Ca²⁺ Signaling Enables Growth Hormone Secretion from Cultured Mice Pituitary Somatotrophs, Facilitating Outgrowth in Hypothalamic Neuron

The hypothalamus regulates anterior pituitary (AP) hormone release via the hypophyseal portal system by secreting specific releasing and inhibiting peptide hormones. Growth hormone (GH) secretion from pituitary somatotrophs (PS) is regulated by a cholinergic mechanism that induces calcium (Ca²⁺) signaling, which in turn triggers vesicular exocytosis and GH release. Given that the transient receptor potential canonical channel 3 (TRPC3) regulates vesicle exocytosis in endocrine and neuroendocrine cells, we investigated its role in GH release from somatotrophs and whether GH release could support neuronal growth in the hypothalamus. Activation of TRPC3 by the diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), resulted in a rapid increase in intracellular Ca²⁺ ([Ca²⁺]_i) in wild type (WT) PS, attenuated by the TRPC3 inhibitor Pyr10, or by the knockout of TRPC3. ELISA assay confirmed that OAG exposure triggered GH release in WT PS. Conditioned media from OAG-stimulated PS promoted significant neurite outgrowth in hypothalamic neuronal cultures, suggesting a role for released GH in this process. Exposure of PS to H₂O₂-induced oxidative damage enhanced store-operated Ca²⁺ entry (SOCE) compared to control cells. Electrophysiological characterization revealed that somatotrophs under increased reactive oxygen species (ROS) conditions exhibited elevated SOCE, whereas cells under more native conditions exhibited receptor-operated Ca²⁺ entry. Furthermore, media from oxidative-stressed PS cultures, followed by the OAG exposure, induced neurite outgrowth, suggesting TRPC3 activation may provide an alternative pathway after ROS-induced damage in GH release. These findings indicate that Ca²⁺ signaling activation may promote GH secretion from control and ROS-induced AP cells, potentially leading to neurite outgrowth in hypothalamic cells. This study may provide insights into restoring neuronal connectivity following brain injury.