Activation of the Melanocortin-4 Receptor Mobilizes Intracellular Free Calcium in Immortalized Hypothalamic Neurons1

BackgroundAt least 4% of childhood obesity is due to mutations in the hypothalamic melanocortin-4 receptor. The melanocortin-4 receptor, a seven transmembrane G-protein-coupled receptor, is important in the regulation of feeding behavior and body weight. The specific pathways of intracellular signaling remain in investigative stages. To further understand its function, we hypothesized that the melanocortin-4 receptor activates the Gαq/phospholipase C signaling pathway, resulting in alterations of cytoplasmic calcium in immortalized hypothalamic (GT1-1) neurons.Materials and methodsChanges in intracellular calcium were measured after loading GT1-1 cells with fura-2-AM. Cells were treated with NDP-αMSH, an α-melanocortin stimulating hormone analogue, and intracellular calcium changes were recorded. Cells treated with NDP-α-MSH were also treated with the melanocortin-4 receptor antagonist, SHU-9119. To assess the specific G-protein subunit involved, GT1-1 neurons were treated with the phospholipase C inhibitor U73122 and its inactive analogue, U73433. Experiments were also performed after inhibition of IP3 receptors with 2-aminoethoxydiphenylborate (2APB). Additional experiments were conducted in a calcium-depleted buffer environment. Data were analyzed by ANOVA with statistical significance of P < 0.05.ResultsAgonist treatment (0.01–1000 nm) of GT1-1 neurons resulted in dose-dependent increases in intracellular calcium. SHU-9119 (0.01–1000 nm) abolished the calcium response. Treatment with U73122 (10 μm) attenuated the calcium response, while U73433 (10 μm) had minimal effect. 2APB (200 μm) inhibited the calcium transient, and the use of calcium-free buffer did not affect the amplitude of the calcium spike.ConclusionsOur study demonstrates that, upon agonist binding, the melanocortin-4 receptor mediates increases in intracellular calcium through the Gαq-protein/phospholipase C dependent signaling pathway. Understanding the physiological importance of calcium signaling by the melanocortin-4 receptor may be important for future development of therapeutic targets.