Figure 4
VPAC receptors signal through canonical and non-canonical pathways in memory Th lymphocytes from HD. (A) Intracellular levels of cAMP under basal conditions and after 1 h treatment with 10 nM of VIP, VPAC1 agonist or VPAC2 agonist was determined in resting- and seven days activated- Th cells by ELISA analysis. Data are the mean ± SEM of duplicate determination of five different HD samples (*p < 0.05, **p < 0.01). Dashed lines represent the basal condition. (B) Western blot analysis of CREB and phosphorylated CREB (p-CREB) under basal conditions and after 1 h treatment with 10 nM of VIP, VPAC1 agonist or VPAC2 agonist was determined in resting- and seven days activated- Th cells. Activating transcription factor-1 (p-ATF1), the other CREB family member, is also shown. Table show the ratio p-CREB/CREB of relative densitometry units. Values are the mean ± SEM of relative densitometry units for each band of five different donors. (C) Upper: Resting- and seven days activated- Th cells were treated with VIP for different times. Rap1 activation was measured by GST-RSD pulldown followed by western blot with anti-Rap1 antibody. Positive control was obtained by treatment of lysates with GTPγS. A representative experiment of five other is shown. Down: Seven days activated Th lymphocytes were treated with VIP, VPAC1 agonist and VPAC2 agonist during 15 min. Rap1 activation was measured by GST-RSD pulldown followed by western blot with anti-Rap1 antibody. Data are the mean ± SEM of duplicate determination of five different donors (*p < 0.05).
