Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.1 (phosphodiesterase)
 18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cellular export of cyclic nucleotides has been observed in various tissues and may represent an elimination pathway for these signaling molecules, in addition to degradation by phosphodiesterases. In the present study we provide evidence that this export is mediated by the multidrug resistance protein isoform MRP5 (gene symbol ABCC5). The transport function of MRP5 was studied in V79 hamster lung fibroblasts transfected with a human MRP5 cDNA. An MRP5-specific antibody detected an overexpression of the glycoprotein of 185 +/- 15 kDa in membranes from MRP5-transfected cells and a low basal expression of hamster Mrp5 in control membranes. ATP-dependent transport of 3',5'-cyclic GMP at a substrate concentration of 1 micrometer was 4-fold higher in membrane vesicles from MRP5-transfected cells than in control membranes. This transport was saturable with a K(m) value of 2.1 micrometer. MRP5-mediated transport was also detected for 3',5'-cyclic AMP at a lower affinity, with a K(m) value of 379 micrometer. A potent inhibition of MRP5-mediated transport was observed by several compounds, known as phosphodiesterase modulators, including trequinsin, with a K(i) of 240 nm, and sildenafil, with a K(i) value of 267 nm. Thus, cyclic nucleotides are physiological substrates for MRP5; moreover, MRP5 may represent a novel pharmacological target for the enhancement of tissue levels of cGMP.
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PMID:The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides. 1089 47

Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3',5'-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4- and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20-600 microm, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A(1) and E(1), the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterase-mediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.
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PMID:Characterization of the MRP4- and MRP5-mediated transport of cyclic nucleotides from intact cells. 1263 26

5'-Fluorouracil (5-FU), used in the treatment of colon and breast cancers, is converted intracellularly to 5'-fluoro-2'-deoxyuridine (5-FUdR) by thymidine phosphorylase and is subsequently phosphorylated by thymidine kinase to 5'-fluoro-2'-dUMP (5-FdUMP). This active metabolite, along with the reduced folate cofactor, 5,10-methylenetetrahydrofolate, forms a stable inhibitory complex with thymidylate synthase that blocks cellular growth. The present study shows that the ATP-dependent multidrug resistance protein-5 (MRP5, ABCC5) confers resistance to 5-FU by transporting the monophosphate metabolites. MRP5- and vector-transfected human embryonic kidney (HEK) cells were employed in these studies. In 3-day cytotoxicity assays, MRP5-transfected cells were approximately 9-fold resistant to 5-FU and 6-thioguanine. Studies with inside-out membrane vesicles prepared from transfected cells showed that MRP5 mediates ATP-dependent transport of 5 micromol/L [(3)H]5-FdUMP, [(3)H]5-FUMP, [(3)H]dUMP, and not [(3)H]5-FUdR, or [(3)H]5-FU. The ATP-dependent transport of 5-FdUMP showed saturation with increasing concentrations and had a K(m) of 1.1 mmol/L and V(max) of 439 pmol/min/mg protein. Uptake of 250 micromol/L 5-FdUMP was inhibited by dUMP, cyclic nucleotide, cyclic guanosine 3',5'-monophosphate, amphiphilic anions such as probenecid, MK571, the phosphodiesterase inhibitors, trequinsin, zaprinast, and sildenafil, and by the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid and glybenclamide. Furthermore, the 5-FU drug sensitivity of HEK-MRP5 cells was partially modulated to that of the HEK-vector by the presence of 40 micromol/L 5-nitro-2-(3-phenylpropylamino)-benzoic acid but not by 2 mmol/L probenecid. Thus, MRP5 transports the monophosphorylated metabolite of this nucleoside and when MRP5 is overexpressed in colorectal and breast tumors, it may contribute to 5-FU drug resistance.
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PMID:The multidrug resistance protein 5 (ABCC5) confers resistance to 5-fluorouracil and transports its monophosphorylated metabolites. 1589 50

