636 Gut 1997; 41: 636–641 Enhanced mucosal permeability and synthase activity in jejunum of deficient Gut: first published as 10.1136/gut.41.5.636 on 1 November 1997. Downloaded from mice

S Komatsu, M B Grisham, J M Russell, D N Granger

Abstract also contributes to the interactions between Background—Recent reports have de- these diVerent cell populations in certain scribed a modulating influence of nitric pathological conditions, such as ischaemia oxide (NO) on intestinal mucosal perme- reperfusion.67 NO mediated cell-cell interac- ability and have implicated a role for mast tions have been described between mast cells, cells in this NO mediated process. leucocytes, and enterocytes in intestinal seg- Aims—To assess further the contribution ments treated with NO synthase inhibitors, G of mast cells to the mucosal permeability such as N -nitro-L- methylester (L- G changes elicited by the NO synthase NAME) or N -monomethyl-L-arginine G 8 (NOS) inhibitor N -nitro-L-arginine (L-NMMA). These inhibitors elicit the de- methylester (L-NAME), using mast cell granulation of mast cells, recruitment of leuco- deficient (W/WV) and mast cell replete cytes, and increased mucosal permeability to mice (+/+). chromium-51 EDTA, responses that are pre- Methods—Chromium-51 EDTA clearance vented by simultaneous treatment with either 59 (from blood to jejunal lumen), jejunal NO donating agents or L-arginine. Mast cell NOS and myeloperoxidase (MPO) activi- stabilising agents such as doxantrazole and ties, and plasma nitrate/nitrite levels were lodoxamide, as well as H1 antago- monitored. nists, also appear to be eVective in blunting the Results—The increased EDTA clearance increased mucosal permeability associated V 10 elicited by intraluminal L-NAME in W/W with NO synthase inhibition. Hence, it has mice (4.4-fold) was significantly greater been proposed that mast cell derived hista- than the response observed in control mine, which is released from degranulated (+/+) mice (1.8-fold). The exacerbated mast cells that are deprived of NO, mediates response in W/Wv mice was greatly attenu- the changes in paracellular permeability in the ated by pretreatment with either dexam- gut after NO synthase inhibition. http://gut.bmj.com/ ethasone (1.3-fold) or the selective The linkage between diminished intestinal inducible NOS inhibitor, aminoguanidine NO production, mast cell degranulation, and (1.4-fold), and partially attenuated by the increased mucosal permeability has been mast cell stabiliser, lodoxamide (2.9-fold). invoked to explain the mucosal barrier dys- 8 Jejunal inducible NOS activity was signifi- function observed after ischaemia reperfusion. cantly higher in W/WV than in +/+ mice, This NO dependent mechanism may also V account for the increased intestinal mucosal

while jejunal MPO was lower in W/W on September 26, 2021 by guest. Protected copyright. mice than in +/+ mice, suggesting that the permeability observed in inflammatory condi- higher inducible NOS in W/WV does not tions that are associated with an enhanced result from the recruitment of inflamma- superoxide mediated inactivation of NO. A tory cells into the gut. The higher induc- limitation of this novel mechanism is that the ible NOS activity in the jejunum of W/WV implication of mast cells as a modulator of the was significantly reduced by dexametha- increased mucosal permeability elicited by NO sone treatment. synthase inhibition is largely derived from Conclusions—Our results suggest that experiments utilising agents that are consid- mast cells normally serve to inhibit induc- ered to act exclusively through mast stabilisa- ible NOS activity tonically in the gut and tion. Since there is published evidence that that inhibitors of NOS elicit a larger challenges the specificity of mast cell stabilisers 11 permeability response when this tonic such as ketotifen, an alternate approach to Department of inhibitory influence is released by mast testing the linkage between mast cells and Physiology, LSU cell depletion. intestinal mucosal permeability is warranted. Medical Center, 1501 (Gut 1997; 41: 636–641) The objective of this study was therefore to Kings Highway, assess further the contribution of mast cells to Shreveport, Louisiana Keywords: aminoguanidine; c-kit; dexamethasone; epi- the increased mucosal permeability elicited by 71130–3932, USA thelium; S Komatsu NO synthase inhibition using mice that are M B Grisham genetically deficient in mast cells. J M Russell D N Granger There is a growing body of evidence that implicates nitric oxide (NO) as a modulator of Methods Correspondence to: the function of diVerent cells that are normally ANIMALS Dr D N Granger. found within the gastrointestinal mucosa, Male (20–30 g) mast cell deficient mice 1 2 V V Accepted for publication including epithelial cells, mast cells, endothe- (WBB6F1/JW/W ;W/W) (n=62) and their 4 June 1997 lial cells, and leucocytes.3–5 It appears that NO age matched controls (WBB6F1/J+/+; +/+) Mast cells and mucosal permeability 637

cats,910was measured in the jejunum of W/WV, +/+ without L-NAME +/+ +/+, and ICR mice. Animals were fasted for 24

