An antisense oligodeoxynucleotide targeted to chromaffin cell scinderin gene decreased scinderin levels and inhibited depolarization-induced cortical F-actin disassembly and exocytosis.
Chromaffin cell secretion requires cortical F-actin disassembly and it has been suggested that scinderin, a Ca2+ dependent F-actin severing protein, controls cortical actin dynamics. An antisense oligodeoxynucleotide targeting the scinderin gene was used to decrease the expression of the protein and access its role in secretion. Treatment with 2 microM scinderin antisense oligodeoxynucleotide for 4 days produced a significant decrease in scinderin expression and its mRNA levels. The expression of gelsolin, another F-actin severing protein, was not affected. Scinderin decrease was accompanied by concomitant and parallel decreases in depolarization-evoked cortical F-actin disassembly and exocytosis. Similar treatment with a mismatched oligodeoxynucleotide produced no effects. Scinderin antisense oligodeoxynucleotide treatment was also a very effective inhibitor of exocytosis in digitonin-permeabilized cells stimulated with increasing concentrations of Ca2+. This ruled out scinderin antisense interference with stimulation-induced depolarization or Ca2+ channel activation. Scinderin antisense treatment decreased the maximum (B(max)) secretory response to Ca2+ without modifying the affinity (K(m)) of the cation for the exocytotic machinery. Moreover, the antisense treatment did not affect norepinephrine uptake or the expression of dopamine ss-hydroxylase, suggesting that the number and function of chromaffin vesicles was not modified. In addition, scinderin antisense treatment did not alter the expression of proteins involved in vesicle-plasma membrane fusion, such as synaptophysin, synaptotagmin or syntaxin, indicating a lack of effects on the fusion machinery components. These observations strongly suggest that scinderin is a key player in the events involved in the secretory process.
Lejen
T
T
Secretory Research Program, Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Canada.
Skolnik
K
K
Rosé
S D
SD
Marcu
M G
MG
Elzagallaai
A
A
Trifaró
J M
JM
D000199:Actins / Q000502:physiology*; D000818:Animals; D002417:Cattle; D002540:Cerebral Cortex / Q000378:metabolism*; D019439:Chromaffin Cells / Q000378:metabolism / Q000502:physiology*; D004594:Electrophysiology; D005089:Exocytosis / Q000187:drug effects*; D018260:Gelsolin; D008840:Microfilament Proteins / Q000235:genetics* / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism
D000199:Actins; D018260:Gelsolin; D008840:Microfilament Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; C065395:scinderin