FINAL ACCEPTED (LCMP-00214-2006) Pulmonary Artery Smooth Muscle Cells from Normal Subjects and Patients with Idiopathic Pulmonary Arterial Hypertension Show Divergent cAMP-mediated Effects on TRPC Expression and Capacitative Ca Entry

Pulmonary vascular remodeling due to overgrowth of pulmonary artery smooth muscle cells (PASMC) is a major cause for the elevated vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Increased cytosolic Ca ([Ca]cyt), resulting from enhanced capacitative Ca entry (CCE) and upregulated transient receptor potential (TRP) channel expression, is involved in stimulating PASMC proliferation. The current study was designed to determine the impact of cyclic AMP, a second messenger that we hypothesized would blunt aspects of PASMC activity, as a possible contributor to IPAH pathophysiology. Short-term (30 min) pretreatment with forskolin (FSK, 10 μM), a direct activator of adenylyl cyclase, in combination with the cyclic nucleotide phosphodiesterase inhibitor isobutylmethylxanthine (IBMX, 200 μM), attenuated CCE in PASMC from normal subjects, patients without pulmonary hypertension (NPH) and patients with IPAH. The FSK-mediated CCE inhibition was independent of protein kinase A (PKA) as the PKA inhibitor H89 negligibly affected the decrease in CCE produced by cAMP. By contrast, longer (4 h) treatment with FSK (with IBMX) attenuated CCE in normal and NPH PASMC, but enhanced CCE in IPAH PASMC. This enhancement of CCE was abolished by PKA inhibition and associated with an upregulaton of TRPC3. In addition, cAMP increased TRPC1 mRNA expression in IPAH (but not in normal or NPH) PASMC, an effect blunted by H89. Furthermore, iloprost, a prostacyclin analog that increases cAMP down-regulated TRPC3 expression in IPAH PASMC and FSKmediated cAMP increase inhibited IPAH PASMC proliferation. Although a rapid rise in cellular cAMP decreases CCE by a PKA-independent mechanism, a sustained cAMP increase inhibits CCE in normal and NPH PASMC but increases CCE via a PKA-dependent pathway in IPAH PASMC. The divergent effect of cAMP on CCE parallels effects on TRPC expression. The Page 2 of 34 (LCMP-00214-2006. R1) 2 results suggest that the combined use of a PKA inhibitor and cAMP-elevating drugs may provide a novel approach for treatment of IPAH.