Biosynthesis of catecholamines in organotypic cultures of peripheral autonomic nervous system: modifications by biopterin and other agents

Abstract

Organotypic cultures of chick-embryo sympathetic ganglion chains maintained in vitro for 3-4 weeks rapidly synthesized catecholamines, as demonstrated by the conversion of L-[U-14C]tyrosine to catechol derivatives and by histofluorescence assay. The biosynthesis of catechols from radioactive L-tyrosine leveled off at 6 hr of incubation and dropped slightly at 10 hr. The addition of DL-alpha-methyl-p-tyrosine to the culture medium did not affect protein synthesis, but produced a complete block in the synthesis of catecholamines from L-tyrosine, with consequent loss of fluorescence in the bodies and proximal processes of adrenergic neurons in 2 hr, and essentially complete loss in 6 hr. Our observations suggest that a major portion of the catecholamines were synthesized in the perikarya and transported via neuronal processes to their terminals. The addition of monoamine oxidase inhibitors to the incubation medium produced a moderate to pronounced increase in fluorescence; reserpine caused a rapid and profound loss of catecholamines. When added to the culture medium, crude biopterin produced an increase in the synthesis of catechol derivatives from radioactive L-tyrosine and a marked increase in fluorescence, beginning in the neuronal perikarya. This effect was completely blocked by DL-alpha-methyl-p-tyrosine. The mechanism of biopterin's action in the synthesis of catecholamines in cultures of sympathetic ganglia is not completely elucidated from these studies, but may be related to the role it plays as cofactor for tyrosine hydrocylase.