Inhibition of NO-synthase and degranulation of rat omental mast cells in vitro

Mast cell amines, platelet-activating factor (PAF), thromboxanes and leukotrienes have been shown to be released during nitric oxide-synthase inhibition in the rat intestine. Mast cells in rat isolated omentum (OMCs) or isolated from the rat peritoneal cavity (PMCs) have been used here to investigate the relationship(s) between these agents. N-nitro-L-arginine methyl ester (L-NAME, 100 μM) caused some degranulation of OMCs, but no enhancement of histamine release from PMCs. PAF (5 μM) and U46619 (1 μM) degranulated OMCs and enhanced histamine release from PMCs. Pre-treatment of the omentum with BN52021 (10 μM) inhibited degranulation of OMCs in response to L-NAME, PAF or U46619. Pretreatment with 1-benzylimidazole (5 or 50 μM) inhibited the effect of L-NAME but not that of PAF. Indomethacin (1 μM) or sodium nitroprusside (10 μM) also inhibited the effects of L-NAME, but nordihydroguaiaretic acid (30 μM) did not. In PMCs BN52021 inhibited PAF-induced, but not U46619-induced, release of histamine. These results suggest that inhibition of nitric oxidesynthase in the omentum by L-NAME allows thromboxanes to release PAF, which in turn degranulates and releases histamine from OMCs.

In the present work we have investigated the Introduction possible relationship(s) between these various Degranulation of mast cells occurs during inhibitors/antagonists and the co-administration ischaemia/reperfusion injury in both the rat of L-NAME. The study has focused on mast cell mesentery and small intestine, 2 and it can be degranulation in the presence of such agents. enhanced by prior administration of N-nitro--Two methods have been used involving, firstly, arginine methyl ester (L-NAME), a NO-synthase mast cells in situ in rat omental milky spots inhibitor, or it can be inhibited by prior adminis-(OMCs) and, secondly, mast cells isolated from tration of sodium nitroprusside (SNP), an NO the rat by peritoneal lavage (PMCs).
donor. Epithelial permeability to 51Cr in rat small intestine is also increased by L-NAME and Methods this is associated with mucosal mast cell degranulation. We have shown recently 4 that the L-Omental mast cells.. Female rats weighing about NAME-induced increase in microvascular perme-250g were killed by inhalation of chloroform ability to injected colloidal carbon in the rat vapour. After opening the abdomen with a small intestine is prevented by pre-treatment with midline incision followed by a lateral subcostal cyproheptadine, a mixed histamine and 5-hydroxyincision, the lesser omenmm was quickly tryptamine receptor antagonist. Other workers removed into a beaker of normal saline (NS) and have shown an increase in albumin leakage in then cut into five pieces of approximately equal the rat small intestine and colon resulting from size. Each piece was quickly but gently spread co-administration of L-NAME and a lipopolyout on a glass microscope slide and then placed saccharide. This could be inhibited by prior in a moist atmosphere at 37C. Specimens were administration of a cyclooxygenase inhibitor, a then flooded either with NS or with NS plus a thromboxane synthase inhibitor, a platelet actiputative antagonist for 5 min. The bathing fluid vating factor (PAF)-receptor antagonist 5 or a 5was then drained away and replaced either by lipoxygenase inhibitor. some fresh NS, NS plus antagonist, NS plus agonist, or NS plus antagonist plus agonist. Incu-2.5 ml NS using a vortex mixer and heated for 10 bation continued for a further 15 min. Specimens min to 60C to release all remaining cellular hiswere then quickly drained, washed briefly with tamine. After cooling, two i ml aliquots of this distilled water and covered with a solution of fluid were assayed for their histamine content as 0.05% toluidine blue in Mcllvaine's buffer at pH4 above. Histamine released during incubation at for 8 min. After a further quick wash in distilled 37C was expressed as a percentage of the total water, each specimen was covered with a glass histamine originally present in the cells, and was coverslip and viewed on the stage of a micro-corrected for the histamine released under scope at x 100 magnification. This protocol was appropriate control conditions. One assay comfound during pilot experiments to give good difprises the results obtained from one 0.5 ml ferential staining of mast cells. The numbers of aliquot of mast cell-rich suspension. Mast cells metachromatically (pink/magenta)-stained and from a minimum of three rats were used for darkly blue-stained mast cells associated with each treatment group. The total numbers of each milky spot were counted. The mean assays performed per treatment were between number of such mast cells per milky spot in seven and 22. Putative agonists used were PMXB, each piece of omentum was then calculated. The L-NAME, PAF or U46619. Putative antagonists numbers of milky spots in each piece of used were SNP and BN52021. omentum varied from 6 to 51, with most being around 25-35.
