The Organic Cation Transporters rOCT1 and hOCT2 Are Inhibited by cGMP

Abstract

The electrogenic cation transporters OCT1 and OCT2 in the basolateral membrane of renal proximal tubules mediate the first step during secretion of organic cations. Previously we demonstrated stimulation and change of selectivity for rat OCT1 (rOCT1) by protein kinase C. Here we investigated the effect of cGMP on cation transport by rOCT1 or human OCT2 (hOCT2) after expression in human embryonic kidney cells (HEK293) or oocytes of Xenopus laevis. In HEK293 cells, uptake was measured by microfluorimetry using the fluorescent cation 4-(4-(dimethyl-amino)styryl)-N-methylpyridinium iodide (ASP + ) as substrate, whereas uptake into Xenopus laevis oocytes was measured with radioactively labelled cations. In addition, ASP +-induced depolarizations of membrane voltages (Vm) were measured in HEK293 cells using the slow whole-cell patch-clamp method. Incubation of rOCT1-expressing HEK293 cells for 10 min with 100 mM 8-Br-cGMP reduced initial ASP + uptake by maximally 78% with an IC50 value of 24 +/- 16 mM. This effect was not abolished by the specific PKG inhibitor KT5823, indicating that a cGMP-dependent kinase is not involved. An inhibition of ASP + uptake by rOCT1 in HEK293 cells was also obtained when the cells were incubated for 10 min with 100 mM cGMP, whereas no effect was obtained when cGMP was given together with ASP +. ASP + (100 mM)-induced depolarizations of Vm were reduced in the presence of 8-Br-cGMP (100 mM) by 44 +/- 11% (n = 6). Since it could be demonstrated that [3H]cGMP is taken up by an endogeneous cyanine863-inhibitable transporter, the effect of cGMP is probably mediated from inside the cell. Uptake measurements with [14C]tetraethylammonium and [3H]2-methyl-4-phenylpyridinium in Xenopus laevis oocytes expressing rOCT1 performed in the absence and presence of 8-Br-cGMP showed that cGMP does not interact directly with the transporter. The data suggest that the inhibition mediated by cGMP observed in HEK293 cells occurs most likely via a mammalian cGMP-binding protein that interacts with OCT1-2 transporters.

Involvement of guanylyl cyclase and cGMP in the regulation of Mrp2-mediated transport in the proximal tubule

“…cGMP has been implicated in the regulation of other transport processes in the kidney, although not directly linked to NO production or to PKC stimulation. For example, Hirsch et al (8 -10) described that cGMP is involved in the regulation of Ca 2ϩ and K ϩ channels in a human proximal tubule cell line, whereas others (28) showed that cGMP is involved in the regulation of the organic cation transporters, rOCT1 and hOCT2. cGMP has also been found to be involved in the regulation of vascular tone in which the ET B receptor is included.…”

Organic Cation Transporter 2 Overexpression May Confer an Increased Risk of Gentamicin-Induced Nephrotoxicity

“…To understand the role of OCTs in gentamicin uptake, HEK293 cells transfected with OCT1, OCT2, or OCT3 were coincubated for 2 min (linear range) with 1 M ASP ϩ (known OCT substrate) in the presence or absence of nonlabeled gentamicin at an extracellular concentration of 10 mM (30). Figure 3A shows that nonlabeled gentamicin reduced OCT2-mediated influx of ASP ϩ by approximately 50% (89.6 Ϯ 17.5 versus 42.8 Ϯ 10.6 pmol/mg of protein; P ϭ 0.02).…”

Remote Communication through Solute Carriers and ATP Binding Cassette Drug Transporter Pathways: An Update on the Remote Sensing and Signaling Hypothesis

“…Pharmacogenetic analysis of the SLC22A7 and pharmacokinetic analysis of its gene products suggest that it plays a role in mediating the balance of intra/extracellular cGMP and consequently regulates this second-messenger signaling pathway (Cropp et al, 2008). In addition, cGMP can serve as an intracellular substrate modulating the expression/activities of other organic ion transporters of the SLC22 family (Schlatter et al, 2002). This cross-talk between different SLC transporters through a transported signaling molecule remains to be explored.…”