Epac (exchange protein directly activated by cAMP) in Pulmonary Artery Smooth Muscle Cells from Patients with Pulmonary Hypertension: Role in Proliferation

Fiona Murray, Ryan Suda, Thao Do, Paul Insel
University of California, San Diego, La Jolla, California, United States

Increased vascular resistance due to sustained contraction and proliferation of pulmonary arterial smooth muscle cells (PASMC) characterize pulmonary arterial hypertension (PAH); decreased adenosine 3’ 5’ cyclic-monophosphate (cAMP) levels contribute to the abnormal tone and remodelling in the pulmonary artery (PA; Murray et al., 2007). In addition to protein kinase A and cyclic nucleotide gated channels, another mediator of cAMP has been identified: exchange protein directly activated by cAMP (Epac, Epac-1 and Epac-2 are 2 isoforms), a guanine nucleotide exchange factor for the GTPases Rap-1 and -2. Activation of Epac can inhibit vasoconstriction and blunt calcium mobilization in vascular smooth muscle cells (Grandoch et al., 2006). We hypothesized that altered Epac expression and Epac-dependent signalling in PASMC contribute to proliferation and remodelling of the PA with PAH.

We isolated PASMC from control- and PAH-patients as described (Yuan et al., 1998) and assayed Epac-1 and Epac-2 mRNA and protein expression using real-time PCR and Western blot [normalized to glyceraldehyde-3-phosphate dehydrogenase]. Epac functional activity was assayed by Rap-1 activation after incubation with an Epac-selective cAMP analogue [8-pCPT-2Me-cAMP (8Me), 50μM, 15min] using a pulldown assay in which GTP-bound Rap-1 was isolated from lysates. [3H]Thymidine incorporation was used to assess serum-stimulated DNA synthesis in control- and PAH-PASMC ± 8Me (50μM, 24hr). The data are expressed as mean ± SEM. Statistical comparisons were performed using paired/unpaired Student’s t-tests, where appropriate, with P<0.05 considered significant.

We detected both Epac-1 and Epac-2 mRNA in PASMC. Comparison of cycle threshold (Ct) levels revealed that Epac-2 is more abundant than Epac-1 (Epac-1, 17 ± 0.7, vs. Epac-2, 11 ± 0.09, n=3, P<0.05). The mRNA expression of both Epac-1 and Epac-2 was decreased in PAH- versus control-PASMC (3.5 ± 0.6, 2.7 ± 0.5-fold, respectively, n=3, P<0.05). Expression of Epac-1 and -2 proteins was also decreased in PAH-PASMC (Epac-1, 41.9 ± 2.5%; Epac-2, 37 ± 1.6% decrease compared to control, n=3, P<0.05). Consistent with these results, 8Me-stimulated Rap-1 activation was blunted in PAH-PASMC compared to control (30.4% ± 2.3, n=3 P<0.05) and 8Me decreased proliferation more in control- than in PAH-PASMC (68.7% ± 4.1 vs. 50.4% ± 3.7 respectively, n=3, P<0.05).

We conclude that PAH-PASMC have decreased Epac-1 and Epac-2 expression, blunted Rap-1 activation and decreased anti-proliferative response to an Epac-selective cAMP analogue. The results suggest that increasing Epac expression may be beneficial for the treatment of PAH by increasing the effects of drugs that raise cAMP levels in PASMC

Grandoch et al., (2006) Naunyn-Schmiedebergs Arch Pharmacol 372: R92
Murray et al., (2007) Am J Physiol- Lung Cell Mol Physiol 292(1): L294-303
Yuan et al., (1998) Circulation 98: 1400-1406