Figure 1. mHypoA-2/10 and -2/12 cells resemble MSH/NPY-sensitive hypothalamic neurons.

A and B, cAMP accumulation was assessed after labeling of mHypoA-2/10 and -2/12 cells with [³H]adenine followed by the purification of [³H]cAMP and [³H]ATP by sequential chromatography. Cells were stimulated with 1 μM MSH (A) and with 1 μM FSK (B) alone or along with 100 nM NPY for 30 minutes at 37°C. Data from 5 independent experiments performed in quadruplicate are presented as means ± SEM. Hash signs indicate a significant difference from MSH-treated (A) or FSK-treated (B) cells. C and D, mHypoA-2/10 (C) and in mHypoA-2/12 (D) cells were serum starved for 16 hours and then stimulated with MSH (1 μM) for the indicated period of time. Cell lysates were subjected to Western blot analysis using a specific antiserum against murine TRH. The same blot was also analyzed with an antiserum against the total ERK-2 protein (t-ERK-2) to control for the total protein amount. One of 3 representative experiments is shown. E, mHypoA-2/10 cells were transfected with a human TRH reporter gene construct, serum starved for 16 hours, and stimulated with MSH (1 μM), NPY (100 nM), or T₃ (10 nM) for various periods of time. Data from at least 4 independent experiments performed in quadruplicate were compiled and are expressed as means ± SEM. Asterisks indicate a significant difference from basal (1.0).