Purification and characterization of neurotrophic factor for retinal cholinergic neurons derived from cultured hippocampal neurons.
A neurotrophic factor that supports the development of cholinergic retinal neurons was purified from media conditioned by a primary culture of embryonic hippocampal neurons. Retinal choline acetyltransferase (ChAT), which is located exclusively in amacrine cells, served as a marker for the development of retinal cholinergic neurons. In a serum-free control culture, retinal neurons from 17-day-old rat fetus displayed little increase in the enzyme activity and a low proportion of neurite-bearing cells (15-20%) within 7 days. The conditioned media, when added to the retinal neuron culture, dose-dependently increased ChAT activity and the number of neurite-bearing cells (40-60%), the maximum ChAT activity being approximately sixfold higher than that in the control. The fraction with these stimulatory activities was purified by Sephadex G-15 column chromatography and two times reverse-phase HPLC. The final fraction showed approximately 3,000-fold higher purification as compared with that in the Sephadex G-15 fraction. Gas-phase protein sequencing analysis of the final fraction yielded a peptide sequence: Tyr-Leu-Leu-Pro-Ala-Gln-Val-Asn-Ile-Asp. A synthetic peptide with this sequence dose-dependently stimulated ChAT activity in the retinal cell culture and dissociated cell culture of the septal nucleus. These findings suggest that the developing hippocampal neurons produce a neurotrophic peptide that stimulates the development of cholinergic neurons.
Department of Pharmacology, Kansai Medical University, Osaka, Japan.
T
T
Yasukura
K
K
Omori
T
T
Mikami
M
M
Inoue
M
M
Uyama
C
C
Inagaki
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002795:Choline O-Acetyltransferase / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D017077:Culture Media, Conditioned; D004305:Dose-Response Relationship, Drug; D006624:Hippocampus / Q000737:chemistry* / Q000166:cytology / Q000196:embryology; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D009474:Neurons / Q000737:chemistry / Q000187:drug effects*; D051381:Rats; D017208:Rats, Wistar; D012160:Retina / Q000166:cytology / Q000187:drug effects* / Q000196:embryology / Q000201:enzymology
D017077:Culture Media, Conditioned; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D002795:Choline O-Acetyltransferase