OCT2 and MATE1 provide bidirectional agmatine transport

Abstract

Agmatine is a biogenic amine (l-arginine metabolite) of potential relevance to several central nervous system (CNS) conditions. The identities of transporters underlying agmatine and polyamine disposition in mammalian systems are not well-defined. The SLC-family organic cation transporters (OCT) OCT1 and OCT2 and multidrug and toxin extrusion transporter-1 (MATE1) are transport systems that may be of importance for the cellular disposition of agmatine and putrescine. We investigated the transport of [(3)H]agmatine and [(3)H]putrescine in human embryonic kidney (HEK293) cells stably transfected with hOCT1, hOCT2, and hMATE1. Agmatine transport by hOCT1 and hOCT2 was concentration-dependent, whereas only hOCT2 demonstrated pH-dependent transport. hOCT2 exhibited a greater affinity for agmatine (K(m) = 1.84 ± 0.38 mM) than did hOCT1 (K(m) = 18.73 ± 4.86 mM). Putrescine accumulation was pH- and concentration-dependent in hOCT2-HEK cells (K(m) = 11.29 ± 4.26 mM) but not hOCT1-HEK cells. Agmatine accumulation, in contrast to putrescine, was significantly enhanced by hMATE1 overexpression, and was saturable (K(m) = 240 ± 31 μM; V(max) = 192 ± 10 pmol/min/mg of protein). Intracellular agmatine was also trans-stimulated (effluxed) from hMATE1-HEK cells in the presence of an inward proton-gradient. The hMATE1-mediated transport of agmatine was inhibited by polyamines, the prototypical substrates MPP+ and paraquat, as well as guanidine and arcaine, but not l-arginine. These results suggest that agmatine disposition may be influenced by hOCT2 and hMATE1, two transporters critical in the renal elimination of xenobiotic compounds.

Structures

Figure 1. Agmatine and Putrescine accumulation in mock-, hOCT1-, and hOCT2-HEK cells

Agmatine (A) or Putrescine (B) accumulation was examined in HEK293 cells. Steady-state agmatine accumulation was significantly greater in hOCT1- and hOCT2-HEK cells as compared to mock cells. Steady-state putrescine accumulation was significantly greater in hOCT2-HEK cells as compared to mock- and hOCT1-HEK cells. Data are expressed as the mean ± SD (n = 3).

Figure 2. Concentration-dependence of agmatine and putrescine accumulation in hOCT2- and hOCT1-HEK cells

The concentration-dependent accumulation of agmatine (AGM) and putrescine (PUT) was examined in (a) hOCT2- and (b) hOCT1-HEK cells (pH 7.4). (a) Agmatine and putrescine accumulation (10 min) was saturable in hOCT2-HEK cells, with hOCT2 displaying a much higher affinity for agmatine (K_m = 1.84 ± 0.38 mM) as compared to putrescine (K_m = 11.29 ± 4.26 mM). (b) Agmatine, in contrast to putrescine, exhibited saturable uptake in hOCT1-HEK cells (K_m = 18.73 ± 4.86 mM). Data are expressed as the mean ± SD and are representative data from two independent experiments done in triplicate (n = 6).

Figure 2. Concentration-dependence of agmatine and putrescine accumulation in hOCT2- and hOCT1-HEK cells

The concentration-dependent accumulation of agmatine (AGM) and putrescine (PUT) was examined in (a) hOCT2- and (b) hOCT1-HEK cells (pH 7.4). (a) Agmatine and putrescine accumulation (10 min) was saturable in hOCT2-HEK cells, with hOCT2 displaying a much higher affinity for agmatine (K_m = 1.84 ± 0.38 mM) as compared to putrescine (K_m = 11.29 ± 4.26 mM). (b) Agmatine, in contrast to putrescine, exhibited saturable uptake in hOCT1-HEK cells (K_m = 18.73 ± 4.86 mM). Data are expressed as the mean ± SD and are representative data from two independent experiments done in triplicate (n = 6).

