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


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.
Section of Gastrointestinal Surgery, Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI 48109-0346, USA.
Erika A
EA
Newman
Bioa-Xin
BX
Chai
Weizhen
W
Zhang
Ji-Yao
JY
Li
John B
JB
Ammori
Michael W
MW
Mulholland
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000502:physiology; D007031:Hypothalamus / Q000166:cytology / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009074:Melanocyte-Stimulating Hormones / Q000494:pharmacology; D051379:Mice; D008822:Mice, Transgenic; D009474:Neurons / Q000378:metabolism*; D044105:Receptor, Melanocortin, Type 4 / Q000037:antagonists & inhibitors / Q000502:physiology*; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000502:physiology; D000521:alpha-MSH / Q000031:analogs & derivatives / Q000494:pharmacology
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D044105:Receptor, Melanocortin, Type 4; D018160:Receptors, Cytoplasmic and Nuclear; C103272:SHU 9119; D000521:alpha-MSH; C027756:MSH, 4-Nle-7-Phe-alpha-; D009074:Melanocyte-Stimulating Hormones; D010738:Type C Phospholipases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium