Abstract
Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are second messengers which regulate many biological processes. They can be eliminated by active efflux transporters, namely the multidrug resistance proteins MRP4 and MRP5. To delineate the role of MRP4/5, we studied arterial smooth muscle cells in which the role of cyclic nucleotide levels on proliferation has been well-established.Methods: Human SMCs were isolated from the coronary artery media from patients. Small interfering RNAs (siRNA) specific for MRP4 mRNA were designed and validated. Adenovirus encoding MRP4 short hairpin RNA (Ad-shMRP4) were used for in vivo studies.Results: MRP4 was over-expressed in serum-induced proliferating SMC as well as in atherosclerotic plaques in human coronary arteries and in neo-intima of injured rat carotid arteries. Inhibition of MRP4 by siRNA blocked VSMC proliferation in vitro. In balloon-injured rat carotid arteries, intima/media ratios were significantly lower in Ad-shMRP4-infected arteries than in Ad-shLuciferase-infected arteries (0.65 ± 0.1 vs 1.05 ± 0.2; p<0.03). In vitro, MRP4 inhibition significantly increased intracellular cAMP and cGMP levels. A PKA inhibitor (PKI) but not the PKG inhibitor (KT5823) completely reversed the anti-proliferative effect of MRP4 inhibition. The level of pCREB increased by 329 ± 18.8% (p=0.003) on MRP4 inhibition. Conclusion: We provide first evidences that MRP4 acts as an independent endogenous regulator of cyclic nucleotides intra-cellular levels in vascular smooth muscle cells
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