L-NAME infusion Gut: first published as 10.1136/gut.41.5.636 on 1 November 1997. Downloaded from W/WV } hours prior to surgery. Anaesthesia was in- 0.30 * duced by intramuscular injection of ketamine A * (150 mg/kg) and xylazine (10 mg/kg), with * supplemental doses of subcutaneous ketamine 0.25 as needed. The right carotid artery and left * × 100 g) jugular vein were cannulated. Systemic arterial 0.20 pressure was measured with a Statham (Ox- nard, California) P23A pressure transducer and recorded with a Grass polygraph DC 0.15 driver amplifier (Grass Instrument Co., Quincy, Massachusetts). A laparotomy was 0.10 performed, and a loop of jejunum (4–5 cm) was isolated; blood vessels were left intact. Inflow and outflow tubes were secured on each 0.05 end of the jejunal segment, and warm phos- Cr-EDTA clearance (ml/min Cr-EDTA

51 phate (10 mM) buVered saline (pH 7.4) was 0 perfused through the segment at a rate of 0 1020 30 40 50 60 70 80 Time 0.5 ml/min. The intestinal segment and ab- (min) dominal contents were kept moist with saline L-NAME infusion soaked gauze and covered with a clear plastic sheet to minimise evaporation and tissue dehy- dration. Body temperature was monitored and 5.0 *† maintained at 36.5–37.5°C by a heat lamp. B 51 4.5 Since Cr-EDTA is rapidly excreted into urine following intravenous injection (unless 4.0 the kidneys are ligated), we continuously

× baseline) infused 35 µCi (11.7 g/ml; 0.2 µCi/min) of the 3.5 * tracer into the jugular vein. This regimen of 51 3.0 Cr-EDTA administration was determined in * initial experiments in which the bolus and 2.5 infusion levels were varied until one was found * to yield a constant plasma level. The appropri- 2.0 ateness of the chosen bolus dose and infusion ‡ ** 51 Cr-EDTA clearance ( Cr-EDTA rate of Cr-EDTA is supported by the stable

1.5 http://gut.bmj.com/

51 clearance values observed over 80 minutes of 1.0 luminal perfusion (fig 1). Thirty minutes were Peak allowed for tissue equilibration of 51Cr-EDTA 0.5 before collecting samples of luminal perfusate (every 10 minutes). Plasma samples (25 µl) 0 V V V V were obtained immediately prior to collection +/+ +/+ W/W W/W W/W W/W ICR + Lod + Dex + AG of the luminal perfusate (time 0) and at the end

of the experiment (at 80 minutes). Plasma on September 26, 2021 by guest. Protected copyright. L-NAME infusion EDTA level for each time point was calculated Figure 1: (A) Time course of mucosal permeability changes in untreated controls (+/+) from an assumed linear curve obtained by 51Cr V (n=5), and elicited by L-NAME in +/+ (n=6), and mast cell deficient (W/W ) mice count of the two plasma samples. 51Cr activity (n=6). (B) L-NAME induced mucosal permeability changes expressed as a ratio of the maximum clearance to baseline value (10–20 minute clearance value) in the following in plasma and luminal perfusate was counted in groups: +/+, W/WV, W/WV pretreated with lodoxamide (Lod) (n=5), W/WV pretreated a 14800 Wizard 3 gamma counter (Wallac, with dexamethasone (Dex) (n=5), and W/WV pretreated with aminoguanidine (AG) Turku, Finland), with automatic correction for (n=6) and ICR mice (n=6). *p<0.05 relative to respective baseline value, †p<0.001 background activity. At the end of the experi- relative to L-NAME-treated +/+, *p<0.05, ‡p<0.001 relative to the peak clearance response of W/WV. ment the jejunal loop was removed, rinsed, and weighed. The plasma to lumen clearance of (n=24) were obtained from Jackson Laborato- 51Cr-EDTA was calculated as: ries (Bar Harbor, Maine). Male ICR mice (n=6), another strain of control mice, were Clearance (ml/min × 100g) = obtained from Harlan Laboratories (Frederick, (CPM, perfusate) × (perfusion rate) × 100 Maryland). Only occasional mast cells (less × than 1% of the number in congenic +/+ mice) (CPM, plasma) (wt of intestinal segment) are observed in the skin of adult W/WV mice, where CPM is counts per minute per ml of and no mast cells have been noted in multiple perfusate or plasma, and wt is weight in grams other tissues of W/WV mice.12 13 Similarly, we of the jejunal segment. The theoretical basis have been unable to find mast cells in the and technical limitations of the 51Cr-EDTA mesentery of 12 week old W/WV mice. clearance technique for measurement of intes- tinal mucosal permeability have been ad- 14 MUCOSAL PERMEABILITY dressed elsewhere. The blood to lumen clearance of 51Cr labelled Stable baseline clearances of 51Cr-EDTA in EDTA, modified for mice from the conven- murine intestine were noted for as long as 80 tional method previously applied in rats and minutes (fig 1A). L-NAME (1 mM, Sigma, St 638 Komatsu, Grisham, Russell, Granger

