Homocysteine transport by human aortic endothelial cells: identification and properties of import systems

Abstract

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Transport of L-homocysteine into and out of the human vascular endothelium is poorly understood. We hypothesized that cultured human aortic endothelial cells (HAEC) would import L-homocysteine on one or more of the L-cysteine transport systems. Inhibitors of the transporters were used to characterize the uptake of [35S]L-homocysteine, [35S]L-homocystine, and [35S]L-cysteine. We found that L-homocysteine uptake is mediated by the sodium-dependent cysteine transport systems X(AG), ASC, and A, and the sodium-independent transport system L. Thus, HAEC utilize multiple cysteine transporters (X(AG) > or = L > ASC > A) to import L-homocysteine. Kinetic analysis supported the uptake results. Michaelis-Menten constants (Km) for the four systems yielded values of 19.0, 27.1, 112, and 1000 microM for systems L, X(AG), ASC, and A, respectively. The binding and uptake of [35S]L-homocystine, the disulfide homodimer of L-homocysteine, was mediated by systems X(AG), L, and ASC but not by system A. In contrast to [35S]L-homocysteine, system x(c) was active for [35S]L-homocystine uptake. A similar pattern was observed for [35S]L-cysteine. Thus, L-homocysteine and L-homocystine found in hyperhomocysteinemic subjects can gain entry into the vascular endothelium by way of multiple L-cysteine transporters.

Fig. 1

Binding and uptake of [³⁵S]L-homocysteine and [³⁵S]L-cysteine by HAEC. Cells were cultured as described in Materials and methods and then incubated with (A) [³⁵S]L-homocysteine or (B) [³⁵S]L-cysteine at the indicated concentrations for 30 min at 37 °C. “Cell associated” is equivalent to “binding and uptake”. Non-specific cell-associated [³⁵S]L-homocysteine or [³⁵S]L-cysteine was determined in the presence of 100-fold molar excess of unlabeled L-homocysteine or L-cysteine, respectively. Specific cell-associated = total cell-associated–non-specific cell-associated. Scatchard plots (insets) were generated from the binding data using Ligand (NIH, Bethesda, MD) software. Each data point is the mean ± SD from three replicates.

Fig. 2

Binding and uptake of [³⁵S]L-homocysteine by HAEC in the presence of increasing concentrations of various L-cysteine transport inhibitors. Confluent cultures of HAEC were incubated with 50 μM [³⁵S]L-homocysteine for 30 min at 37 °C, in the presence of either 0.1–5.0 mM ABH (inhibitor of system X_AG) (A); 0.1–5.0 mM MeAiB (inhibitor of system A) (B); 0.1–5.0 mM BCH (inhibitor of system L) (C); 0.1–5.0 mM Q (inhibitor of system x_c) (D); or 0.1–5.0 mM Ser (inhibitor of system ASC and/or LAT) (E). Non-specific binding (NS) was determined in the presence of 100-fold molar excess of unlabeled L-homocysteine. Results are expressed as percent of the control L-homocysteine uptake. Each data point is the mean ± SD from three replicates. *p ≤ 0.05, significantly different from control (C).

