Calcium and GTP-gamma-S as single effectors of secretion from permeabilized rat mast cells: requirements for ATP

Treatment with metabolic inhibitors and addition of exogenous MgATP exerted different effects on secretion from streptolysin-O-permeabilized mast cells, responding to calcium and GTP-gamma-S as single effectors (i.e., independently of each other). These effects were also strongly dependent on the experimental conditions. Thus cells, triggered by Ca2+ at the time of permeabilization, did not require MgATP, but after metabolic inhibition rapidly became absolutely dependent on its provision, requiring high (> mM) concentrations. AMP-PNP was not effective. After longer treatment with metabolic inhibitors, the absolute dependence on MgATP was also exhibited by cells responding to dual effectors (i.e., Ca2+ and GTP-gamma-S applied together). In contrast, calcium independent secretion due to GTP-gamma-S was more resistant to metabolic inhibition, exhibiting no absolute requirements for MgATP. Once the responsiveness to GTP-gamma-S had been lost, it could not be restored by addition of MgATP. MgATP, in fact, inhibited the response of permeabilized cells to GTP-gamma-S. This effect could be mimicked by AMP-PNP. When permeabilized cells were washed before triggering, MgATP (0.1-1 mM concentration range) was no longer inhibitory but stimulatory. These differences between Ca(2+)- and GTP-gamma-S-induced responses indicate that ATP utilization is essential to the calcium, but not to the guanine nucleotide, pathway to secretion. The rate of the response to calcium/MgATP was much slower in the absence than in the presence of GTP-gamma-S. The onset of secretion occurred after an initial delay. This lag phase was abolished by addition of GTP-gamma-S, suggesting that a GTP-binding protein may control a reaction which constitutes a rate-limiting step in the secretory process.