2.00 * the carotid artery. Plasma nitrate and nitrite levels were also measured in dexamethasone or

+/+ V Gut: first published as 10.1136/gut.41.5.636 on 1 November 1997. Downloaded from 1.75 V aminoguanidine pretreated W/W mice under W/W basal conditions. * W/WV + DEX 1.50 V ** W/W + AG STATISTICS The data were analysed using an analysis of 1.25 ** variance with ScheVé’s (post hoc) test. When 1.00 only two groups were compared, the paired or unpaired Student’s t test was used where 0.75 † appropriate. All values are reported as means (SEM). Statistical significance was set at 0.50 p<0.05.

0.25 Results

NO synthase activity (pmol/mg protein) MUCOSAL PERMEABILITY 0 The blood to lumen clearance of 51Cr labelled Total NOS Inducible NOS Constitutive NOS EDTA was significantly increased by the infu- V sion of L-NAME in W/W mice, but not in +/+ Figure 2: (NOS) activity in control (+/+) mice (n=7), mast cell deficient (W/WV) mice (n=8), W/WV mice pretreated with dexamethasone (DEX) (n=8), mice (fig 1A). Figure 1B shows the peak and W/WV mice pretreated with aminoguanidine (AG) (n=6). *p<0.05, **p<0.001 changes in L-NAME induced EDTA clearance relative to +/+, †p<0.05 relative to W/WV. (normalised to its baseline value) in the diVer- Louis, Missouri) was perfused into the intesti- ent groups of animals. Although L-NAME caused a small increase in EDTA clearance nal lumen for 60 minutes, which was preceded × by a 20 minute period of control perfusion (1.8 baseline) in control (+/+) mice, a more substantial increase (4.4× baseline) was elicited without L-NAME (baseline measurements). V Preparations with a baseline clearance greater by L-NAME in W/W mice. This L-NAME than 0.2 ml/min/100 g were discarded. Lower induced increase in the mucosal permeability observed in W/WV mice was slightly attenuated concentrations of L-NAME (0.1–0.5 mM) in × the gut lumen did not elicit an increase in 51Cr- by lodoxamide (2.9 baseline) and greatly EDTA clearance in control mice (unpublished attenuated by pretreatment with either dexam- data), as shown in previous studies.89 It has ethasone or aminoguanidine. The magnitude of the increase in EDTA clearance elicited by been reported that concentrations of L-NAME as high as 1 mM do not increase the permeabil- L-NAME in another strain of mast cell replete ity of monolayers of rat intestinal epithelial mice (ICR) was similar to that observed in +/+ cells.10 controls. In another series of experiments, the mast Systemic arterial pressure was unaVected by http://gut.bmj.com/ cell stabiliser lodoxamide (15 mg/kg), was infusion of L-NAME in all groups studied. administered intravenously as a bolus at 15 minutes before the start of the experiment, in JEJUNAL NOS ACTIVITY W/WV mice.10 Other animals (W/WV) were pre- Total NOS activity under baseline conditions was significantly higher in the jejunum of treated with either dexamethasone (1 mg/kg V intraperitoneally, Sigma, St Louis, Missouri) W/W mice than in (+/+) control mice (fig 2). This diVerence between W/WV and +/+ mice