Statistics: Bonferroni's test was used for compar-Compounds tested as possible agonists were: ing several groups with one control group. L-NAME, an NO-synthase inhibitor; 7 PAF, a known inflammatory mediator; 8 U46619, a Chemicals used: L-NAME, PMXB, PAF, U46619, thromboxane-mimetic; 9 and polymixin B BZI, NDGA, compound 48/80, and OPT were all (PMXB) and compound 48/80, which are both obtained from Sigma Chemical Co. Ltd (Poole, known to degranulate mast cells. Compounds UK); SNP was obtained from David Bull Laboraused as possible antagonists were: indomethacin, tories (Warwick, UK); indomethacin from Merck, a cyclooxygenase inhibitor; 11 SNP, an NO Sharpe & Dohme Ltd (Hoddesdon, UK); tolui-12 donor; 1-benzylimidazole (BZI), a thromboxdine blue (batch 9244890D) from BDH (Poole, ane synthase inhibitor; BN52021, a specific PAF. UK). BN52021 was a gift from Dr P. Braquet, receptor antagonist; 4 and nordihydroguaiaretc Institut Henri Beaufour (Le Plessis-Robinson, acid (NDGA), a 5-1ipoxygenase inhibitor. 5 France). L-NAME, PMXB, compound 48/80, BZI, SNP and indomethacin were used as aqueous Histamine release from peritoneal mast cells: solutions, indomethacin being dissolved with the Female rats were killed as above. Then 40 ml of aid of a little NaiCO. NDGA and BN52021 were NS at 37C was injected into each peritoneal dissolved initially in dimethylsulphoxide, the find cavity and left for 15 min, massaging the concentration of solvent being <0.5%. U46619, abdomen gently from time to time. Approxas a concentrate in methyl acetate, and PAF, as a imately 35 ml of mast cell rich-fluid was recoverconcentrate in chloroform, were diluted with a able from each animal. After spinning the fluid at little ethanol before final dilution with NS. OPT 670 x g, the resultant cell pellet was re-suspended was dissolved in analytical grade methanol. in NS and then spun again for a total of three times. Finally the pellet was re-suspended in 5 ml Results NS, and 0.5 ml aliquots of mast cell-rich suspension were placed in each of 10  min, at which time histamine release was or U46619 (0.1 l,tM), however, were without a stopped by adding 1.5 ml ice-cold NS and significant effect. OMCs that were either subplacing the tubes in melting ice. After spinning stantially or completely degranulated by such the tubes at 670 x g for 10 min, two 1 ml alitreatments stained darkly blue. The ratio of quots from each supernatant were removed and .numbers of metachromatically stained OMCs to assayed fluorimetrically for histamine using, 1% darkly blue-stained OMCs (M/DB) in the milky phthaldialdehyde (OPT) as a fluorophore.  2). Of these antagonists, however, only indomethacin (Fig. 1) gave significant protection against the disappearance of pink/magentastained OMCs in NS-treated omenta during incubation at 37C, and this was accompanied by a significant increase in the M/DB ratio (to 10.5 _ _ _ 0.9). There was also a significant increase in the M/DB ratio after pre-treatment with SNP (to 12.6 _ _ _ 1.0), but with this drug the increase in the number of metachromatically stained OMCs per milky spot did not reach a conventionai level of statistical significance (Fig. 1). Pre-treatment of the omentum with NDGA (3 or 30 l.tM) did not reduce the effects of L-NAME significantly (Fig.  2). In contrast, pre-treatment with BN52021 (10 l.tM) significantly reduced the degranulating effects of PAF (Fig. 3), whereas a lower concentration (1 l.tM) was not significantly effective. Pre-treatment of the omentum with BZI (50 l.tM) had no effect on the response to PAF. Pre-treatment with BN52021 (10 l.tM), however, significantly reduced the degranulating effects of U46619 (Fig. 3).
PMCs responded to the addition of PMXB, PAF or U46619 with a significant increase in histamine release (Fig. 4). SNP, on the other hand, significantly decreased the release of histamine from PMCs (Fig. 4). In contrast to its mast celldegranulating effect in OMCs (see above), L-NAME exerted no significant effect on the release of histamine from PMCs (Fig. 4). However, pretreatment of PMCs with BN52021 (10 l.tM) significantl attenuated the histamine-releasing effect of PAF, but not that of U46619 (Fig. 4). In the presence of L-NAME (100 l.tM) histamine release in response to incubation with PAF or U46619 was increased but only slightly (Fig. 4) both anti-inflammatory and pro-inflammatory effects, depending upon the circumstances. 7 '18 In acute gastrointestinal injury there is substantial evidence to su&gest that NO has an anti-inflammatory effect, whereas a pro-inflammatory effect is sometimes more evident during inflammation occurring elsewhere in the body. 8 It has been shown previously that administration of L-NAME, which would be expected to have inhibited NO-synthase, caused leakage of albumin and of injected colloidal carboff' from rat small intestines and mesentery that apparently was due to a release of certain mast cell-derived amines. In the present experiments the application of L-NAME caused degranulation of OMCs in the milky spots. In contrast, pre-treatment with SNP, an NO donor, counteracted this degranulation ( Fig. 2), suggesting that where OMCs are concerned, both exogenous and endogenous NO can be protective. However, in which of the various omental cell type(s) the synthesis of NO was actually being blocked by added L-NAME remains unknown. In contrast, L-NAME did not increase histamine release from PMCs (Fig. 4).
