D2 Receptors Mediate Dopamine Suppression of irANF Release and Pro-ANF mRNA Expression of Rat Hypothalamic Neurons in Culture

Abstract

Although N-terminal truncated forms of atrial natriuretic factor (ANF) are produced and released from rat hypothalamic neurons, the intrahypothalamic regulation of these processes remains unclear. Employing a well-characterized hypothalamic cell culture system, we report here that dopamine, mediating through D2 receptors, inhibits the synthesis and release of ANF. In long-term cultures of hypothalamic neurons, daily treatment for 4 days with quinpirole, a D2 agonist, significantly suppressed the basal irANF release in a time-related and a dose-dependent manner. The ED50 and Emax of the drugs were 9.1 × 10-8M and 10-5M, respectively. This effect of quinpirole was mimicked by 10-7M of dopamine, a physiological ligand for D2 receptor. Furthermore, the suppressing effects of both quinpirole and dopamine were abolished by sulpiride, a D2 antagonist. Whereas 10-6M of forskolin treatment consistently enhanced the release of irANF through activating the adenylyl cyclase-cAMP system, this stimulatory effect was suppressed by quinpirole in a dose-related manner. In addition, the application of pertussis toxin, a bacterial toxin which inactivated Gi protein activity, reversed the suppressing effect of quinpirole or dopamine on irANF release. These immunoassay findings were accompanied by corresponding changes in the abundance of pro-ANF mRNA in the cultures as determined by colorimetric Northern blot analysis. By combining the techniques of in situ hybridization and immunocytochemistry, the mRNA of D2 receptor was colocalized with irANF at a single cell level by double fluorescent staining. We thus conclude that the release and gene expression of ANF in rat hypothalamic neurons are directly suppressed by dopamine acting through its D2 receptors on ANF neurons. This inhibitory effect is likely to be mediated, at least in part, through Gi protein-induced suppression of the adenylyl cyclase-cAMP system.