24 hours before the experiment, or with the on September 26, 2021 by guest. Protected copyright. was more evident for iNOS activity. The selective inducible nitric oxide synthase V (iNOS) inhibitor, aminoguanidine.15–17 Amino- elevated iNOS in W/W mice was significantly guanidine was administered in the drinking attenuated by pretreatment with dexametha- water (18.5 mg/l) for three days before the sone, but not by aminoguanidine pretreatment. experiment, a regimen which has been shown The activity of constitutive NOS (cNOS) in to blunt the increased plasma nitrate and mouse jejunum did not diVer between the vari- nitrite levels normally elicited by ous experimental groups. peptidoglycan/polysaccharide in rats.18 JEJUNAL MPO ACTIVITY Tissue MPO activity in jejunum of W/WV mice NOS AND MYELOPEROXIDASE (MPO) ACTIVITIES (6.52 (0.38) U/g) was significantly lower V In additional groups of fasted (24 hours) W/W (p<0.001) than that of +/+ mice (11.31 (0.53) and +/+ mice, samples of jejunum were U/g). harvested and quickly frozen for later measurement of NOS or MPO activity. Jejunal PLASMA NITRATE AND NITRITE NOS activity was determined using the The plasma level of nitrite/nitrate (NO /NO ) 5 2 3 method of Kurose et al. MPO activity was in W/WV mice was significantly higher than that 18 determined using the method of Grisham et al measured in control (+/+) mice. The higher V in jejunal samples of untreated W/W and +/+ V levels of plasma NO2/NO3 in W/W mice were mice. not significantly attenuated by pretreatment with either dexamethasone or aminoguanidine PLASMA NITRITE AND NITRATE LEVELS (fig 3). The absence of a change in plasma V Plasma nitrite and nitrate levels in W/W and NO2/NO3 in the face of a profound reduction in +/+ mice were measured using the method of iNOS activity after dexamethasone treatment Grisham et al.19 Animals were fasted for 24 may reflect a diVerential sensitivity of various hours, and a blood sample was collected from organs to dexamethasone. We measured NOS Mast cells and mucosal permeability 639

225 * the magnitude of the L-NAME induced increase in 51Cr-EDTA clearance elicited in the 200 * * jejunum of control mice, that is, +/+ (1.8-fold) Gut: first published as 10.1136/gut.41.5.636 on 1 November 1997. Downloaded from 175 and ICR (2.5-fold), is significantly lower than that previously observed in rat and cat 150 intestine.910 Although a definitive explanation 125 for the diVerent responses to L-NAME is not 100 available, two possibilities warrant considera- tion. Firstly, it appears likely that the luminal 75 route of L-NAME administration used in our 50 study achieves a lower concentration of the NO synthase inhibitor in the mucosa than that 25 Plasma nitrate and nitrite (µM) achieved following intravascular (intravenous 0 or intra-arterial) administration. Another V V V +/+ W/W W/W W/W possibility is that mice respond less vigorously + Dex + AG to NO synthase inhibition (or have a lower Figure 3: Plasma levels of nitrite and nitrate in control activity of the ) than other species (rat (+/+) (n=6), mast cell deficient (W/WV) (n=6), and or cat). This possibility seems less plausible W/WV mice pretreated with either dexamethasone (Dex) (n=6) or aminoguanidine (AG) (n=6). *p<0.01 relative since we have noted in a few rat intestine to +/+. preparations that luminal L-NAME elicits an increased 51Cr-EDTA clearance comparable to activity in the jejunum but not in other tissues, that observed in mouse intestine (data not which may contribute more significantly to shown).

plasma NO2/NO3 in mast cell deficient mice. There is published evidence that implicates This possibility, however, is not supported by mast cells in the enhanced epithelial barrier previously published work which demonstrates permeability associated with inhibition of NO that dexamethasone (1 mg/kg) inhibits iNOS synthase.810 Kanwar et al10 have shown that

activity to a comparable extent in diVerent tis- mast cell stabilisers or histamine H1 receptor sues of the rat after endotoxin treatment.20 antagonists greatly attenuate the increased Hence, an alternative explanation for our find- mucosal permeability normally elicited by ings of a sustained elevation in plasma L-NAME, and that chronic dexamethasone

NO2/NO3 and accompanying reduction in treatment, which depletes mucosal mast cells, iNOS after dexamethasone or aminoguanidine also prevents the L-NAME induced rise in

treatment is that the increased plasma NO2/ mucosal permeability. These observations sug-

NO3 level in mast cell deficient mice is not gest that mast cell derived histamine mediates related to the elevated jejunal iNOS activity. the increased mucosal permeability caused by

