Two specific methods, Northern blot analysis using a 50 nucleotides probe to the conserved region of the nerve growth factor (NGF) gene, and enzyme immunoassay using a monoclonal biotinylated rat anti-NGF IgG-avidin conjugated peroxidase system, were used to demonstrate the production and secretion of NGF by mouse striated muscle cell line G8-1. Calcium ionophore, A23187 (0.1-1 μM), forskolin (0.1-100 μM) and dibutyryl cyclic AMP (0.1-10 mM) strongly decreased the secretion of ir-NGF. The level of NGF mRNA was decreased by veratridine, A23187, forskolin and cyclic AMP but not by cyclic GMP. Consequently, we conclude that the secretion of NGF molecules paralleled the changes of NGF mRNA levels in the cells induced by all agents tested. Carmamylcholine also decreased the level of NGF mRNA. Immunoblot analysis suggested that denatured ir-NGF molecules exist in a higher molecular weight form (22 KDa) than those of mouse submaxillary gland (13 KDa). Both Ca2+- and cAMP mediated mechanisms contribute to the decreased production of NGF mRNA in the cells and the consequent inhibition of secretion of NGF molecules. Finally, molecular cloning of NGF of G8-1 cells was conducted and confirmed the structure of the gene that consists of 1, 3, and 4 exons deleting exon 2. Thus, G8-1 NGF is derived from transcript B.
ASJC Scopus subject areas
- Cellular and Molecular Neuroscience
- Cell Biology