Multidrug resistance protein 4 mediates cAMP efflux from rat preglomerular vascular smooth muscle cells

Abstract

SUMMARY1. Our previous studies show that stimulation of adenylyl cyclase in preglomerular vascular smooth muscle cells (PGVSMCs) increases extracellular 3',5'-cAMP; however, the mechanism by which PGVSMCs transport intracellular 3',5'-cAMP into the extracellular milieu is unknown. 2.We hypothesize that multidrug resistance protein 4 (MRP4) is the primary transporter mediating efflux of intracellular 3',5'-cAMP from PGVSMCs.3. Both RT-PCR and real-time PCR detected MRP4 mRNA in PGVSMCs in culture. Moreover, Western blotting using an antibody specific for MRP4 gave rise to a 150 kDa signal consistent with the presence of MRP4 protein in PGVSMCs. 4.Specifically-designed siRNA reduced MRP4 mRNA expression by 71% (p=0.0075) and MRP4 protein by 80% (p=0.0004). 5.Isoproterenol (1 μmol/L) increased intracellular 3',5'-cAMP, which resulted in efflux of 3',5'-cAMP into the medium. siRNA knockdown of MRP4 significantly reduced basal extracellular 3',5'-cAMP and nearly abolished isoproterenol-induced increases in extracellular 3',5'-cAMP (p=0.0143, interaction between isoproterenol and MRP4 siRNA in 2-factor analysis of variance). In isoproterenol-treated cells, MRP4 siRNA decreased the ratio of extracellular 3',5'-cAMP to intracellular 3',5'-cAMP by 72% (p=0.0019). 6.We conclude that MRP4 is the dominant 3',5'-cAMP transporter in PGVSMCs.

Cannabinoid Receptor Activation Correlates with the Proapoptotic Action of the β2-Adrenergic Agonist (R,R′)-4-Methoxy-1-Naphthylfenoterol in HepG2 Hepatocarcinoma Cells

“…The lack of effect of ␤ 2 -AR agonists on cAMP accumulation may be caused by the low level of ␤ 2 -AR protein expression in these cells. It is also possible that the intracellular accumulation of cAMP may be reduced by MRP4/5-mediated export (Cheng et al, 2010). In this study, the presence of the MRP inhibitor probenecid had no effect on isoproterenol-mediated increase in cAMP accumulation in HepG2 cells, and the overall expression levels for MRP4/5 proteins in these cells were found to be below the detection limit.…”

“…1B). Because of the ability of cyclic nucleotides to move across the cell membrane via the drug efflux pump MRP4 (ABCC4) and MRP5 (ABCC5) (Wielinga et al, 2003;Cheng et al, 2010), we investigated whether the active export of cAMP accounted for the apparent lack of effect of isoproterenol and fenoterol compounds on intracellular cAMP accumulation. HepG2 cells were pretreated with the MRP inhibitor probenecid (Copsel et al, 2011), followed by agonist stimulation.…”

2′-AMP and 3′-AMP Inhibit Proliferation of Preglomerular Vascular Smooth Muscle Cells and Glomerular Mesangial Cells via A2B Receptors

“…However, in the present study these compounds are applied extracellularly. Although cAMPs are actively pumped out of cells (Barber and Butcher, 1983;Deeley et al, 2006;Li et al, 2007;Cheng et al, 2010), because both 2Ј,3Ј-cAMP and 3Ј-AMP are very hydrophilic, they are not likely to diffuse across cell membranes into cells. Because the effects on mitochondria and the P site of adenylyl cyclase would require an intracellular action of 2Ј,3Ј-cAMP and 3Ј-AMP, it seems unlikely that these mechanisms are involved.…”

Mammalian Cyclic Nucleotide Phosphodiesterases: Molecular Mechanisms and Physiological Functions

“…However, the cN-binding properties of GAFs (named for cGMP-binding PDEs, Anabaena adenylyl cyclase, and Escherichia coli FhlA) in some PDEs can sequester cN (129,198). Although cNs are extruded from some cells by certain multianion transporters, the impact of this process on lowering cN levels is suggested to be small (21,64,211). Other processes may lower cN levels in particular cells.…”