Biosynthesis of immunoreactive somatostatin by hypothalamic neurons in culture

The neuronal biosynthesis of somatostatin-like immunoreactivity (SLI) was investigated using mechanically dispersed neonatal rat hypothalamic cells kept in culture for up to 6 wk. Immunohistochemically, SLI was specifically localized to a small subpopulation of parvicellular neurons and their cell processes. By radioimmunoassay the cellular SLI content declined steadily during the first 2 wk in culture (nadir value of 60 fmol/dish at day 15) but then increased progressively to reach a maximum value of 381 fmol/dish at day 46. Gel chromatographic analysis showed this immunoreactivity to consist of forms corresponding to tetradecapeptide somatostatin (S-14), somatostatin-28 (S-28), and a 15,000-mol-wt molecule. After incubation of the cells with [3H]phenylalanine, the cellular extracts, purified by adsorption to C18 silica, contained material that bound specifically to an immobilized antisomatostatin antibody. Analysis by gel chromatography and high performance liquid chromatography of the specifically bound label provided evidence for the presence of labeled S-14, S-28, and the 15,000-mol-wt molecule. Pulse-chase experiments (20-min pulse, 20-min chase) demonstrated a transfer of radioactivity from the 15,000-mol-wt form to material corresponding to S-14 as well as to S-28. These studies demonstrate that cultured hypothalamic neurons are capable of synthesizing three somatostatin-like peptides (15,000-mol-wt SLI, S-28, S-14), one of which (15,000-mol-wt SLI) serve as a biosynthetic precursor for both S-28 and S-14. This in vitro system should provide a powerful tool for further investigation of the biosynthesis and regulation of biosynthesis of somatostatin in the hypothalamus.