Multidrug resistance protein-5 (MRP5, ABCC5) is a member of the ATP-binding cassette transporter superfamily that effluxes a broad range of natural and xenobiotic compounds such as cyclic GMP, antiviral compounds, and cancer chemotherapeutic agents including nucleoside-based drugs, antifolate agents and platinum compounds. In cellular assays, MRP5 transfectants are less fluorescent after incubation with 5-chloromethylfluorescein diacetate (CMFDA). The present study examines the uptake of a close fluorescent analog, carboxydichlorofluorescein (CDCF), and drug substrates into inside-out membrane vesicles prepared from MRP transfected cells. MRP5-mediated uptake of CDCF was ATP-dependent and GSH-independent and possessed a Km of 12 microM and a Vmax of 56 pmol/min/mg prot. Comparison of kinetic parameters with drug substrates such as methotrexate (MTX), pemetrexed (Alimta), and the metabolite of 5-fluorouracil, 5-fluorodeoxyuridine monophosphate (5-FdUMP) (Km values of 0.3-1.3 mM) indicated that MRP5 has a 25-100-fold higher affinity for CDCF than for these drugs and that they share a common transport binding site. In addition, the potency of MRP5 inhibitors such as probenecid, MK571, and the phosphodiesterase 5 inhibitors correlated well between the uptake of CDCF and MTX. A survey of CDCF uptake by other MRPs revealed that MRP2 (ABCC2) also demonstrated ATP-dependent uptake with a Km of 19 microM and Vmax of 95.5 pmol/min/mg prot, while MRP1 (ABCC1) and MRP4 (ABCC4) had little to no uptake. Taken together, these data indicate that CDCF is a useful fluorescent drug surrogate with which to measure ATP-dependent MRP5-mediated transport.
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PMID:Kinetic validation of the use of carboxydichlorofluorescein as a drug surrogate for MRP5-mediated transport. 1633 12

The second messengers cAMP and cGMP can be degraded by specific members of the phosphodiesterase superfamily or by active efflux transporters, namely the multidrug resistance-associated proteins (MRPs) MRP4 and MRP5. To determine the role of MRP4 and MRP5 in cell signaling, we studied arterial SMCs, in which the effects of cyclic nucleotide levels on SMC proliferation have been well established. We found that MRP4, but not MRP5, was upregulated during proliferation of isolated human coronary artery SMCs and following injury of rat carotid arteries in vivo. MRP4 inhibition significantly increased intracellular cAMP and cGMP levels and was sufficient to block proliferation and to prevent neointimal growth in injured rat carotid arteries. The antiproliferative effect of MRP4 inhibition was related to PKA/CREB pathway activation. Here we provide what we believe to be the first evidence that MRP4 acts as an independent endogenous regulator of intracellular cyclic nucleotide levels and as a mediator of cAMP-dependent signal transduction to the nucleus. We also identify MRP4 inhibition as a potentially new way of preventing abnormal VSMC proliferation.
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PMID:Multidrug resistance-associated protein 4 regulates cAMP-dependent signaling pathways and controls human and rat SMC proliferation. 1863 20

This chapter addresses cNMP hydrolysis by phosphodiesterases (PDEs) and export by multidrug resistance associated proteins (MRPs). Both mechanisms are well-established for the canonical cNMPs, cAMP, and cGMP. Increasing evidence shows that non-canonical cNMPs (specifically cCMP, cUMP) are also PDE and MRP substrates. Hydrolysis of cUMP is achieved by PDE 3A, 3B, and 9A, which possibly explains the cUMP-degrading activities previously reported for heart, adipose tissue, and brain. Regarding cCMP, the only known "conventional" (class I) PDE that hydrolyzes cCMP is PDE7A. Older reports describe cCMP-degrading PDE-like activities in mammalian tissues, bacteria, and plants, but the molecular identity of these enzymes is not clear. High K M and V max values, insensitivity to common inhibitors, and unusually broad substrate specificities indicate that these activities probably do not represent class I PDEs. Moreover, the older results have to be interpreted with caution, since the historical analytical methods were not as reliable as modern highly sensitive and specific techniques like HPLC-MS/MS. Besides PDEs, the transporters MRP4 and 5 are of major importance for cAMP and cGMP disposal. Additionally, both MRPs also export cUMP, while cCMP is only exported by MRP5. Much less data are available for the non-canonical cNMPs, cIMP, cXMP, and cTMP. None of these cNMPs has been examined as MRP substrate. It was shown, however, that they are hydrolyzed by several conventional class I PDEs. Finally, this chapter reveals that there are still large gaps in our knowledge about PDE and MRP activities for canonical and non-canonical cNMPs. Future research should perform a comprehensive characterization of the known PDEs and MRPs with the physiologically most important cNMP substrates.
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PMID:Inactivation of Non-canonical Cyclic Nucleotides: Hydrolysis and Transport. 2820 55