Regulation of cAMP/PKA signaling pathways in receptor and agonist dependent manner.
(A) Receptor coupling to adenylyl cyclase. SST, L-803087 and SB-205607 displayed significant inhibition of FSK stimulated cAMP in comparison to control. Data is representative of three independent experiments and presented as % inhibition upon treatment as indicated. (B) Concentration dependent inhibition of cAMP in HEK-293 cells. Treatment of cells with FSK alone was taken as 0% inhibition and treatment with forskolin and SST (1 µM) was considered as 100% inhibition. Note the significant increase in the efficiency of cAMP inhibition upon treatment with SST (10−12–10−6 M) in combination with SB-205607 (10 nM). (C and D) PKA phosphorylation in mono-and/or cotransfected cells. HEK-293 cells expressing SSTR4 and/or δOR were treated for 15 min at 37°C as indicated and cell lysate prepared was subjected to western blot analysis. In monotransfected cells, the status of phospho-PKA was comparable to basal upon receptor specific activation (C). In cotransfected cells, significant inhibition of PKA phosphorylation was observed which was further enhanced upon combined agonist treatment as indicated (D). Densitometric analysis for phospho-PKA was performed by using β-actin or total as loading control and data analysis was done by using ANOVA and post hoc Dunnett’s to compare against basal level (*, p<0.05).