Biosynthesis of Immunoreactive Somatostatin by Hypothalamic Neurons

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 (S14), 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 Cl8 silica, contained material that bound specifically to an immobilized antisomatostatin antibody. Analysis by gel chromatography and high performance liquiid 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) serves as a biosynthetic precursor for both S-28 and S-14. This in vitro system should provide a poverful tool for further investigaAddress all correspondence to Dr. Patel, Room M3-10. Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, H3A lAl, Canada. Dr. H. H. Zingg is a Fellow of the Canadian Medical Research Council. Received for publication 20 April 1982 and in revised form 29 July 1982. J. Clin. Invest. ©) The American Society for Clinical Investi tion of the biosynthesis and regulation of biosynthesis of somatostatin in the hypothalamus.