Immortalized cells from the rat suprachiasmatic nucleus express functional melatonin receptors
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abstract
Immortalized SCN2.2 cells retain most biochemical and biophysical characteristics of the native rat SCN including the expression of clock genes and circadian regulatory proteins, and its distinctive pacemaker function. This study assessed the expression and signaling of MT(1) and MT(2) melatonin receptors in SCN2.2 cells. SCN2.2 cells express MT(1) and MT(2) receptors mRNA as detected by RT-PCR. In situ hybridization with digoxigenin-labeled probes demonstrated that mRNA for MT(1) and MT(2) melatonin receptors is expressed mostly in cells with neuronal-like morphology, representing 10.8+/-2.2% and 9.8+/-0.2%, respectively, of the SCN2.2 cell population. MT(1) and MT(2) melatonin receptor proteins are expressed in both rat SCN2.2 cells and rat SCN tissue as demonstrated by Western blot analysis with specific receptor antiserum. Melatonin (0.1-100 nM) inhibited forskolin (20 microM)-stimulated cAMP formation in a dose-dependent manner and this effect was blocked by the competitive melatonin receptor antagonist luzindole (100-1000 nM). Furthermore, melatonin (1 nM) stimulated protein kinase C (PKC) activity by approximately 2-fold. The selective MT(2) receptor antagonist 4P-PDOT (100 nM) blocked this effect, indicating that the melatonin-mediated increase in PKC activity occurs through activation of MT(2) melatonin receptors. We conclude that SCN2.2 cells express functional melatonin receptors, providing an in vitro model to unveil the melatonin signaling pathway(s) involved in the regulation of circadian rhythms.