Fig. 3

Binding and uptake of [³⁵S]L-homocysteine, [³⁵S]L-homocystine, and [³⁵S]L-cysteine by HAEC in the presence of L-cysteine transport inhibitors. (A) Inhibition of [³⁵S]L-homocysteine binding and uptake. Confluent cultures of HAEC were incubated with 50 μM [³⁵S]L-homocysteine for 30 min at 37 °C, and the effect of each cysteine transport inhibitor on [³⁵S]L-homocysteine binding and uptake is shown. All inhibitors: BCH (system L), Q (system x_c), ABH (X_AG), MeAiB (system A), and Ser (system ASC) were used at a final concentration of 1.0 mM. Non-specific binding (NS) of [³⁵S]L-homocysteine was determined in the presence of 100-fold molar excess of unlabeled L-homocysteine. Results are expressed as percent of the control L-homocysteine uptake. Each data point is the mean ± SD from three replicates. *p ≤0.05, significantly different from control (C). (B) Inhibition of [³⁵S]L-homocystine binding and uptake. HAEC were incubated with 25 μM [³⁵S]L-homocystine for 30 min at 37 °C, and the same concentrations of cysteine transport inhibitors were used as described in (A). Non-specific binding (NS) of [³⁵S]L-homocystine was determined in the presence of 100-fold molar excess of unlabeled L-homocystine. Results are expressed as percent of the control L-homocystine uptake. Each data point is the mean ± SD from three replicates. *p ≤0.05, significantly different from control (C). (C) Inhibition of [³⁵S]L-cysteine binding and uptake. HAEC were incubated with 50 μM [³⁵S]L-cysteine for 30 min at 37 °C, and the same concentrations of cysteine transport inhibitors were used as in described in (A). Non-specific binding (NS) of [³⁵S]L-cysteine was determined in the presence of 100-fold molar excess of unlabeled L-cysteine. Results are expressed as percent of the control L-cysteine uptake. Each data point is the mean ± SD from three replicates. *p ≤0.05, significantly different from control (C). (D) Inhibition of [³⁵S]L-homocysteine binding and uptake in sodium-free medium. Confluent cultures of HAEC were incubated with 50 μM [³⁵S]L-homocysteine for 30 min at 37 °C in sodium-free medium. The sodium-free media contained: 100 mM choline chloride, 2.68 mM KCl, 0.90 mM CaCl₂, 0.50 mM MgCl₂, 10 mM KH₂PO₄, and 5.5 mM D-glucose, pH 7.4. No inhibition was observed in the presence of the system x_c inhibitor Q (1.0 mM final concentration), as was also observed in sodium-containing media in (A). Nearly complete inhibition of L-homocysteine binding and uptake was observed in the presence of the system L inhibitor BCH (1.0 mM final concentration). Non-specific binding (NS) of [³⁵S]L-homocysteine was determined in the presence of 100-fold molar excess of unlabeled L-homocysteine in sodium-free medium. Results are expressed as percent of the control L-homocysteine uptake. Each data point is the mean ± SD from three replicates. *p ≤0.05, significantly different from control.

Fig. 4

Total inhibition of binding and uptake of [³⁵S]L-homocysteine by HAEC. Confluent cultures of HAEC were incubated with 50 μM [³⁵S]L-homocysteine for 30 min at 37 °C. Complete inhibition of [³⁵S]L-homocysteine binding and uptake was observed in sodium-free medium in the presence of 1.0 mM BCH as previously observed (Fig. 3D). In sodium-containing medium, a cocktail of all four inhibitors (BCH, ABH, MeAiB, and Ser all at 1.0 mM final concentration) was used, and binding and uptake was inhibited 88%. In sodium-containing medium combinations of ABH + BCH, MeAiB + BCH, and Ser + BCH, resulted in inhibitions of 53, 41, and 48%, respectively. Non-specific binding (NS) of [³⁵S]L-homocysteine was determined in the presence of 100-fold molar excess of unlabeled L-homocysteine in sodium-containing medium. Results are expressed as percent of the control L-homocysteine uptake. Each data point is the mean ± SD from three replicates. *p ≤0.05, significantly different from control.

Fig. 5

Kinetics of L-homocysteine binding and uptake by HAEC. (A) Confluent cultures of HAEC were incubated with [³⁵S]L-homocysteine in concentrations ranging from 5 to 32 μM. The data were normalized to DNA content in each well and plotted as picamole L-Hcy/μg DNA. (B) Initial binding and uptake rates were plotted as a function of L-homocysteine concentration. (C) Lineweaver–Burk plot of the data was analyzed using Prism (GraphPad, San Diego, CA) software from which K_m = 21.3 ± 0.9 μM and V_max = 13.1 ± 1.8 pmol L-Hcy/μg DNA min were derived. Each data point is the mean ± SD from three replicates.

Fig. 6

Kinetics of L-homocysteine binding and uptake by systems X_AG, A, ASC, and L in HAEC. Systems X_AG, A, and ASC were studied in sodium-containing medium and a cocktail of inhibitors as described in Table 1. System L was studied in sodium-free medium in the absence of inhibitors. The initial-rate data and Lineweaver–Burk plots (insets) are shown for system X_AG (A), system A (B), system ASC (C), and system L (D). The data were analyzed using Prism (GraphPad, San Diego, CA) software to determine the “best-fit” model and to compute the kinetic constants which are summarized in Table 2.