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Department of Pharmacology and Therapeutics, Louisiana State University Medical Center, Shreveport, Louisiana 71130
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENT
FOOTNOTES
LITERATURE CITED
ABSTRACT 
Although reabsorption across the apical (AP) membrane of the renal proximal tubule cell plays a vital role in the conservation of plasma 5-methyltetrahydrofolate, basolateral (BL) membrane-directed secretory pathways may also be important in regulating the urinary excretion of folate. Folate transport proteins, folate receptor and the reduced folate carrier have been implicated in the renal conservation of folate across the AP membrane, but their role in BL membrane-directed folate transport has not been studied. 5-Methyltetrahydrofolate transport across the AP and BL membranes of human proximal tubule cells was studied in cells grown on membrane inserts to allow optimum differentiation of AP and BL domains. Colchicine, an inhibitor of vesicular-mediated endocytosis, inhibited AP binding and AP-directed transport without affecting BL transport. Probenecid, an inhibitor of anion exchange, did not affect binding, but inhibited both AP and BL-directed transport with a greater effect on BL transport. Folic acid abolished AP binding of 5-methyltetrahydrofolate, but diminished AP-mediated transport by only 50%. These data suggest that both the folate receptor and the reduced folate carrier participate in AP uptake of folates by human kidney cells, but that BL-mediated uptake occurs primarily by the reduced folate carrier. Folate transport from the secretory direction occurred as readily as that from the reabsorptive direction, indicating that altered secretion could mediate excess urinary folate excretion.
KEY WORDS: humans · folate receptor · reduced folate carrier · urinary folate excretion · endocytosisINTRODUCTION
The specific cellular accumulation of folate is generally thought to occur via two different protein-mediated pathways. The first folate transport system is binding protein-mediated, occurring via different isoforms of the folate receptor (Ross et al. 1994
, Wang et al. 1992). The second specific transport system involves a reduced folate carrier anion exchange system (Henderson et al. 1980, Westerhof et al. 1991). These folate transport pathways have generated considerable interest since cellular folate accumulation is crucial for the synthesis of DNA and RNA and, hence, for normal cellular growth. Numerous studies have also shown that selective expression or overexpression of these proteins may be linked with neoplastic growth (Sirotnak 1985, Weitman et al. 1992). In addition, the folate transport proteins are important therapeutically since they are involved in cellular accumulation of antifolates such as methotrexate (Antony 1992, Henderson et al. 1980). Specific chemotherapeutic targeting has been shown by Low and coworkers, who have delivered pharmacologically important, impermeant macromolecules into the cellular cytoplasm via the folate receptor-mediated endocytic pathway (Lee et al. 1995, Wang et al. 1995).
In addition to their roles in growth and malignancy, or as potential targets of cancer chemotherapy, the folate transport proteins could play a vital role in the overall homeostasis of folate by controlling the urinary excretion of folate. Plasma 5-methyltetrahydrofolate (5-CH3-H4-PteGlu)5 is freely filtered at the glomerulus and is reabsorbed in the proximal tubule (Goresky et al. 1963). Previous studies have shown an abundant expression of the folate receptor in the apical (AP) surface of the proximal tubule cell (Corrocher et al. 1985, Selhub and Franklin 1984). After the initial binding to the AP membrane folate receptor, folate appears to be rapidly internalized in endocytic vesicles distinct from clathrin-coated pits (Hjelle et al. 1991, Selhub et al. 1987). The internalized vesicles transfer folate to the cytoplasm, and the folate receptors recycle back to the AP membrane by dense apical tubules (Birn et al. 1993). Several studies have also shown the involvement of the reduced folate carrier in the AP membrane subsequent to or distinct from the folate receptor-mediated initial binding step (Kamen et al. 1991, Rothberg et al. 1990). In addition to reabsorbing folate from the tubular lumen, the proximal tubule cells also secrete folate in vivo (Eisenga and McMartin 1987, Williams and Huang 1982). The mechanism of secretory folate transport has not been studied. Also, the extent of basolateral (BL)-to-AP transport of 5-CH3-H4PteGlu has not been directly compared with that occurring from the AP-to-BL direction.