L-NAME. The assumption that NO synthase http://gut.bmj.com/ Discussion inhibition promotes mast cell degranulation is There is a growing body of evidence that supported by the observation that mast cell implicates nitric oxide as a modulator of the protease II activity in plasma is increased in restrictive properties of the paracellular path- animals receiving L-NAME, compared with way between intestinal epithelial cells.8 Using control plasma. Additional support for a blood to lumen clearance of 51Cr-EDTA as a linkage between mast cells and NO production measure of intestinal mucosal permeability, is provided by intravital microscopic studies of

910 5 on September 26, 2021 by guest. Protected copyright. Kubes and associates have demonstrated rat mesentery, in which L-NAME or ischaemia that intravascular administration of NO syn- reperfusion7 induced mast cell degranulation is thase inhibitors such as L-NAME elicit large blunted by coadministration of NO donating (fivefold to sixfold) increases in mucosal agents (for example, spermine NO). permeability of both cat and rat intestine. They The results of the present study are inconsist- also showed that this eVect of L-NAME was ent with the prevailing view that mast cell prod- completely reversed by simultaneous adminis- ucts (for example, histamine) are responsible for tration of either nitroprusside or L-arginine, but the increased intestinal mucosal permeability could not be mimicked by administration of that is associated with inhibition of NO produc- the inactive enantiomer, D-NAME. Based on tion. Surprisingly, we found that L-NAME elic- these observations, the authors proposed that its a substantially larger increase in 51Cr-EDTA nitric oxide produced in the gut mucosa exerts clearance in jejunal segments of mast cell a tonic inhibitory influence on the passive per- deficient mice (W/WV) compared with their meation of solutes into the bowel lumen, mast cell replete counterparts (+/+ mice). This presumably through a direct (or indirect) finding contrasts the profound protective action action of NO on epithelial junctions. Support of mast cell stabilisers, such as lodoxamide and for an indirect action is provided by experi- doxantrazole, on L-NAME induced mucosal ments demonstrating that L-NAME does not permeability changes observed in rat small increase 51Cr-EDTA clearance across monolay- intestine.10 Our findings in mast cell deficient ers of cultured rat intestinal epithelial cells.10 mice would argue in favour of a role for mast Our observation that perfusion of the lumen cells in producing factors that protect the intes- of mouse jejunum with L-NAME elicits an tinal mucosa against the permeability increasing increased blood to lumen clearance of 51Cr- eVects of NO synthase inhibition. EDTA provides additional support for the view While a definitive explanation is not readily that acute inhibition of NO production leads to available for the discrepancy between our find- an increased mucosal permeability. However, ings in mast cell deficient mice and previously 640 Komatsu, Grisham, Russell, Granger

published work on NO synthase inhibition in including inhibition of diamine oxidase.22 23 normal intestine treated with mast cell stabilis- Alternatively, a negative feedback to iNOS by

ers, some mechanistic insights may be afforded NO24 may be released by selective inhibition of Gut: first published as 10.1136/gut.41.5.636 on 1 November 1997. Downloaded from by the responses of other variables measured in cNOS with a relatively lower dose of 21 our experiments. The clearance response L-NAME, with the elevated NO levels medi- elicited by L-NAME in mast cell deficient mice ating the rise in mucosal permeability. was slightly but significantly attenuated by pre- The induction of iNOS in mast cell deficient treatment with the widely used mast cell stabi- mice is not likely to result from the recruitment liser lodoxamide, suggesting that this agent of inflammatory cells into the jejunum inas- may exert an action on the mucosal barrier that much as tissue MPO activity was significantly is independent of mast cells. While such lower in W/WV than in control (+/+) mice. non-specific actions of mast cell stabilisers Another consideration relates to the mutations could account, at least in part, for our at the W/c-kit locus25 26 in W/WV mice, which contradictory results, adaptive responses re- results either in an absence of c-kit lated to the chronic absence of mast cells in the kinase receptor on cell surfaces or in the intestinal mucosa may contribute more signifi- expression of receptors that are notably defi- cantly to the larger permeability response cient in tyrosine kinase activity.26–28 Thus, it is observed in the mutant mice. conceivable that inactivation of the receptor We noted that jejunal MPO (an index of tis- tyrosine kinase encoded by c-kit could induce sue granulocyte count) was reduced, while iNOS expression. However, this possibility jejunal iNOS (but not cNOS) activity was appears unlikely in view of recent reports dem- greatly elevated in mast cell deficient mice, onstrating that inhibition of tyrosine kinase compared with their mast cell replete counter- attenuates the induction of iNOS expression, parts. The profound increase in jejunal iNOS indicating that tyrosine kinase is necessary for activity was accompanied by a large increase in iNOS expression.29 30 A more tenable explana- plasma nitrate/nitrite concentration, an index tion for the increased jejunal iNOS activity in of total body NO production. The relatively W/WV mice is that mast cells produce factors low jejunal MPO activity in W/WV mice that suppress the gene for iNOS. Indeed, suggests that an accumulation of activated Dileepan et al31 showed that mast cell granules granulocytes is an unlikely explanation for the inhibited lipopolysaccharide (LPS) or inter- greater 51Cr-EDTA clearance response in these feron ã induced nitric oxide production in mutants. However, the greatly elevated iNOS peritoneal macrophages, suggesting an inhibi- – – V activity (and plasma NO2 /NO3 )inW/W mice tory eVect of mast cell products on iNOS may relate more directly to the exaggerated expression. Furthermore, it has been shown 51Cr-EDTA clearance response noted in these that mast cell derived factors such as inter- animals. leukin (IL) 4,32 IL-13, or transforming growth 33 34 35

