Pulmonary arterial hypertension (PAH) is characterized by increased proliferation of pulmonary arterial smooth muscle cells (PASMCs), which leads to remodelling of the pulmonary artery and increased pulmonary artery pressure. Multiple risk factors, such as female bias, circulating co-mitogens and hypoxia, lead to the progression of PAH. Decreased levels of cyclic nucleotides, both cAMP and cGMP, in PASMC contribute to the abnormal tone and remodelling in the pulmonary vasculature seen with PAH. Phosphodiesterases (PDEs) catalyse the hydrolysis of cAMP and cGMP. We have shown that an increase in the expression of PDE1 isoforms (PDE1A and PDE1C), accounts, at least in part, for lower agonist-induced cAMP levels and increased proliferation of PASMC isolated from PAH patients. Recently, novel selective PDE1 inhibitors have been developed and tested in phase II clinical trials for central nervous system disorders. We aimed to investigate the regulation of PDE1 isoforms in human PASMC and provide evidence for the repurposing of such PDE1 inhibitors for PAH. Data are presented as means ± S.E.M., compared by ANOVA. Real-time PCR showed that PDE1A and PDE1C increase in response to serotonin (5-HT, 10 µM, 24 hr, 4.2 ± 0.3 and 2.9 ± 0.2 fold-increase respectively, n=3, p<0.01) and hypoxia (72 hours 1% O2, 7.6 ± 0.9 and 59 ± 11 respectively, n= 3, p<0.05) and are higher expressed in female- PASMC compared to male-PASMC (3.5 ± 1.4 and 3.6 ± 0.6 fold-increase in female vs. male-PASMC respectively, n=3, p<0.05). Of interest, increased PDE1C protein expression (western blot) and translocation to the nucleus correlated with the proliferation of PASMC, whereas arresting PASMC in the G1 phase of the cell cycle inhibited PDE1C expression. Inhibiting PDE1 activity, with 16K (10 µM), decreased the proliferation (MTS assay) of normoxic and hypoxic PASMC (14% ± 2.9 and 21% ± 2.4 decrease respectively, n=3, p<0.05) and decreased the expression of PCNA (n=3). 16K increased cAMP levels (151 ± 8.6 pmol/mg protein for forskolin vs. 224 ± 16.8 pmol/mg protein for forskolin + 16K, n=3, p<0.01, cAMP ELISA assay) and phospho-VASP in PASMC (n=3). In rat pulmonary artery preconstricted with phenylephrine, PDE1 inhibition (1nM-10 µM) induced a concentration-dependent relaxation, which was independent of an intact endothelium (control 25.3 ± 5.2%, n=5 vs. endothelium-denuded 20.1 ± 9.2%, n=6, performed in accordance with the United Kingdom Home Office regulations and approved by Aberdeen University Ethics Committee). Taken together, these data show that the increased expression of PDE1 isoforms in PAH could be attributed to multiple risk factors and that selective PDE1 inhibitors could be novel drugs for the disease.