The organic cation transporter OCT2 mediates the uptake of beta-adrenoceptor antagonists across the apical membrane of renal LLC-PK(1) cell monolayers

Abstract

Previous studies have shown that beta-adrenoceptor antagonists may be substrates of organic cation transporters in kidney and lung. In this study we examined the transport of the beta-adrenoreceptor antagonists propranolol and metoprolol, in renal LLC-PK(1) cell monolayers. Experiments with BCECF (2', 7'-bis(2carboxyethyl)-5(6)-carboxyfluorescein) loaded LLC-PK(1) cell monolayers demonstrated that metoprolol and propranolol flux across the basolateral membrane was consistent with non-ionic diffusion. Flux across the apical membrane consisted of both non-ionic diffusion and the uptake of the cationic form of the beta-adrenoceptor antagonists. Uptake of the cationic form of metoprolol across the apical membrane was Na(+)-independent, electrogenic and sensitive to external pH. Furthermore, uptake was sensitive to inhibition by Decynium-22 and the organic cations TEA (tetraethylammonium) and MPP(+) (1-methyl 4-phenylpyridinium). These results, allied with the apical location of the uptake mechanism suggest that beta-adrenoceptor antagonists may be substrates for the organic cation transporter, OCT2. To confirm beta-adrenoceptor antagonists as substrates for OCT2, we demonstrate, in cells transiently transfected with an epitope tagged version of hOCT2 (hOCT2-V5):(1) Decynium-22 sensitive [(14)C]-propranolol uptake, (2) cis-inhibition of OCT2 by a range of beta-adrenoceptor antagonists and (3) metoprolol induced intracellular acidification.

Figure 1

The effect of 10 mM metoprolol upon intracellular pH. Intracellular pH was measured in monolayers of LLC-PK₁ cells loaded with BCECF. (a) The effect of basolateral addition of metoprolol at a basolateral perfusion pH of 8.4, apical perfusion pH was constant at pH 7.4. A single trace representative of eight separate experiments. (b) The effect of apical addition of metoprolol at an apical perfusion pH of 8.4, basolateral perfusion pH was constant at pH 7.4. A single trace representative of eight separate experiments.

Figure 2

The effect of a range of β-adrenoceptor antagonists upon intracellular pH or upon hOCT2-V5 mediated cimetidine uptake. β-adrenoceptor antagonists (all 10 mM except propranolol (1 mM)) were added to the apical membrane at a perfusate pH of 8.4. The basolateral pH was held constant at pH 7.4. Results are the mean±s.e.mean of four separate determinations. (b) To test the relative permeabilities of neutral weak base (B) and the conjugate weak acid (BH⁺) forms of metoprolol across the apical and basolateral membranes of LLC-PK₁ cells, cell monolayers were loaded with metoprolol (7.69 mM) at pH 7.8 for 40 min. Intracellular pH was measured under four conditions; (1) in the absence of an imposed pH or metoprolol gradient (pH 7.8, 7.69 mM metoprolol), (2) in the presence of an inwardly directed [BH⁺] gradient (pH 7.4 19.1 mM metoprolol), (3) an outwardly directed [BH⁺] gradient (pH 8.2, 3.12 mM metoprolol) and (4) an outwardly directed [B] gradient (pH 7.4, 7.69 mM metoprolol). A single trace representative of three separate experiments.

Figure 3

Metoprolol uptake is Na⁺ dependent. The effect of apical addition of 10 mM metoprolol at an apical perfusate pH 8.4 upon intracellular pH, was measured in the presence and absence of Na⁺ in both perfusates. The basolateral perfusate pH was constant at pH 7.4. A single trace representative of three separate experiments. (b) Metoprolol uptake is electrogenic. The effect of apical challenge with 10 mM metoprolol at pH 8.4 was measured under control (4.5 mM) or high K⁺ (40 mM) conditions. [K⁺] was changed in both apical and basolateral perfusates simultaneously. The basolateral perfusate pH was constant at pH 7.4. The data are the mean±s.e.mean of four separate determinations. ^*P<0.05, ^**P<0.01, compared to control.

Figure 4

Pharmacological characterization of metoprolol uptake. (a) The effect of the organic cation transport inhibitor Decynium-22 (2 μM) was tested upon the pH response to either 10 mM metoprolol or 20 mM NH₄Cl. Both compounds were added to the apical perfusate at pH 8.4. The basolateral pH was held constant at pH 7.4. Results are the mean±s.e.mean of four separate determinations. ^*P<0.05, ^***P<0.001 compared to control condition. (b) The effect of apical addition of various organic cations (20 mM) upon the pH response to a 10 mM metoprolol challenge (pH 8.4). The basolateral pH was held constant at pH 7.4. Results are the mean±s.e.mean of four separate determinations, ^*P<0.05, ^**P<0.01 compared to control.

Figure 5

The effect of metoprolol upon intracellular pH was dependent upon extracellular pH. The effect of a 10 mM apical metoprolol challenge was assayed at either an apical pH of 7.4 or 8.4. The basolateral pH was held constant at 7.4. Results are the mean±s.e.mean of four separate determinations, ^**P<0.01, ^***P<0.001 compared to pH 7.4.

Figure 6

MPP⁺ uptake into hOCT2-V5 transfected mIMCD3 cells. (a) The dose response relationship between hOCT2-V5 mediated MPP⁺ uptake and MPP⁺ concentration, analysis of the data gave an apparent K_m of 22.2±1.8 μM. The results are the mean±s.e.mean of six separate determinations. (b) Initial rates of MPP⁺ uptake (25 μM) into native and hOCT2-V5 transfected cells measured in the presence and absence of 500 nM Decynium-22. The results are the mean±s.e.mean of nine separate determinations, ^*P<0.05 compared to native cells in the control condition.

Figure 7

The effect of metoprolol upon [³H]-cimetidine uptake into native and hOCT2-V5 transfected mIMCD3 cells. Initial rates of cimetidine uptake (100 μM) were measured at pH 7.4 in the absence and presence of 1 mM metoprolol. Results are the mean±s.e.mean of 12 separate determinations, ^***P<0.001 compared to native cells in the control condition. (b) The effect of a range of β-adrenoceptor antagonists (all at 1 mM) upon hOCT2-V5 mediated [³H]-cimetidine uptake (100 μM) measured at pH 7.4. Uptake is expressed as per cent of the control condition. Results are the mean±s.e.mean of eight separate determinations, ^*P<0.05, ^**P<0.01 compared to control cells.

Figure 8

The uptake of propranolol (75 μM) into native and hOCT2-V5 transfected cells. Initial rates of propranolol uptake were measured at pH 7.4 in the absence and presence of 500 nM Decynium-22. Results are the mean±s.e.mean of nine separate determinations, ^***P<0.001 compared to native cells in the control condition. To investigate the effect of 10 mM metoprolol upon intracellular pH in Hela cells transiently transfected with hOCT2-V5 intracellular pH was measured in subconfluent monolayers of cells loaded with BCECF. (b) The effect of metoprolol challenge upon Hela cells transfected with hOCT2-V5 at a perfusion pH of 8.4 and subsequent recovery at pH 7.4. A single trace representative of five separate experiments. (c) The effect of an identical metoprolol challenge upon intracellular pH in mock-transfected Hela cells. A single trace representative of five separate experiments.