Regulation of the preprosomatostatin gene by cyclic-AMP in cerebrocortical neurons.

The gene coding for preprosomatostatin (ppSom), the molecular precursor of somatostatin (Som), is regulated at the level of transcription by calcium ions and cyclic-AMP [F. Baldino, S. Fitzpatrick-McElligott, T. O'Kane, I. Gozes, Hormonal regulation of somatostatin, Synapse 2 (1988) 317-325; M.R. Montminy, M.J. Low, L. Tapia-Arancibia, Cyclic AMP regulates somatostatin mRNA accumulation in primary diencephalic cultures and in transfected fibroblast cells, J. Neurosci. 6 (1986) 1171-1176.], or by agents which increase intracellular levels of cAMP directly, such as forskolin [M.R. Montminy, M.J. Low, L. Tapia-Arancibia, Cyclic AMP regulates somatostatin mRNA accumulation in primary diencephalic cultures and in transfected fibroblast cells, J. Neurosci. 6 (1986) 1171-1176.]. Transcriptional induction of the ppSom gene as examined in PC12 cells, transfected fibroblasts and primary diencephalic cultures, requires the highly conserved cAMP response element (CRE), which confers gene responsiveness to cAMP [M. Comb, N. Mermod, S.E. Hyman, Proteins bound at adjacent DNA elements act synergistically to regulate human proenkephalin cAMP inducible transcription, EMBO J. 7 (1988) 3793-3805; T. Tsukada, J.S. Fink, G. Mandel, Identification of a region in the human vasoactive intestinal polypeptide gene responsible for regulation by cyclic AMP, J. Biol. Chem. 262 (1987) 8743-8747.]. The ppSom gene is subject to stringent regulation during cerebrocortical development in vivo; however, little information is available regarding ppSom gene regulation by neurotransmitters or second-messengers in cortical neurons. We used primary cerebrocortical cell cultures from fetal mice to examine the dose-response and time-course of ppSom gene expression in response to the cyclic-AMP analogs, dibutyrl-cAMP (dbcAMP), and 8-bromo-cAMP (8-BrcAMP). We report a dose-response for both analogs in the range of 0.1-10 mM. Dose-response studies using agents which directly stimulate intracellular cAMP synthesis (forskolin) or inhibit its breakdown (3-isobutyl 1-methyl xanthine) were also performed. We observed an apparent synergistic effect on ppSom expression when used in combination. An increase in ppSom mRNA levels was observed by 4 h, with a maximal response at 12-24 h. No change in ppSom mRNA levels was observed in response to phorbol myristate acetate (PMA). Our findings confirm the specificity of ppSom gene regulation by cAMP and Ca2+ ions, and demonstrate the utility of using primary cerebrocortical cultures for the study of somatostatin gene expression by neurotransmitters and second-messengers as a model of human neurologic disorders.