A temperature-sensitive step in exocytosis.

M.A Bittner,R.W Holz

doi:10.1016/s0021-9258(18)41989-8

M.A Bittner, R.W Holz

Open Access

https://doi.org/10.1016/s0021-9258(18)41989-8

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Abstract

We have examined the temperature sensitivity of exocytosis in digitonin-permeabilized chromaffin cells. The time course of secretion is markedly slowed by incubating the cells at 18 degrees C rather than 27 degrees C. We have previously shown that secretion has both ATP-dependent and ATP-independent components (Holz, R. W., Bittner, M. A., Peppers, S. C., Senter, R. A., and Eberhard, D. A. (1989) J. Biol. Chem. 264, 5412-5419). Reducing the temperature has no effect on ATP-independent secretion. However, cold (18 degrees C) greatly slows the ability of ATP to stimulate secretion. The ATP-requiring priming step itself is not affected by reducing the temperature since an effect of ATP can be seen after permeabilization at 18 degrees C if the cells are subsequently stimulated to secrete at 27 degrees C. When cells are permeabilized at 27 degrees C with ATP and then stimulated by Ca2+ in the absence of ATP, the secretion which was primed by ATP during the permeabilization step is inhibited 75% at 18 degrees C. Similar results are seen when ATP-dependent priming is enhanced by low concentrations of Ca2+. Thus, the temperature-sensitive step occurs after ATP and Ca2+ act to prime the cell. The temperature-sensitive step is likely to be overall rate-limiting step during the later phase of secretion, when the ATP-dependent priming process is limiting.

Highlights

At the endof an experiment, the incubation solutiownas removed, andthe cells were lysed with 1% Triton X-100
We have previously shown that Ca2+-dependent secretion absence of MgATP
Whecnells are permeabilized in the presence of Ca2+, there is a large secretory response that does not require ATP; aftseerveral minutes thisresponse slows and an ATP-dependent secretory responseis revealed

Summary

Introduction

At the endof an experiment, the incubation solutiownas removed, andthe cells were lysed with 1% Triton X-100. Cells were incubated for an additional 2 min in KGEP without digitonin in the continuing absence of MgATP a t 18 "C. The qualitative difference in temperature sensitivity between the two phases of secretion suggested that a unique step in the ATP-dependent pathway is inhibited by cold.

Results

Conclusion