Figure 6.

Roles of PTX-sensitive G proteins, tyrosine kinases, PI3-kinases and phospholipase C on hGHSR-1a-mediated activation of ERK1/2. CHO cells were transiently transfected with the plasmid encoding hGHSR-1a (30 μg/5 × 10⁶ cells). (a) Effects of pertussis toxin (PTX) treatment on hGHSR-1a-mediated activation of ERK1/2. Cells were pretreated overnight with or without 100 ng ml⁻¹ PTX. Cells serum-starved for 1 h were then stimulated with ghrelin (100 nM, 5 min). (b) Effects of tyrosine kinases inhibitors on hGHSR-1a-mediated activation of ERK1/2. Cells were preincubated for 1 h with vehicle (0.1% DMSO) or the tyrosine kinase inhibitor tyrphostin 23 (Tyr. 23, 100 μM or 200 μM, prepared in DMSO) in serum-free medium. Cells were then stimulated with ghrelin (100 nM, 5 min). (c) Cells were preincubated for 1 h with vehicle (0.1% DMSO) or the tyrosine kinase inhibitor genistein (50 μM, prepared in DMSO) in serum-free medium. Cells were then stimulated with ghrelin (100 nM, 5 min). (d) Cells were preincubated for 1 h with vehicle (0.1% DMSO) or the tyrosine kinase inhibitor genistein (50 μM, prepared in DMSO) in serum-free medium. Cells were then stimulated with 2 ng ml⁻¹ IGF-1 for 5 min. (e) Cells were preincubated for 1 h with vehicle (0.1% DMSO) or the c-Src kinase family-specific tyrphostin PP2 (1, 5 and 10 μM, prepared in DMSO) or PP3, an inactive homolog to PP2 (5 and 10 μM, prepared in DMSO) in serum-free medium. Cells were then stimulated with ghrelin (100 nM, 5 min). (f) Effects of the PI3Ks inhibitor on hGHSR-1a-mediated ERK1/2 activation. Cells serum-starved for 30 min were then preincubated for 30 min with vehicle (0.1% DMSO) or the PI3Ks inhibitor wortmannin (Wort., 100 μM, prepared in DMSO) in serum-free medium. Cells were then stimulated with ghrelin (100 nM, 5 min). (g) Effects of the PLC inhibitor on hGHSR-1a-mediated activation of ERK1/2. Cells were pretreated for 1 h with vehicle (0.1% DMSO) or the PLC inhibitor U-73122 (10 and 20 μM, prepared in DMSO) or U-73343, an inactive homolog to U-73122 (10 and 20 μM, prepared in DMSO) in serum-free medium. Cells were then stimulated with ghrelin (100 nM, 5 min). Phosphorylated ERK1/2 (pERK1/2) and total ERK1/2 (ERK1/2) were detected by immunoblotting described in Figure 2. All blots are representative of two to four experiments with similar results.