Profiling the mRNA expression of G-protein coupled receptors in human pulmonary artery smooth muscle cells to identify new targets for pulmonary arterial hypertension

Nakon Aroonsakool, Daniel McDonald, Paul Insel, Fiona Murray. University of California, San Diego, la Jolla, CA, USA.

Pulmonary arterial hypertension (PAH) is a disease associated with increased vascular resistance due to the increased proliferation of pulmonary artery smooth muscle cells (PASMC). PAH has a poor prognosis and remains difficult to treat because few PASMC-selective vasodilators are available. G-protein coupled receptors (GPCRs), which constitute the largest family of membrane receptors in the human genome, coordinate cellular signalling in response to extracellular stimuli. Plasma membrane localization, tissue distribution and impact on physiological functions make GPCRs attractive pharmacological targets for PAH via their ability to tailor downstream signalling in a pulmonary-specific manner. In particular, GPCRs that couple to Gαs or Gαi could selectively increase the accumulation of the second messenger cyclic AMP (cAMP) in PASMC; cAMP attenuates vascular tone and proliferation of PASMC (Murray et al., 2007). We hypothesized that defining the profile and function of highly or uniquely expressed GPCRs in human PASMC, in particular those that regulate cAMP, could uncover new targets for PAH.

Using an unbiased approach, a GPCR RT-PCR array (Applied Biosystems), we initiated studies to define the GPCR mRNA profile in PASMC. We found that isolated human PASMC (n = 3, Murray et al., 2007) express 135 GPCRs, which could be classified according to their G-protein coupling (The 2009 BJP Guide to Receptors and Channels); >50 GPCRs detected have the potential to regulate cAMP accumulation. Based on mRNA expression, the two most abundant Gαs- and Gαi-coupled GPCRs were the adenosine A2B and vasoactive intestinal peptide 1 receptors (VPAC1) and the lysophosphatidic acid 1 (LPA1) and oxytocin receptors, respectively. We initially focused on the VPAC1 and the oxytocin receptors since limited data were available regarding their role in PASMC. Data are expressed as mean ± SEM. Statistical comparisons were performed using paired Student’s t-tests, with P<0.05 considered significant. We found that VPAC1 protein is expressed in the membrane fraction of PASMC and that its activation by VIP (0.1-100 nM, 10 min) significantly increases cAMP accumulation (70 +/- 6 % to 333 +/- 30 %, n = 3, P<0.05); moreover, incubation with VIP (100 nM, 24 hr) decreases serum-induced PASMC proliferation (40 +/- 3 %, n = 3, P<0.001). In parallel, we found the oxytocin receptor protein is also expressed on PASMC membranes and that oxytocin (0.1 nM-10 μM, 30 min) dose-dependently decreases cAMP accumulation (12 +/- 2 % to 40 +/- 5 %, n = 3, P<0.05) in response to forskolin (10 μM, 10 min) and that oxytocin (10 μM, 24 hr) promotes PASMC proliferation in serum-free media by 21 +/- 5 % (n = 3, P<0.001).

These data demonstrate that PASMC express a large number of GPCRs and provide evidence that profiling GPCR expression can identify previously underappreciated GPCRs that contribute to PASMC function. Thus, efforts to define GPCR expression in PASMC, in particular GPCRs that regulate cAMP, may identify novel targets to lower pulmonary vascular tone in patients with PAH.

Murray et al., (2007), Am J Physiol- Lung Cell Mol Physiol 292(1): L294-303