The Effects of G-Protein Activators, Mastoparan and Compound 48/80, on Serotonin Secretion and Signaling Pathway in Human Platelets

Abstract

Mastoparan and compound 48/80 have been found to accelerate guanine nucleotide

exchange and GTPase activity of purified GTP-binding protein. These compounds can

directly activate secretory processes of mast cells, pancreatic islets and adrenal chromaffin

cells by penetrating through plasma membranes and directly stimulate membrane

GTPase activity and stimulates PLC-mediated events without mediating via receptor

binding. This study aims to examine whether these compounds affect secretion of both intact

and permeabilized human platelets, and examine the subtype of G-protein signaling. [³H]-serotonin labeled platelets were pre-incubated for 5 min and were activated with various

concentrations of mastoparan and compound 48/80 for 3 min at room temperature or

preincubated with streptolysin O (SLO) for 2 min before activation. The amounts of [³H]5-HT

release were determined by liquid scintillation counting. Mastoparan was found to produce a

concentration-dependent increase in 5-HT release from intact platelets with an EC50 of 20 μM.. The maximal secretion was obtained at the concentration of 60 μM .. Similarly, compound

48/80 caused a concentration-dependent increase in 5-HT release with maximal secretion

obtained at the concentration of 400 μg/ml.. Permeabilized platelets with streptolysin O

significantly increase serotonin secretion. To investigate whether the observed stimulation of

serotonin secretion is mediated through the Gi subunit of G-protein, the G-protein blocking

agents (e.g. Gi-sensitive pertussis toxin, benzalkonium chloride, a selective Gi inhibitor, and

daunomycin, a lipid bilayer stabilizer) were used. Mastoparan- and compound 48/80-induced

secretion was inhibited by preincubation with pertussis toxin only in SLO-permeabilized

platelets whereas benzalkonium chloride and daunomycin did not affect rnastoparan- and

compound 48/80-induced secretion in both intact and SLO-permeabilized platelets. The

results from this study suggested that mastoparan- and compound 48/80 promoted secretion

by mechanisms involved neither the stimulation of Gi-subtype of G-protein nor interfering

with lipid bilayer of the membranes. The secretory event may result either from a direct

fusogenic action and/or from the stimulation of putative exocytosis-linked G-protein, Ge ..

Their mechanisms on small GTPase proteins, on Ge and on membrane perturbation in human

platelets remain to be elucidated.