Increased Vasopressin Secretion from Hypothalamic Cultures Following Administration of Exogenous Vasopressin mRNA

Abstract

In Brattleboro rats, exogenous vasopressin (VP) mRNA can be accumulated, transported, and translated by magnocellular neurons. To determine whether this phenomenon may also occur in magnocellular neurons of normal rats, dispersed hypothalamic neuronal cultures of fetal Sprague–Dawley rats were exposed to VP mRNA. The cultures were maintained in either control medium or medium containing the cAMP elevating drugs, IBMX (3-isobutyl-1-methylxanthine), and forskolin for 23 or 30 days of culture to induce VP synthesis and secretion. Following removal of the IBMX and forskolin at Day 23, VP secretion into the medium declined to baseline by 30 daysin vitro,but administration of VP mRNA to these cultures on Day 30 resulted in a 5-fold increase in VP content of the medium (P=0.005) after 6 h and a 2.5-fold increase after 24 h (P=0.002). Administration of VP mRNA to the cultures treated continuously with IBMX and forskolin also resulted in a small increase in VP secretion which did not reach significance after either 6 or 24 h. When cultures prepared with continuous I/F were exposed to antisense VP mRNA, VP secretion into the media was decreased by 58%, and VP immunoreactive perikayra were difficult to observe. This demonstrates that the increase in VP release observed after the addition of sense VPmRNA did not reflect a nonspecific effect of the addition of mRNA to the culture medium. Autoradiography of cultures administered3H- or32P-VP mRNA for 24 h revealed silver grains associated with varicosities, perikarya, and neuritic processes of neurophysin (NP)-positive, but not NP-negative neurons. These results suggest that exogenously administered mRNA has access to cell translation systems in cultured hypothalamic neurons as well as magnocellular neurons of Brattleboro rats.