Activation of the melanocortin-4 receptor mobilizes intracellular free calcium in immortalized hypothalamic neurons.

Abstract

BACKGROUND:

At 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 Galphaq/phospholipase C signaling pathway, resulting in alterations of cytoplasmic calcium in immortalized hypothalamic (GT1-1) neurons.

MATERIALS AND METHODS:

Changes in intracellular calcium were measured after loading GT1-1 cells with fura-2-AM. Cells were treated with NDP-alphaMSH, an alpha-melanocortin stimulating hormone analogue, and intracellular calcium changes were recorded. Cells treated with NDP-alpha-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.

RESULTS:

Agonist 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 microm) attenuated the calcium response, while U73433 (10 microm) had minimal effect. 2APB (200 microm) inhibited the calcium transient, and the use of calcium-free buffer did not affect the amplitude of the calcium spike.

CONCLUSIONS:

Our study demonstrates that, upon agonist binding, the melanocortin-4 receptor mediates increases in intracellular calcium through the Galphaq-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.

• Authors: Erika A Newman, Bioa-Xin Chai, Weizhen Zhang, Ji-Yao Li, John B Ammori, Michael W Mulholland
• Journal: The Journal of surgical research (J Surg Res) Vol. 132 Issue 2 Pg. 201-7 (May 15 2006) ISSN: 0022-4804 [Print] United States
• PMID: 16580690 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• Calcium Channels
• Inositol 1,4,5-Trisphosphate Receptors
• Receptor, Melanocortin, Type 4
• Receptors, Cytoplasmic and Nuclear
• SHU 9119
• alpha-MSH
• MSH, 4-Nle-7-Phe-alpha-
• Melanocyte-Stimulating Hormones
• Type C Phospholipases
• GTP-Binding Protein alpha Subunits, Gq-G11
• Calcium

Topics

• Animals
• Calcium (metabolism)
• Calcium Channels
• Cell Line, Transformed
• Cell Line, Tumor
• GTP-Binding Protein alpha Subunits, Gq-G11 (physiology)
• Hypothalamus (cytology, metabolism)
• Inositol 1,4,5-Trisphosphate Receptors
• Melanocyte-Stimulating Hormones (pharmacology)
• Mice
• Mice, Transgenic
• Neurons (metabolism)
• Receptor, Melanocortin, Type 4 (antagonists & inhibitors, physiology)
• Receptors, Cytoplasmic and Nuclear (antagonists & inhibitors)
• Signal Transduction
• Type C Phospholipases (antagonists & inhibitors, physiology)
• alpha-MSH (analogs & derivatives, pharmacology)