Altered folate balance such as a decrease in the level of plasma 5-CH3-H4PteGlu has been observed during pregnancy (Landon and Hytten 1971) and alcohol ingestion (Eisenga et al. 1989, McMartin et al. 1986). Although the exact mechanism is not clearly understood, excess urinary excretion of folate appears to play a role in the depletion of plasma 5-CH3-H4PteGlu. Such urinary loss could result from an alteration in the proximal tubule reabsorptive or secretory folate transport pathways. A clear understanding of the cellular bidirectional folate transport pathways, including the role played by the folate receptor and the reduced folate carrier, is essential to delineate the mechanisms of physiologic or pharmacologic alterations in urinary folate excretion. Such mechanistic studies require a system that allows for distinction between the AP and BL components of the proximal tubule cell, which are highly polarized in vivo. In culture, proximal tubule cells have been shown to possess folate transport pathways (McMartin et al. 1992) and to maintain domain-specific nutrient transport across the epithelium (Middleton et al. 1989). Traditional proximal tubule cell culture systems, in which cells are grown on plastic, do not allow assessment of bidirectional folate transport. However, human proximal tubule (HPT) cells grown on microporous membrane inserts have been shown to involve both the AP and BL domains in mediating the cellular transport of folic acid (Morshed and McMartin 1996a). These results were suggestive of folate receptor-mediated folate transport. However, 5-CH3-H4PteGlu, the chief plasma form of folate, is a reduced folate and could be transported by both the folate receptor and the reduced folate carrier. The studies presented here examine the relative roles of the folate receptor and the reduced folate carrier in the AP-directed and BL-directed uptake of 5-CH3-H4PteGlu by the HPT cell. The studies suggest a membrane domain-specific distribution of the transport of folate by the folate receptor and the reduced folate carrier, through which the reabsorptive and secretory folate transport pathways may regulate overall folate homeostasis in the kidney.
MATERIALS AND METHODS
,5,7,9-3H]-(6S)-5-CH3-H4PteGlu (1.18 Tbq/mmol) was obtained as a solution in 2-mercaptoethanol (20 g/L) from Moravek Biochemicals (Brea, CA). These chemicals were used at purity above 98% as judged by the use of a high-performance liquid chromatography assay (Eisenga et al. 1989). When deterioration occurred, samples were purified before use as previously described for unlabeled folates (McMartin et al. 1981). [Carboxyl-14C]-inulin (62.9 GBq/g), was obtained from DuPont-New England Nuclear (Boston, MA). All other chemicals unless noted below were obtained from Sigma Chemical Co (St. Louis, MO).
Cell Culture.
Human proximal tubule (HPT) cells were isolated from kidneys that were removed because of renal adenocarcinoma or trauma, or from kidneys unable to be used in transplantation. Immediately after the surgery, adjacent healthy tissue from the outer cortical region was removed; tissue was remote from any pathologic alterations as judged by the surgical pathologist. No record was kept of the patient, and the studies were exempted by the Institutional Review Board for Human Research (Louisiana State University Medical Center). HPT cells were isolated by the enzyme dissociation method using a collagenase-DNAase mixture as described previously (Blackburn et al. 1988). Isolated cells were regularly cultured on collagen-coated plastic surfaces in a serum-free mixture of Dulbecco's modified Eagle's medium-Ham's F-12 medium (50:50 by volume, GIBCO, Grand Island, NY) with the following additions per L: selenium (5 µg), insulin (5 mg), transferrin (5 mg), hydrocortisone (36 µg), epidermal growth factor (10 µg, Collaborative Research, Bedford, MA), triiodothyronine (4 ng, Sigma Chemical), and 2 mmol glutamine (GIBCO). The folate concentration of this medium, determined microbiologically, was 3 µmol/L (McMartin et al. 1992). For transport studies, confluent monolayers of HPT cells were subcultured by detaching with trypsin-EDTA (0.5 and 0.2 g/L, respectively) and seeding in fresh medium in a 1:1 ratio in membrane inserts (Millicell-PCF; pore size 0.4 µm; diameter 12 mm; Millipore, Bedford, MA) that were coated with bovine dermal collagen (Celltrix Labs, Palo Alto, CA). The inserts were placed in tissue culture wells (12-well plates, Costar, Cambridge, MA), growth media (above) was placed in the AP (0.3 mL) and BL (1.5 mL) chambers and the plates containing the inserts were incubated at 37°C in a humidified atmosphere containing 5% CO2 . These inserts acted as a permeable support allowing optimum differentiation of HPT cellular AP and BL membrane domains to represent a remarkably tight epithelium. Upon confluency, the cell monolayer effectively separated the AP and BL media as judged by the negligible leakage of paracellular probe, inulin (Morshed and McMartin 1995). Cells were fed fresh growth medium every 2-3 d until confluency (about 7 d), which was determined by monitoring development of transepithelial electrical resistance using an EVOM Voltohmmeter (World Precision Instruments, New Haven, CT). 5-CH3-H4PteGlu transport studies were conducted using these primary cultured cells at passages 4-8.