Figure 3. pH-dependence of agmatine, putrescine, and TEA accumulation in hOCT1- and hOCT2-HEK cells

The pH-dependent accumulation (10 min) of (a) agmatine, (b) putrescine, or (c) TEA was investigated in hOCT1- and hOCT2-HEK cells. Agmatine accumulation was markedly enhanced in hOCT2-HEK cells with increasing extracellular pH, exhibiting maximal uptake at pH 9.0, between pH 6.0 to 9.0. Putrescine accumulation was also enhanced in hOCT2-HEK cells at extracellular pH 8.0, 8.5, and 9.0 (compared to uptake at pH 7.4). Agmatine and putrescine accumulation was not influenced by changes in extracellular pH in hOCT1-HEK cells. TEA accumulation was increased in both hOCT1- and hOCT2-HEK cells with increasing extracellular pH. Values in all three studies are corrected for mock cellular uptake, which was not influenced by extracellular pH. Data are expressed as the mean ± SD and represent pooled data from two independent experiments done in triplicate (n = 6)

Figure 3. pH-dependence of agmatine, putrescine, and TEA accumulation in hOCT1- and hOCT2-HEK cells

The pH-dependent accumulation (10 min) of (a) agmatine, (b) putrescine, or (c) TEA was investigated in hOCT1- and hOCT2-HEK cells. Agmatine accumulation was markedly enhanced in hOCT2-HEK cells with increasing extracellular pH, exhibiting maximal uptake at pH 9.0, between pH 6.0 to 9.0. Putrescine accumulation was also enhanced in hOCT2-HEK cells at extracellular pH 8.0, 8.5, and 9.0 (compared to uptake at pH 7.4). Agmatine and putrescine accumulation was not influenced by changes in extracellular pH in hOCT1-HEK cells. TEA accumulation was increased in both hOCT1- and hOCT2-HEK cells with increasing extracellular pH. Values in all three studies are corrected for mock cellular uptake, which was not influenced by extracellular pH. Data are expressed as the mean ± SD and represent pooled data from two independent experiments done in triplicate (n = 6)

Figure 4

Figure 4a. Agmatine accumulation in mock- and hMATE1-HEK cells Agmatine (10 nM) accumulation was examined in mock- and hMATE1-HEK293 cells. Agmatine accumulation was significantly enhanced in hMATE1-HEK cells, as compared to mock cells. Data are expressed as the mean ± SD and represent pooled data from three independent experiments done in triplicate (n = 9) (*p < 0.05). Figure 4b. Putrescine accumulation in mock- and hMATE1-HEK cells Putrescine (10 nM) accumulation was examined in mock- and hMATE1-HEK293 cells. Putrescine accumulation was nearly identical in mock- and hMATE1-HEK cells. Interestingly, putrescine accumulation was more robust as compared to agmatine accumulation in either cell type. Data are expressed as the mean ± SD (n = 3).

Figure 5. Concentration-dependent agmatine accumulation in hMATE1-HEK cells

The concentration-dependent accumulation of agmatine was examined in hMATE1-HEK cells (pH 8.0). Agmatine accumulation (10 min) was saturable, displaying high-affinity, low-capacity transport by hMATE1 (K_m=240 ± 31 μM; V_max=192 ± 10 pmol/min/mg protein). Final values are corrected for mock agmatine uptake at each corresponding concentration. Data are expressed as the mean ± SD and represent pooled data from three independent experiments done in triplicate (n = 9).

Figure 6

Figure 6a. pH-dependent agmatine accumulation in hMATE1-HEK cells The pH-dependent accumulation of agmatine (10 nM) was examined in hMATE1-HEK cells. Agmatine accumulation (10 min) was significantly influenced by extracellular pH in hMATE1-HEK cells, increasing from pH 6.0 to 8.0, after which uptake decreased. Data are expressed as the mean ± SD and represent pooled data from two independent experiments done in triplicate (n = 6). Figure 6b. TRANS-stimulation of agmatine in hMATE1-HEK cells Trans-stimulation of agmatine was examined in hMATE1-HEK cells. Intracellular agmatine was trans-stimulated in the presence of an inward-proton gradient (extracellular pH 6.0), but was unchanged when the extracellular pH was kept at 8.0. Data are expressed as the mean ± SD and represent pooled data from three independent experiments done in groups of eight (n = 24) (*p < 0.05 compared to control, time zero).

Figure 7. Proposed mechanism of agmatine transport in tissues known to express OCT1, OCT2, and MATE1

The following schematic is a proposed mechanism of agmatine transport in tissues (i.e., kidney and liver) widely-recognized to express OCT1, OCT2, and MATE1. Organic cation transporter (OCT) 1 and 2 mediate the facilitated influx transport of organic cations at the basolateral membrane of hepatocytes and renal proximal tubule cells, respectively. The multidrug and toxic compound extrusion (MATE) transporter 1, an H+/cation antiporter, is critical in the efflux elimination of various organic cations from the brush-border and canalicular membrane of the kidney and liver, respectively. The results of the present study suggest that the divalent cation agmatine (AGM) may be eliminated by OCT2-mediated uptake and MATE1-mediated secretion in the renal tubules. (Descriptions in parentheses on the graphic refer to equivalent structures in the liver).