Tercer Paso

4.3.1.1 Histamine release from human GI mast cells

The mast cells isolated from human colon and stomach were structurally intact as judged under light microscopy. They exhibited high viability (>97 %), low

spontaneous release o f histamine ( < 1 0 %) and effective dose-dependent histamine release when stimulated with anti-human IgE [Fig 4.3,4.4].

The histamine release from human colonic mast cells stimulated with anti-human IgE (1 0 0-1 0 , 0 0 0 fold dilutions) showed mucosal mast cells to be more responsive than submucosa/muscle mast cells, reaching maximal releases o f 42.0 ± 1 . 3 (HCM) and 24.0 ± 3.9 % (HCS) [Fig 4.3].

The same stimulus on human gastric mast cells also released histamine dose- dependently. The HGM and HGS mast cells had similar responsivity, reaching maximal releases of 38.1 ± 1.5 (HGM) and 34.2 ± 4.0 % (HGS) [Fig 4.4].

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4.3.1.2 Effects o f sulphasalazine and its metabolites on isolated HCM and HCS

mast cells

5-ASA [Fig 4.5,4.6] produced comparable dose-dependent inhibition of histamine release from HCM and HCS mast cells stimulated with an optimal amount of anti­ human IgE (200 fold dilution). The inhibition of histamine release was similar when the cells were preincubated with the drug for 1 0 min before challenge, compared to when the drug was added simultaneously with the immunological stimulus.

In contrast to the effect of 5-ASA, neither sulphasalazine [Fig 4.7,4.8] nor sulphapyridine [Fig 4.9,4.10] had any effect on immunologically stimulated histamine release from HCM and HCS mast cells, while DSCG, used as a positive control, produced effective dose-dependent inhibition of this release.

In contrast to sulphasalazine’s ineffectiveness on GI mast cells stimulated with optimal amounts o f anti-human IgE, the drug markedly potentiated the release induced by a suboptimal (10,000 fold dilution) amount o f the ligand [Fig 4.11,4.12]. This effect was most marked, in the case of the HCS cells, when the preparations were preincubated with the drug for 10 min before challenge. Under these conditions, the histamine release from HCM and HCS mast cells was increased, in the presence o f lO'"* M sulphasalazine, from 13.1 ± 2.0 to 30.0 ± 5.8 % and 10.4 ± 4.1 to 21.3 ± 5.0 %, respectively. Neither sulphapyridine nor 5-ASA had any effect under these conditions [Table 4.1].

This striking potentiation was further studied using an optimum concentration o f sulphasalazine (10^ M) and a range of anti-human IgE dilutions (100-10,000 fold dilutions). Sulphasalazine increased histamine release of HCM mast cells from 7.5 ± 2.5 (anti-human IgE 10,000 fold dilution) to 15.0 ± 4.9 % (anti-human IgE 10,000 fold dilution + sulphasalazine (10'"^ M)). This potentiation was only apparent at high dilutions of anti-IgE and, as reported above, disappeared at lower dilutions. A similar effect was observed with HCS mast cells, and the release increased from 6.5 ± 1 . 8 (anti-human IgE 10,000 fold dilution) to 13.0 ± 3.4 % (anti-human IgE 10,000 fold

4.3.1.3 Effects o f sulphasalazine and its metabolites on isolated HGM mast cells

5-ASA [Fig 4.15] produced a dose-dependent inhibition of histamine release from HGM mast cells stimulated with an optimal amount o f anti-human IgE (200 fold dilution). The inhibition was negligible in the concentration range 10'^ M to 10'^ M and a marked increase was then observed in the 10'^ M to 10'^ M range, reaching a maximal inhibition o f 62.5 ± 3.0 %. This attenuation of histamine release was identical when the cells were preincubated with the drug for 1 0 minutes before challenge and when the drug was added simultaneously with the immunological stimulus.

As found with the study on human colonic mast cells, neither sulphasalazine [Fig 4.16] nor sulphapyridine [Fig 4.17] had any effect on immunologically-stimulated histamine release from HGM mast cells, while DSCG, used as positive control, produced effective dose-dependent inhibition o f this release [Fig 4.16,4.17].

In HGM mast cells, sulphasalazine again strikingly potentiated the histamine release induced by a suboptimal (10,000 fold dilution) amount o f the ligand [Fig 4.18]. Under these conditions, the histamine release from HGM mast cells was increased, in the presence o f 10*^ M sulphasalazine, from 9.2 ± 2.8 to 18.8 ± 3.0 %. In contrast, sulphapyridine and 5-ASA had no effect under these conditions [Table 4.2].

