Figure 5
From: ML290 is a biased allosteric agonist at the relaxin receptor RXFP1
The role of G proteins, βγ subunits and PI3-kinase in ML290-mediated cAMP and cGMP accumulation in human primary vascular cells. ML290 (10 μM, 30 min) increased cAMP accumulation in HCAECs ((A) n = 5), HUVECs ((B) n = 8), HUASMCs ((C) n = 8) and HUVSMCs ((D) n = 4). Treatment with the Gαs inhibitor NF449 (10 μM, 30 min) of HCAECs ((A) n = 3) and HUVECs ((B) n = 4) abolished ML290-mediated cAMP accumulation (30 min) whereas in HUASMCs ((C) n = 4) and HUVSMCs ((D) n = 4) it reduced the maximum cAMP response. The Gαi/GαOB inhibitor NF023 (10 μM, 30 min) in HCAECs ((A) n = 4) and HUVECs ((B) n = 4), HUASMCs ((C) n = 4) and HUVSMCs ((D) n = 4) had no effect on ML290-mediated cAMP accumulation. Pre-treatment of HCAECs ((A) n = 3) and HUVECs ((B) n = 3) with the Gβγ inhibitors mSIRK (5 μM, 30 min) and gallein (50 μM, 45 min) and the PI3-kinase inhibitor Wortmannin (100 nM, 30 min) had no effect on ML290-mediated cAMP accumulation whereas pre-treatment of HUASMCs ((C) n = 3) or HUVSMCs ((D) n = 3) with both Gβγ inhibitors and the PI3-kinase inhibitor reduced the maximum cAMP response to ML290. ML290 (10 μM, 30 min) also increased cGMP accumulation in HCAECs ((E) n = 3), HUVECs ((F) n = 4), HUASMCs ((G) n = 3) and HUVSMCs ((H) n = 3). NF449 (10 μM, 30 min) pre-treatment of HCAECs ((E) n = 4); HUVECs ((F) n = 3), HUASMCs ((G) n = 6) and HUVSMCs ((H) n = 4) reduced the maximum ML290-mediated cGMP response. Pretreatment with NF023 (10 μM, 30 min) of HCAECs ((E) n = 3), HUVECs ((F) n = 3), HUASMCs ((G) n = 4) and HUVSMCs ((H) n = 4) had no effect on ML290-mediated cGMP accumulation. Pretreatment with the Gβγ inhibitors mSIRK (5 μM, 30 min) and gallein (50 μM, 45 min) and the PI3-kinase inhibitor Wortmannin (100 nM, 30 min) had no effect on cGMP accumulation in HCAECs ((E) n = 3) and HUVECs ((F) n = 3) whereas in HUASMCs ((G) n = 5) and HUVSMCs ((H) n = 4), it reduced the maximum response. Pre-treatment of HUASMCs ((C,G) n = 4–11) and HUVSMCs ((D,H) n = 4–5) with mSIRK control peptide L9A (5 μM, 30 min) had no significant effect on ML290-mediated cAMP or cGMP accumulation. Statistical significance was assessed using a one-way ANOVA with a Dunnet’s post-hoc test compared to ML290 alone: **p < 0.01 and *p < 0.05. Data shown are mean ± SEM of ‘n’ independent experiments.
