Involvement of SNAP-25 in TRH-induced exocytosis in pituitary GH4C1 cells.
The synaptic membrane protein synaptosomal-associated protein (SNAP-25) has recently been implicated as one of the key proteins involved in exocytotic membrane fusion in neurons. However, the role of SNAP-25 in pituitary hormone release is not known. In this study, we determined that SNAP-25 is involved in regulated exocytosis in the clonal pituitary cell line GH4C1. SNAP-25 messenger RNA and protein were detected in GH4C1 cells by RT-PCR and immunoblot analysis, respectively. Immunofluorescence analysis indicated that SNAP-25 protein was localized in the plasma membrane. Next, to determine the function of SNAP-25 in GH4C1 cells, specific inhibitors of SNAP-25, botulinum neurotoxin (BoNT)/A or /E, and antisense SNAP-25 oligonucleotide were used. Neither BoNT/A nor BoNT/E affected thyrotropin-releasing hormone (TRH)-induced cytosolic Ca2+ increase, but both inhibited TRH-induced exocytosis. Moreover, they dose-dependently inhibited TRH-induced prolactin release. The introduction of antisense oligonucleotide into the cells also inhibited TRH-induced prolactin release. These results suggest that SNAP-25 is involved in regulated exocytosis in GH4C1 cells.
Masumoto
N
N
Department of Obstetrics and Gynecology, Osaka University Medical School, Japan.
Ikebuchi
Y
Y
Matsuoka
T
T
Tasaka
K
K
Miyake
A
A
Murata
Y
Y
D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002999:Clone Cells; D004305:Dose-Response Relationship, Drug; D005089:Exocytosis; D006801:Humans; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D010902:Pituitary Gland / Q000378:metabolism*; D011388:Prolactin / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D016376:Oligonucleotides, Antisense; C494208:SNAP25 protein, human; D050825:Synaptosomal-Associated Protein 25; D013973:Thyrotropin-Releasing Hormone; D011388:Prolactin; D002118:Calcium