This striking potentiation was further studied using an optimum concentration of sulphasalazine and a range of anti-human IgE dilutions (100-10,000 fold dilutions). Sulphasalazine increased histamine release of HGM mast cells from 5.4 ± 4.7 (anti­ human IgE 10,000 fold dilution) to 12.4 ± 2.6 % (anti-human IgE 10,000 fold dilution + sulphasalazine (10'"^ M)). This potentiation was again observed only at high dilutions of anti-IgE and disappeared at lower dilutions [Fig 4.19].

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4.3.2 Effects of H2-receptor antagonists and a cytoprotectant on histamine release

from histaminergic cells

4.3.2.1 Histamine release from freshly isolated histaminocytes induced by H2'

receptor antagonists and a cytoprotectant, misoprostol

The H2-receptor antagonists famotidine and nizatidine (5x10'^^ M-10'^ M) had no stimulatory effect on histamine release from any o f the cell types employed in this study. The cytoprotectant misoprostol caused striking histamine release at concentrations above 10‘® M, which suggests this drug may be cytotoxic under these conditions [Table 4.3].

4.3.2.2 Effects o f H2-receptor antagonists and the cytoprotectant misoprostol on

histamine release from RPMCs

Famotidine and nizatidine produced dose-dependent inhibition of histamine release from RPMCs stimulated with an optimal amount o f anti-rat IgE [Fig 4.20,4.21]. Some evidence of tachyphylaxis was observed, particularly with lower concentrations of famotidine. The maximal inhibitions achieved were 43.3 ± 5.6 (famotidine, 10'^ M, 0 min preincubation), 30.9 ±6. 1 (famotidine, 10'^ M, 10 min preincubation), 47.7 ± 3.7 (nizatidine, 10'^ M, 0 min preincubation) and 50.8 ± 5.2 % (nizatidine, 10'^ M, 10 min preincubation).

The inhibition produced by misoprostol was bell shaped; very low concentrations (10 pM) produced modest inhibition (20-30 %), while higher concentrations (>1 nM) produced a negligible effect [Fig 4.22].

histamine release from human colonic mast cells

Famotidine and nizatidine produced dose-dependent inhibition of histamine release from HCM and HCS mast cells stimulated with an optimal amount o f anti-human IgE [Fig 4.23-4.25]. The effect was independent of the preincubation period (0 min and 1 0 min) and there was no evidence o f tachyphylaxis.

The maximal inhibitions achieved by the compounds on HCM mast cells were 31.0 ± 2.5 (famotidine, 10"^ M, 0 min preincubation), 25.0 ± 2.0 (famotidine, 10'^ M, 10 min preincubation), 45,5 ± 4 .1 (nizatidine, 10"^ M, 0 min preincubation) and 50.4 ± 5.8 % (nizatidine, lO'^M, 10 min preincubation) [Fig 4.23,4.24].

The maximal inhibitions achieved by the compounds on HCS mast cells were 45.1 ± 2.1 (famotidine, 10'^ M, 5 min preincubation) and 39.8 ± 3.5 % (nizatidine, 10"^ M, 5 min preincubation) [Fig 4.25].

The cytoprotectant misoprostol produced a bell shaped inhibition curve with both cell types. At very low concentrations o f the drug (0.1 nM) there was significant inhibition 50.8 ± 6 . 8 (HCM, 0 min preincubation), 35.8 ± 4.2 (HCM, 10 min preincubation) and 63.4 ± 4.9 % (HCS, 5 min preincubation), whereas higher concentrations (>100 nM) had a negligible effect [Fig 4.26,4.27].

4.3.2.4 Effects o f H2-receptor antagonists and the cytoprotectant misoprostol on

histamine release from human basophil leukocytes

In these experiments famotidine and nizatidine again produced dose-dependent inhibition of histamine release from human basophil leukocytes stimulated with anti­ human IgE (500-1,000 fold dilution). The maximal inhibitions achieved were 23.3 ± 3.1 (famotidine, 10'^ M) and 53.0 ± 2.4 % (nizatidine, 10'^ M) after 5 min preincubation [Fig 4.28].

149

In contrast to the bell shaped effects o f misoprostol on RPMCs and human colonic mast cells, misoprostol showed increasing dose-dependent inhibition o f histamine release from basophils, reaching a maximum of 55.0 ± 3.3 % (5 min preincubation) at the highest test concentration (1 |iM ) [Fig 4.29].

4.4 Discussion

This study has investigated the effect o f clinically relevant compounds on histamine release from various histaminocytes.