Membrane potential changes induced by antigen

The resting membrane potential in RBL-2H3 cells has been reported to be between -60 to -90mV by several groups [57, 125, 126]. Membrane potential is affected by changes in [K^Jo but not by ouabain, a NaVK^ ATPase inhibitor, indicating that, in contrast to rat peritoneal mast cells, the resting membrane potential in RBL-2H3 cells is not determined by the NaVK"^ ATPase pump [125]. In addition, no potassium channel has been detected in rat peritoneal mast cells [137, 222, 223].

In this study, RBL-2H3 cells were depolarised by KCl, RbCl and gramicidin ( Fig. 4.1 and 4.2) and the depolarisation by Ipg/m l gramicidin was used as a standard to quantify the observed data.

The antigen-induced depolarisation has been studied in RBL-2H3 cells by various groups, using different methods [53, 57, 111, 129]. Following antigen stimulation, these cells were depolarised and this depolarisation is followed by a partial repolarisation which is inhibited by quinidine, a channel blocker [53]. The induced depolarisation is likely to be due to Ca^"^ influx, since it is inhibited by any procedure that inhibits Ca^"^ influx including; application of extracellular La^^, prior depolarisation by high buffer, addition of excess DNP-Lys (hapten) and ATP-depletion [128, 130]. The observation that A23187 induced depolarisation in RBL-2H3 cells (Fig. 4.8) also suggests a relationship between Ca^^ influx and depolarisation. However, in the absence of [Ca^'^]o, antigen still depolarises RBL-2H3 cells, indicating that Na"^ influx through CRAC channels may be responsible for the depolarisation in the absence of [Ca^"^]o [57, 111, 148, 150]. This result is consistent with the lack of depolarisation in the Ca^^/Na^-free buffer (Fig. 4.6). The repolarisation phase is also greatly inhibited in Ca^^-free buffer, suggesting an important role of the increase in [Ca^^]i in producing the repolarisation phase.

In this study, membrane potential changes were monitored, using bis-oxonol, a fluorescent dye. The antigen-triggered depolarisation is dose-dependent and starts after a delay of 15 seconds and reaches a peak at 1 minute (Fig. 4.3). This response shows different kinetics to those reported by Labrecque et al. [53]. The depolarisation is followed by a partial repolarisation which is also antigen dose-dependent, though in the reverse direction; i.e. the repolarisation phase decreased with increasing concentration of antigen. The repolarisation phase of the antigen-induced membrane potential changes has

been suggested to be the result of the opening of channels, since it can be inhibited by Ba^^ and quinidine [53].

However, it could be argued that the repolarisation phase might not be a genuine phenomenon but rather the result of exocytosis and loss of the dye bound to the cell membrane. It is very difficult to establish that this is not the case, because in any condition in which the stimulated secretion is inhibited, depolarisation is also inhibited. In the absence of depolarisation, the repolarisation phase cannot be observed.

The repolarisation phase has been reported with antigen stimulation of RBL-2H3 cells in free solution, and after the pretreatment of the cells with 5mM 4-AP [53]; these are conditions in which secretion induced by antigen is greatly inhibited, indicating that the repolarisation phase is not a consequence to secretion.

La^^ (lOOpM) can immediately abrogate antigen-stimulated secretion, when it is added to the cell suspension. If La^'*’ is added 5min before cell stimulation, the antigen- induced depolarisation is inhibited. However, when La^^ is added at the peak of antigen- induced depolarisation, which abrogates secretion induced by antigen, the repolarisation phase still can be observed. This also suggests that the antigen-stimulated depolarisation precedes secretion.

Moreover, the increase in antigen concentration causes a progressive reduction in repolarisation (which reaches a maximum decrease with antigen excess), while the rate of secretion increases with higher concentrations of antigen. If secretion and the observed reduction in bis-oxonol fluorescence follow a cause and effect relationship, they should have the same dose-response curve. However, the curves are in opposite directions. This further suggests that the repolarisation phase (reduction in fluorescence of bis-oxonol) is not the result of antigen-induced secretion. One possible explanation as to why antigen excess reduces repolarisation is that Ca^^ influx continues to rise during this phase so that

depolarisation dominates and becomes too large to be offset by opening of channels.

It seems that the repolarisation has some relationship with the decline in [Ca^^], following the initial rise, although the time course for [Ca^^]j decline in a cell suspension (20-30min) is much longer than that for the repolarisation phase (8-9min) [111]. Thus, the time course of the change in [Ca^^]i response is more prolonged than that of the membrane potential changes initiated by antigen.

It appears that the depolarisation and repolarisation are in a balanced condition at an antigen concentration of O.lng/ml DNP-HSA. In high concentrations of antigen, depolarisation is dominant and cells do not fully repolarise to the resting membrane

potential. In low concentrations of antigen, repolarisation is more dominant and antigen at very low concentrations may cause a hyperpolarisation. These results are compatible with the effect of quinidine on the antigen-induced 8-hexosaminidase release: quinidine is

more potent against lower concentrations of antigen than the higher concentrations of antigen.