FIGURE 5.
Dual coupling of the MC4R to Gs and Gi/o proteins in hypothalamic GT1-7 cells. A, cAMP accumulation in GT1-7 cells was assessed after labeling of the cells with [3H]adenine followed by the purification of [3H]cAMP and [3H]ATP by sequential chromatography and expressed as the ratio of [3H]cAMP/([3H]cAMP + [3H]ATP). Cells were treated or not with 50 ng/ml PTX for 16 -24 h at 37 °C and then stimulated with 1 μm α-MSH or 5 μm forskolin for 30 min at 37 °C. Results are expressed as the mean ± S.E. of five independent experiments carried out in triplicate. Asterisks indicate a significant (**, p < 0.01) difference between PTX-treated and non-treated cells. B, cAMP accumulation in GT1-7 cells was assessed as described above. Cells were treated or not with 50 ng/ml PTX for 16–24 h at 37 °C and then stimulated with 100 nm AGRP for 30 min at 37 °C. Results are expressed in percentage of untreated and non-stimulated cells. Basal cAMP accumulation of each condition was set to 100%. Asterisks indicate a significant (*, p < 0.05) difference between PTX-treated and non-treated cells. C, cAMP accumulation in GT1-7 cells was assessed as described above and presented as percentage of forskolin-promoted cAMP accumulation. Cells were treated or not with 50 ng/ml PTX for 16–24 h at 37 °C and then stimulated with increasing concentrations of AGRP alone or together with 500 nm HSO-14 in the presence of 5 μm forskolin for 30 min at 37 °C. Results are expressed as a percentage of forskolin-induced cAMP accumulation in the absence of AGRP and represent the mean ± S.E. of five independent experiments carried out in triplicate. Asterisks indicate a significant (*, p < 0.05) difference between PTX-treated and non-treated cells. D, expression analysis of adenylyl cyclase isoforms (AC1–7) in GT1-7 cells was performed by RTQ-PCR and is given relative to the expression of β-actin. Results are expressed as the mean ± S.D. of two independent experiments carried out in duplicate. E, cAMP accumulation in GT1-7 cells was assessed as described above. Cells were pretreated or not with 10 μm gallein for 15 min at 37 °C and then stimulated with 5 μm forskolin alone or along with 100 nm AGRP for 30 min at 37 °C. Results of four experiments are compiled and expressed in the percentage of untreated and non-stimulated cells. cAMP accumulation of with forskolin (alone)-treated cells was set to 100%. Asterisks indicate a significant (*, p < 0.05) difference between AGRP-treated and non-treated cells.