This observation is similar to that of other workers who used A/-monomethyl-t-arginine as 19 an NO-synthase inhibitor. Perhaps resting PMCs have too low a cytoplasmic calcium ion concentration to permit activation of the constitutive form of NO-synthase. If so, then L-NAME would have no enzyme activity to inhibit. However, under certain circumstances, such as during stirring or incubation with lipopolysaccharide, PMCs in vitro do respond to treatment with L-NAME or other NO-synthase inhibitors with an enhanced release of histamine. 9 In the present experiments, however, L-NAME only slightly augmented the release of histamine that was produced in PMCs by adding PAF or U46619 (Fig. 4). The reasons for these discrepancies are presently unknown.
Successful antagonism of the degranulating effect of L-NAME on OMCs by pre-incubation with indomethacin, BZI, or BN52021 (Fig. 2) suggests that such degranulation may have been in response to the release of endogenous thromboxane A2 (TXA2) and PAF; but from which of the many cell types to be found within the omentum this release occurs is unknown. It is possible that different cell types may be responsible for secretion of the two agents. Since the degranulating effect of exogenously applied U46619 in OMCs, although not PMCs, was fully blocked by pre-treatment with BN52021, it is likely that the exogenous thromboxane-mimetic was first releasing PAF from the omentum and, therefore, that most of the OMC degranulation seen was due to this PAF, rather than to any direct effect that U46619 also can exert upon OMCs. The degranulating effect of exogenous PAF was blocked by pre-treatment of the omentum with BN52021, as would be expected, but it was not blocked by pre-treatment with BZI. This suggests, therefore, that in the presence of L-NAME there may be a release of wxa 2 within the omentum, which in turn releases substantial quantities of endogenous PAF. It may be the degranulating effects of the latter substance, therefore, that were being observed in incubated omenta in the present experiments.
Pre-treatment of the isolated omentum with NDGA in the present work did not affect the extent of L-NAME-induced degranulation of OMCs. This observation contrasts with a previous report that BW A137C, another 5-1ipoxygenase inhibitor, could ameliorate the combined effects of L-NAME and lipopolysaccharide in provoking the vascular leakage of albumin, 6 as also did indomethacin, BZI and BN52021. 5 By being able to secrete leukotrienes as well as prostaglandin D2, mucosal mast cells would appear to be phenotypically rather different from the connective tissue variety, since the latter can secrete only minute amounts of leukotrienes. 2 Moreover, OMCs appear to be of the connective tissue type, since Fig. 1 shows that they were degranulated in response to an application of compound 48/80, as were mesenteric and peritoneal mast cells in other earlier experiments, 21 whereas mucosal mast cells were not. 21 However, we are aware that NDGA, unlike BW A137C, has non-specific antioxidant properties. This might account for its lack of effect in the present experiments.
The protective effect of indomethacin on omental mast cells (Fig. 2) is interesting, since inhibiting cyclooxygenase with this drug would be expected to reduce the availability of both prostaglandins and wxa 2. Prostaglandins, however, appear to lack a regulatory effect on 22 mast cells. Consequently, only the effect of inhibiting wxa 2 production with indomethacin is likely to be evident in the isolated omentum. The overall effect of the drug, therefore, would be a reduced degranulation, an effect similar to that of BZI (Fig. 2), which was used to inhibit the synthesis of wxa 2 selectively. It appears that metabolic processes in the mast cell are finely balanced, but they can be pushed by various pharmacological means either towards cellular protection, for example, by inhibiting wxa 2 synthesis, or towards cellular damage, by inhibiting NO synth-esis. It seems likely that mucosal and omental mast cells react in pharmacologically similar ways. We hope to investigate this hypothesis further, but meanwhile we may conclude that the effects of L-NAME on mast cell degranulation in the rat intestine, mesentery and omentum are rather similar processes, involving a destabilizing influence of endogenous wxa 2 and PAF, possibly acting sequentially.