INDICE DE CONFIANZA DEL COMERCIO MINORISTA

5.4.1.1 The characterisation of the hyperosmolar response

5.4.1.1.1 Dose response

O ver a 10 min period of incubation, non-purified and purified mast cells achieved mean maximal histamine releases of 2.6 ± 0.8 % and 4.1 ± 1.7 % respectively at the highest mannitol (0.2-1.0 M) concentrations tested (Table 5.1). Immunologic activation (anti-lgE, 1/3000 - 1/100 dilution. Table 5.2), elicited maximal values of 9.8 ± 0.6 % and 11.3 ± 4.1 % for non-purified and purified suspensions, respectively. Since there was little difference between purified and non-purified preperations, all sub sequent inve stigatio ns employed non-purified dispersed lung mast cells.

5.4.1.1.2 The kinetics of the release process

Mannitol (0.1-1.0 M)-induced histamine release from DL mast cells was extremely time-dependent and was minimal for 5, 10 and 20 min incubation periods, thereafter increasing to a maximum of ca 20 % (1 M mannitol, 45 min incubation. Fig. 5.1). The kinetics of anti-lgE (1/100 dilution. Fig. 5.2)- induced release were much more rapid and the release process was approximately 80 % complete within 10 min. In order to elicit a reasonable level of histamine release (> 15 %), a challenge time of 45 min (unless otherwise stated) was chosen for subsequent experiments. This incubation period was used for both modes of stimulation.

5.4.1.1.3 The effect of temperature

The optimum for mannitol (1.0 M)-induced amine liberation was 37 °C (Fig. 5.3). Additionally, release was also observed for 30 and 60 min incubation periods at 0, 25 and 45 °C. Under immunologic conditions (anti-lgE, 1/100 dilution. Fig. 5.4), histam ine secretion was maximal under physiological conditions and markedly depressed at extremes of temperature.

5.4.1.1.4 The effect of pH

H yperosm olar-induced histam ine release was affected by changes in extracellular pH, with maximal secretion being seen at pH extremes (pH = 6.5 and 8.5, Fig. 5.5). With anti-lgE, release was maximal and virtually equivalent at pH values of 7.0 and 7.5 (Fig. 5.6).

5.4.1.1.5 The effect of calcium

At low concentrations of mannitol (0.2, 0.4 M), histamine release in calcium- free buffer was com pletely negated (Fig. 5.7). Thereafter, an increase in mannitol concentration produced a dose-dependent increase in histamine secretion in this medium. Additionally, the release observed in FHT and EDTA buffers was very similar. A distinctly more Ca^+ - dependent result was seen when anti-lgE (1/100 dilution. Fig. 5.8) was tested on DL mast cells.

5.4.1.1.6 The effect of metabolic Inhibitors

The metabolic inhibitors 2DG and AA were employed to determine whether m ediator release under hyperosm olar conditions occurs via cytotoxic or no n-cyto toxic m echanism s (Table 5.3 a-d). R elease was e sse n tia lly unaffected by these agents fo r all incubation periods tested. This observation indicates that m annitol-induced amine secretion in DL mast cells occurs by A TP -independent, cytotoxic m echanism s. C onversely, release initiated by anti-lgE (1/100 dilution) was shown to be inhibited in the presence of 2DG and AA, suggesting that IgE-mediated release in DL mast cells follows an ATP-dependent, non-cytotoxic pathway (Table 5.4).

5.4.2 BAL data

5.4.2.1 The characterisation of the hyperosmolar response

5.4.2.1.1 Dose response

Human BAL mast cells were responsive to the effects of mannitol (0.1-1.0 M, Fig. 5.9). The diuretic induced a dose-dependent release from this cell type after 10 min incubation with a maximum secretion of ca 35 % at the highest concentration tested. BAL mast cells also responded to anti-lgE (1/3000 - 1/50 dilution, 10 min incubation. Fig. 5.10), producing a maximal histamine release of ca 30 % at 1/100 dilution.

5.4.2.1.2 The kinetics of release

The time-course of mannitol (1.0 M)-induced amine liberation was biphasic (Fig. 5.11). Initially, there was a rapid release of histamine which reached a m axim um at 10 min of challenge. S ubsequent release was slow, yet progressive, reaching a value of ca 30 % at 60 min challenge. Histamine secretion under immunologic conditions (anti-lgE, 1/100 dilution, Fig. 5.12) was extremely rapid, being essentially complete after two min.

5.4.2.1.3 The effect of temperature

The tem perature dependency of m annitol-induced histam ine release from BAL mast cells was sim ilar to that seen in DL mast cells (Fig. 5.13). The

optimum for mannitol (0.8 M, 10 min challenge)-induced amine secretion was 37 °C, but release (> 10 %) was seen at 25 °C. With anti-lgE (1/100 dilution, 10 min incubation, Fig. 5.14), maximal release was recorded under ph ysiolog ica l con dition s, w ith little se cre tio n docum ented at o th e r tem peratures.

5.4.2.1.4 The effect of pH

Mannitol (0.1-1.0 M, 10 min incubation)-induced histamine release from the BAL mast cell was observed to be maximal at extremes of pH (pH = 8.5 and 6.5, Fig. 5.15). Conversely, the secretion initiated by anti-lgE (1/100 dilution, 10 min incubation. Fig. 5.16) was greatest under physiological conditions (pH = 7.0 and 7.5).

5.4.2.1.5 The effect of calcium

At low mannitol concentrations at 10 min incubation (0.2, 0.4 M), release in buffers lacking Ca2+ ions was negated, but at higher diuretic levels, dose- reliant increments of histamine release were documented (Fig. 5.17). This secretion was furthe r enhanced in buffers containing EDTA. However, m axim al secretion was observed in the presence of ph ysio lo g ica l concentrations of the cation. A similar pattern of release was recorded with anti-lgE (1/100 dilution, 10 min challenge. Fig. 5.18).

5.4.2.1.6 The effect of metabolic inhibitors

The release induced by m annitol (0.2-1.0 M, 10 min challenge) was unaffected by simple omission of glucose (-G) from the medium (Fig. 5.19). Release was diminished, however, in the presence of 2DG, especially at low er concentrations (0.2, 0.4 M) of the diuretic. Secretion was also attenuated, though not abolished, in buffers containing 2DG and AA. In this system, a dose-dependent increase in amine liberation was recorded. This result indicates that a com ponent of hyperosm olar-induced histam ine release in BAL mast cells is cytotoxic. When anti-lgE (1/100 dilution, 10 min incubation) was tested, it was seen that release was dim inished in the presence of 2DG and effectively negated when 2DG and AA were employed in combination (Fig. 5.20).