromi transmission, ausing loal release of vasoative neuropeptides in the gut. 29 In the rabbit esophagus, stimulation of sensory afferent neurons with luminally applied apsaiin inreases blood flow. 4,5 Calitonin gene-related peptide was reently impliated as an important mediator of inreased esophageal blood flow after luminal hallenge with sodium deoxyholate at ph 7.7 However, it is less lear whether this mehanism plays an important role in the aid-indued inrease in blood flow. Sandler et al. 6 showed that the apsaiinindued inrease in anine esophageal blood flow is sensitive to neuronal blokade by topial luminal appliation of lidoaine, whereas the aid-indued inrease in blood flow is resistant to lidoaine. They onluded that the aid-indued inrease in esophageal blood flow is a nonneurall y mediated phenomenon. Out present findings that the aid-indued inrease in esophageal blood flow is not ompletely bloked by TTX at a dose that bloked vagally mediated responses are onsistent with this hypothesis. We have shown that the aute aid-indued inrease in esophageal blood flow was insensitive to TTX pretreatment, but the response was not sustained and blood flow rapidly returned to baseline. Prolonged hypotension also attenuated the response to aid suh that values in the aid-perfused hypotensive group were similar to the normal saline-perfused ontrol group. Therefore, the hypotension indued by TTX ould aount for the lak of a sustained inrease in blood flow beause hypotension alone ould prevent this response. However, the role and signifiane of a population of reently demonstrated TTX-resistant sensory afferent fibers have not been examined. 3 Evidene onsistent with a role for mast ells and histamine in the regulation of gastri blood flow was provided by Wallae et ae 4 in apsaiin-indued hyperemia in parasitized rats. In this study, the hyperemi response to apsaiin was attenuated by the mast ell stabilizer doxantrazole or the HI-reeptor antagonist pyrilamine. To date, the role of esophageal mast ells in modulating blood flow during injury has not been examined. Previous studies in our laboratory have shown that exposure of the opossum distal esophagus to 5 or 1 mmol/l HCl is assoiated with mast ell degranulation and luminal histamine release. 8 Although mast ells in the opossum esophageal muosa may be more abundant

7 January 1996 MAST CELL REGULATION OF ESOPHAGEAL BLOOD FLOW 127 than in humans, reent evidene that mast ells are inreased in esophagitis and esophageal metaplasia suggests they may play a role in the pathogenesis of esophageal inflammation in humans. 31 The present findings suggest that mast ell-derived histamine is largely responsible for early aid-indued inreases in esophageal blood flow and that this works via an NO synthase-dependent mehanism. This is onsistent with endothelial-dependent generation of NO, whih has been impliated in the mehanism of ation of a number of vasoative mediators, inluding histamine. 32 The mehanism by whih aid ativates mast ells is not known. It is possible that aid diffusion aross the epithelium into the muosa diretly perturbs mast ell membrane potentials and stimulates degranulation. Alternatively, apsaiin-sensitive afferent neurons have been shown to release mediator in low-ph environments. 33 Aidifiation of the esophageal muosa may stimulate the release of neuropeptide mediators that are in turn apable of stimulating mast ell mediator release. The ability of the esophageal muosa to withstand repeated exposure to reflux of gastri ontents depends in part on intrinsi defense mehanisms. The present study examined the role of mast ells and nerves in mediating the inrease in esophageal blood flow aused by luminal exposure to aid. Our findings provide evidene that mast ells play a entral role in the response of the esophageal miroirulation to luminal aid in the opossum model. Referenes 1. Johnson LF, Demeester TR. Twenty-four-hour ph monitoring of the distal esophagus. A quantitative measure of gastroesophageal reflux. Am J Gastroenterol, 1974;62: Dodds WJ, Hogan WJ, Helm JF, Dent J. Pathogenesis of reflux esophagitis. Gastroenterology 1981; 81: Orlando RC. Esophageal epithelial defenses against aid injury. Am J GastroenteroI1994;89:S48-S Trad KS, Harmon JW, Femiola MT, Hakki Fl, Rao JS, Dziki AJ, Bass BL. Evidene for a role of apsaiin-sensitive muosal afferent nerves in the regulation of esophageal blood flow in rabbits (abstr). Gastroenterology 199; 98:A Bass BL, Trad KS, Harmon JW, Hakki Fl. Capsaiin-sensitive nerves mediate esophageal muosal protetion. Surgery 1991; 11: Sandler AD, Shmidt C, Rihardson K, Murray J, Maher JW. Regulation of distal esophageal muosal blood flow: the roles of nitri oxide and substane P. Surgery 1993; 114: MKie LD, Dunkin BJ, Pennanen MF, Dunlap KW, Harmon JW, Bass BL. Esophageal muosal blood flow: a entral role for alitonin gene-related peptide. Surgery 1994; 116: Barlay RL, Paterson WG, Dinda PK, Morris GP. The role of mast ells and histamine in aute aid-indued esophageal muosal injury (AIEMI) (abstr). Gastroenterology 1992; 12:A Feldman MJ, Morris GP, Dinda PK, Paterson WG. 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8 128 FELDMAN ET AL. GASTROENTEROLOGY Vol. 11. No.1 speialized intestinal metaplasia (abstr). Gastroenterology 1995; 18:A Ryan US. Avdonin PV. Posin EY. Popov EG. Danilov SM. Tkahuk VA. Influene of vasoative agents on ytoplasmi free alium in vasular endothelial ells. J Appl PhysioI1988;65: Geppetti p. Tramontana M. Evangelista S. Renzi D. Maggi CA. Fuso BM. del Biano E. Differential effet on neuropeptide release of different onentrations of hydrogen ions on afferent and intrinsi neurons of the rat stomah. Gastroenterology 1991; 11: Reeived January Aepted August Address requests for reprints to: William G. Paterson. M.D. Gastrointestinal Division. Hotel Dieu Hospital. 166 Brok Street. Kingston. Ontario K7L 5G2. Canada. Fax: (613) Supported by grants from Physiians' Servies Inorporated Foundation of Ontario (91-15) and the Medial Researh Counil of Canada (MA 9978). M. Feldman was supported by a Proter & Gamble Pharmaeutials Canada Studentship. The authors thank David Miller and Gail Pringle for tehnial assistane.