title: Facilitation of Ca(2+)-dependent exocytosis by Rac1-GTPase in bovine chromaffin cells.
abstract: Rho family GTPases are primary mediators of cytoskeletal reorganization, although they have also been reported to regulate cell secretion. Yet, the extent to which Rho family GTPases are activated by secretory stimuli in neural and neuroendocrine cells remains unknown. In bovine adrenal chromaffin cells, we found Rac1, but not Cdc42, to be rapidly and selectively activated by secretory stimuli using an assay selective for the activated GTPases. To examine effects of activated Rac1 on secretion, constitutively active mutants of Rac1 (Rac1-V12, Rac1-L61) were transiently expressed in adrenal chromaffin cells. These mutants facilitated secretory responses elicited from populations of intact and digitonin-permeabilized cells as well as from cells under whole cell patch clamp. A dominant negative Rac1 mutant (Rac1-N17) produced no effect on secretion. Expression of RhoGDI, a negative regulator of Rac1, inhibited secretory responses while overexpression of effectors of Rac1, notably, p21-activated kinase (Pak1) and actin depolymerization factor (ADF) promoted evoked secretion. In addition, expression of effector domain mutants of Rac1-V12 that exhibit reduced activation of the cytoskeletal regulators Pak1 and Partner of Rac1 (POR1) resulted in a loss of Rac1-V12-mediated enhancement of evoked secretion. These findings suggest that Rac1, in part, functions to modulate secretion through actions on the cytoskeleton. Consistent with this hypothesis, the actin modifying drugs phalloidin and jasplakinolide enhanced secretion, while latrunculin-A inhibited secretion and eliminated the secretory effects of Rac1-V12. In summary, Rac1 was activated by secretory stimuli and modulated the secretory pathway downstream of Ca2+ influx, partly through regulation of cytoskeletal organization.
authors.affiliation: Department of Physiology, University of Michigan, Ann Arbor 48109, USA.
authors.forename: Quanwen
authors.initials: Q
authors.lastname: Li
authors.forename: Chi S
authors.initials: CS
authors.lastname: Ho
authors.forename: Vlad
authors.initials: V
authors.lastname: Marinescu
authors.forename: Humaa
authors.initials: H
authors.lastname: Bhatti
authors.forename: Gary M
authors.initials: GM
authors.lastname: Bokoch
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Ernst
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Holz
authors.forename: Edward L
authors.initials: EL
authors.lastname: Stuenkel
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002417:Cattle; D002458:Cell Fractionation; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000378:metabolism / Q000502:physiology*; D003599:Cytoskeleton / Q000502:physiology; D005089:Exocytosis / Q000235:genetics / Q000502:physiology*; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000096:biosynthesis; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007150:Immunohistochemistry; D008564:Membrane Potentials / Q000502:physiology; D009154:Mutation / Q000235:genetics / Q000502:physiology; D018408:Patch-Clamp Techniques; D010590:Phalloidine; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism / Q000502:physiology; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000502:physiology*; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
chemical_list: D000199:Actins; D020730:Guanine Nucleotide Dissociation Inhibitors; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D006153:Guanosine Diphosphate; D010590:Phalloidine; D006160:Guanosine Triphosphate; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D002118:Calcium