Repression of serotonin secretion by an endogenous Ca2(+)-activated protease in electropermeabilized bovine platelets.
Micromolar levels of free calcium ions added to the extracellular medium elicit secretion of serotonin from electropermeabilized bovine platelets in the presence of millimolar levels of Mg-ATP. Such Ca2(+)-dependent secretion of serotonin was almost completely impaired when the permeabilized platelets were preincubated for 1 min at 35 degrees C in 100 microM Ca2+ without Mg-ATP. The half-maximal effect was observed with about 45 microM Ca2+ in the preincubation medium. Inhibitors of serine-thiol protease, such as leupeptin and antipain, suppressed the impairment of the secretion of serotonin by the preincubation with Ca2+. Electron microscopic observation revealed that disorganization of the cytoskeletal structures, in particular of the membrane undercoat and the network of microfilaments, accompanied the impairment of secretion of serotonin. Microfilaments were also found to be dissociated from dense granules that contained serotonin. These morphological changes were also suppressed when antipain was included in the Ca2(+)-preincubation medium. Coincident with these morphological changes, the following biochemical changes were observed in 100 microM Ca2+ but not in the presence of Ca2+ and antipain. The amount of Triton-insoluble cytoskeleton and the acto-myosin content of the dense-granule fraction were markedly decreased. The decrease in Triton-insoluble cytoskeletons was quantitatively correlated with the degree of impairment of secretion of serotonin. Immunoblot analysis of EGTA extracts of the cells showed that the 240-kDa spectrin in platelets was degraded to a 235-kDa fragment, and a 260-kDa actin-binding protein (ABP) in platelets was partially degraded to 190- and 110-kDa components.(ABSTRACT TRUNCATED AT 250 WORDS)
Morimoto
T
T
Department of Biology, Faculty of Science, Osaka University.
Oho
C
C
Ueda
M
M
Ogihara
S
S
Takisawa
H
H
D000818:Animals; D000976:Antipain; D001792:Blood Platelets / Q000201:enzymology* / Q000648:ultrastructure; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000201:enzymology; D003599:Cytoskeleton / Q000737:chemistry; D003851:Deoxyribonucleases; D010450:Endopeptidases / Q000097:blood / Q000378:metabolism / Q000502:physiology*; D004789:Enzyme Activation; D005089:Exocytosis; D066298:In Vitro Techniques; D008840:Microfilament Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D010539:Permeability; D012701:Serotonin / Q000378:metabolism*; D013049:Spectrin / Q000276:immunology; D013347:Subcellular Fractions / Q000201:enzymology
D008840:Microfilament Proteins; D013049:Spectrin; D012701:Serotonin; D000976:Antipain; D003851:Deoxyribonucleases; D010450:Endopeptidases; D002118:Calcium