Supplementary Figure 8: AC superactivation and cAMP levels in HEK V1bR–μ-receptor cells.
(A) The time course of the development of AC superactivation was examined after treatment of HEK–V1bR–μ-receptor cells with 100 nM AVP. Cells were collected at indicated times and stimulated with 0.3 μM forskolin. Significant increases in cAMP levels were detected after 4 h of AVP incubation (one-way ANOVA was followed by two-sided pairwise t-tests with Holm’s method for P value adjustment. F6,77 = 5.882, P < 0.0001). *P < 0.0001 vs. 0 h, (n = 12,12,12,15,6 for 0, 1, 2, 4, 24 h independent experiments). (B) AVP did not have an acute effect on cAMP levels in HEK–V1bR–μ-receptor cells. (n = 9). (C) Acute inhibition of cAMP levels by morphine was not changed by AVP in HEK–V1bR–μ-receptor cells. n = 6 independent experiments. (D) AC superactivation by 100 nM AVP was not sensitive to the PKC inhibitor Ro 31-8220 (50 nM). Two-way ANOVA, F1,33 = 2.713, P = 0.109. n = 9, 9, 9, 10. (E) AVP induced AC superactivation in the presence of 1 μM naloxone in HEK-V1bR- μ-receptor cells. For control samples, PBS was used. The cells were incubated for 24 h and forskolin (1 μM)-induced cAMP production was measured. In two-way ANOVA and Tukey’s test, no difference was detected between AVP and AVP plus naloxone (P = 0.380, n = 12). Graphs represent the mean ± S.E.M.
