ASSOCIATION OF ALPHA(S)-SUBUNIT OF THE G(S) PROTEIN WITH MICROFILAMENTS AND MICROTUBULES - IMPLICATION DURING ADRENOCORTICOTROPIN STIMULATION IN RAT ADRENAL GLOMERULOSA CELLS

The aim of the present study was to investigate if and how microfilame nts and microtubules could be involved in the early events of ACTH act ion. In primary cultures of rat glomerulosa cells, a 30-min preincubat ion with either 10 mu M colchicine (a microtubule-disrupting agent) or 10 mu M cytochalasin B (a microfilament-disrupting agent) decreased A CTH-induced cAMP production. Moreover, colchicine decreased cAMP produ ction induced by fluoroaluminate (a nonspecific activator of all G pro teins), but not of forskolin (which directly activates adenylyl cyclas e). These results indicate that microtubules appear to be essential fo r the G(s) protein activation. In contrast, cytochalasin B decreased t he stimulating effect of both fluoroaluminate and forskolin, indicatin g that microfilaments may be involved in both fluoroaluminate and fors kolin, indicating that micrfilaments may be involved in both G(s) and adenylyl cyclase activations. Analyses of microfilament- and microtubu le-enriched fractions and immunoprecipitation of actin and tubulin ind icated that the alpha(s)-subunit of the G(s) protein was associated wi th both structures. Stimulation of cells with ACTH induced a rapid inc rease (within 1 min) in the levels of microfilaments, microtubules, an d alpha(s) associated with the membrane. In addition, ACTH stimulation of cAMP production was very sensitive to Ca2+, without any stimulatio n in Ca2+-free medium. Under these conditions, actin filaments were sh ort and formed a dense network. These observations suggest that the Ca 2+-free medium stabilized the actin fibers in such a way that activati on by ACTH failed, further documenting the importance of microfilament s in cAMP production.