Our observations that plasma NO2/NO3 is factor â inhibit the expression of iNOS. http://gut.bmj.com/ greatly elevated and that jejunal iNOS activity In summary, the findings of this study, cou- is increased fivefold in W/WV mice suggests pled with published reports, suggest that the that chronic mast cell deficiency is associated modulatory role of mast cells in mediating the with an accelerated rate of NO production in increased mucosal permeability that is elicited the gut (and possibly other tissues). Whether or by L-NAME diVers between conditions of not this enhanced NO generation is causally acute versus chronic suppression of mast cell related to the exaggerated mucosal permeabil- function. Our results support the view that ity response to L-NAME in mast cell deficient c-kit (and presumably mast cells) play an on September 26, 2021 by guest. Protected copyright. mice remains unclear. However, some support important role in maintaining epithelial barrier for this possibility is provided by the observa- integrity in the small intestine. However, the tion that pretreatment of W/WV mice with dex- possibility cannot be excluded that other cells amethasone, which abolished the rise in iNOS mediate this process, since the depletion of normally associated with mast cell depletion, c-kit receptor tyrosine kinase aVects diVerent resulted in an attenuated 51Cr-EDTA clearance cell populations, such as interstitial cells of 36 response to L-NAME. Similarly, treatment Cajal, to regulate gastrointestinal motility, as with aminoguanidine, which is a more selective well as haemopoietic cells. 15–17 competitive inhibitor of iNOS (L-NAME is more specific for cNOS),21 also blunts the rise Supported by a grant from the National Institutes of Health in mucosal permeability that is elicited by (P01 DK43785). V L-NAME in W/W mice. Overall, these find- 1 Brown JF, Tepperman BL, Hanson PJ, Whittle BJR, ings suggest that mast cells normally serve to Moncada S. DiVerential distribution of nitric oxide inhibit iNOS activity tonically in the gut and synthase between cell fractions isolated from the rat gastric mucosa. Biochem Biophys Res Commun 1992; 184: 680–5. that L-NAME elicits a larger permeability 2 Salvemini D, Masini E, Pistelli A, Mannaioni PF, Vane JR. response when this tonic inhibitory influence is Nitric oxide: a regulatory mediator of mast cell reactivity. J Cardiovasc Pharmacol 1991; 17 (suppl 3): S258–64. released by mast cell depletion. Nonetheless, it 3 Kubes P, Suzuki M, Granger DN. Nitric oxide: an remains unclear how iNOS inhibitors such as endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci USA 1991; 88: 4651–5. aminoguanidine and dexamethasone act to 4 Kurose I, Kubes P, Wolf R, Anderson DC, Paulson JC, protect against the actions of another inhibitor Miyasaka M, et al. Inhibition of nitric oxide production: mechanism of vascular albumin leakage. Circ Res 1993; 73: of NOS—that is, L-NAME. It is conceivable 164–71. 5 Kurose I, Wolf R, Grisham MB, Granger DN. EVects of an that the beneficial actions of aminoguanidine endogenous inhibitor of nitric oxide synthesis on postcapil- and dexamethasone are unrelated to iNOS lary venules. Am J Physiol 1995; 268: H2224–31. inhibition. Indeed, it has been shown that 6 Kubes P. Ischemia/reperfusion in the feline small intestine: a role for nitric oxide. Am J Physiol (Gastrointest Liver Physiol) aminoguanidine has other biological actions, 1993; 264: G143–9. Mast cells and mucosal permeability 641

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