Cytoskeletal inhibitors impair Ca2+ elevations via neuropeptide Y and other Gi-coupled receptors

Neuropeptide Y, alpha 2-adrenoceptors, thrombin and certain muscarinic acetylcholine receptors can couple to elevations of intracellular free Ca2+ concentrations via Gi-proteins. We have studied the effects of inhibitors of microtubules (colchicine, nocodazole, vinblastine) and microfilaments (cytochalasin B, cytochalasin D) on these effects in human erythroleukemia (HEL) cells. Both types of inhibitors reduced neuropeptide Y-, adrenaline- (via alpha 2A-adrenoceptors) and thrombin-stimulated Ca2+ elevations while the inactive analog beta-lumicolchicine was without inhibitory effects. Similarly, in SK-N-MC cells vinblastine inhibited neuropeptide Y and carbachol (via muscarinic receptors) stimulated Ca2+ elevations. In HEL cells the inhibitory effects of the microfilament inhibitor cytochalasin D and the microtubule inhibitor colchicine were not additive. Colchicine, nocodazole or cytochalasin D did not affect the binding of the agonist neuropeptide Y. On the other hand, neuropeptide Y and thrombin significantly stimulated GTP gamma S binding in the absence but not in the presence of colchicine, vinblastine or cytochalasin B. This was not due to sequestration of G-protein alpha-subunits, since nocodazole did not affect the distribution of immunodetectable Gi alpha 1/2 or Gi alpha 3 between membrane and cytosolic fractions. We conclude that disruption of microfilaments or microtubules impairs Ca2+ elevations by neuropeptide Y and other Gi-coupled receptors by inhibiting receptor/Gi-protein interaction; this does not involve impairment of agonist binding to the receptor or redistribution of Gi-protein alpha-subunit

Translocation of microfilament-associated inhibitory guanine-nucleotide-binding proteins to the plasma membrane in myeloid differentiated human leukemia (HL-60) cells

The cytoskeletal localization of inhibitory guanine-nucleotide-binding (Gi) proteins and the coupling of these proteins to formyl peptide receptors were studied in myeloid differentiated human leukemia (HL-60) cells. Treatment of HL-60 cells with cytochalasin B or botulinum C2 toxin, which leads to the disruption of microfilaments, increased the binding of the stable GTP analogue guanosine 5'[gamma-thio]triphosphate (GTPS[S]) to permeabilized cells by about 30%. In contrast, the microtubule-disrupting agents colchicine and vinblastine, and cytochalasin B treatment of isolated HL-60 membranes did not affect GTP[S] binding. The stimulatory effect of cytochalasin B treatment was concentration and time dependent, with maximal increases observed at 5 micrograms/ml cytochalasin B and an incubation time of 10 min, and was counteracted by the F-actin-stabilizing toxin phalloidin. Cytochalasin B treatment increased the amount of G proteins activated by chemoattractant receptors by about 25%. Furthermore, the number of Gi-protein-coupled receptors for the chemoattractant, N-formyl-Met-Leu-Phe, was increased by about 25% upon cytochalasin B treatment. Based on these functional data, which suggest an association of G proteins with actin filaments, the Triton X-100 (1%)-insoluble cytoskeleton was analyzed for the presence of G proteins. Gia subunits were detected in the cytoskeleton preparations, both by specific antisera and by pertussis-toxin -catalyzed ADP-ribosylation. Cytochalasin B pretreatment depleted the cytoskeleton in Gialpha, with an approximately 20% concomitant increase in membrane Gialpha content. In conclusion, evidence is presented that part of the cellular Gia is localized at actin filaments in HL-60 cells. After filament disruption, these Gia subunits seem to be translocated to the plasma membrance, where they can productively interact with chemoattractant receptor