LIGHT AND ELECTRON-MICROSCOPIC STUDY OF CHANGES IN THE ORGANIZATION OF THE CORTICAL ACTIN CYTOSKELETON DURING CHROMAFFIN CELL SECRETION

Chromaffin cells cultured for 2 days were incubated in the absence or presence of 10 mu M nicotine for 40 sec. Resting and stimulated cells were fixed and either prepared for fluorescence microscopy or treated with Triton X-100 to obtain cytoskeletons for ultrastructural studies. Electron microscopy of cytoskeletons revealed the presence of polygon al areas devoid of actin filaments only in nicotinic receptor-stimulat ed cells. Staining of these cytoskeleton preparations with rhodamine-p halloidin, a probe for filamentous actin, produced fluorescent pattern s and three-dimensional images similar to those obtained from resting or stimulated intact cells prepared directly for fluorescence microsco py. Moreover, the percentage of stimulated cells showing disrupted cyt oskeleton at the electron microscopic level was similar to the percent age of stimulated cells showing patched rhodamine fluorescence at the fluorescence microscopic level. In addition, cells stimulated with nic otine for 40 sec showed a fivefold increase in amine output and a sign ificant decrease in F-actin levels. These results provide the first ul trastructural evidence for nicotinic receptor-evoked chromaffin cell F -actin disassembly and show that the rhodamine-phalloidin-unstained ar eas observed in fluorescence microscopy represent the areas devoid of filamentous actin observed at the electron microscopic level.