5-CH3-H4PteGlu uptake studies.
The binding, transport and transepithelial transfer of 5-CH3-H4PteGlu by HPT cells grown on inserts were measured from the AP to BL (A-B) and BL to AP (B-A) directions, which are the in vitro counterparts of the reabsorptive (lumenal-to-serosal) and secretory pathways (serosal-to-lumenal), respectively. The 5-CH3-H4PteGlu uptake studies on insert-grown HPT cells were conducted using slight modifications of procedures recently published (McMartin et al. 1992, Morshed and McMartin 1995). Briefly, the growth media from both chambers were removed, and the AP and BL chambers were rinsed three times with a pH 7.4 assay buffer that contained (in mmol/L) NaCl, 107; KCl, 5.3; CaCl2 , 1.9; MgCl2 , 1.0; NaHCO3 , 26.2; D-glucose, 7.0; and N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES), 20. The washed inserts were then transferred into fresh 24-well plates, 0.3 and 0.6 mL of the pH 7.4 assay buffer was added to the AP and BL chambers, respectively, and the cells were incubated for 1 h at 37°C to attain the tight monolayer condition (Morshed and McMartin 1995). 5-CH3-H4PteGlu uptake studies in the A-B and B-A directions were then initiated by adding the labeled substrate to the appropriate chamber using a small volume of buffer, which assured that the tight monolayer integrity was not disturbed (Morshed and McMartin 1995). Paracellular permeability was determined by co-incubating [3H]-5-CH3-H4PteGlu with [14C]-inulin (5.55 GBq/L incubation media). Paracellular transfer of inulin into the opposite compartment (i.e., into BL when added in the AP compartment or vice-versa) in intact inserts remained below 1% of the initial label for up to 4 h. Samples that showed excess amounts of inulin transfer indicated that the cell layer had become leaky, possibly because of pipette puncture, and such samples were excluded from the analyses.
). Finally, the cells were solubilized for analysis of cellular transport by twice adding 0.5 mL of Triton X-100 (1 g/L phosphate buffered saline, pH 7.4) for 15 min each time. Protein content in the solubilized cells was determined by the commercial Bio-Rad method (Bio-Rad, Richmond, CA).
). Briefly, after washing the confluent HPT monolayers, the inserts were transferred to 24-well plates, preincubated for 1 h with different concentrations of colchicine by adding 0.3 and 0.6 mL of colchicine-containing assay buffer to the AP and BL chambers, respectively. 5-CH3-H4PteGlu uptake studies in the A-B and B-A directions were then initiated by adding the labeled substrate to the appropriate chamber and incubating for up to 4 h as per the protocol above.
, Pritchard and Miller 1993) and indirectly through the possibly negative effect of PteGlu in the above-described studies. Transport experiments were run for 15 and 60 min following the same protocol as described above in that the uptake experiments were initiated by adding the labeled 5-CH3-H4PteGlu and probenecid simultaneously to the appropriate chamber (AP or BL).
Statistics.
Statistical comparisons between two treatment means were conducted by Student's t-test with P < 0.05 as the level of significance. Group data for multiple comparisons were analyzed by analysis of variance followed by Duncan's multiple range test (Keppel 1982) with P < 0.05 as the level of significance. Values in the text are means ± SEM.
RESULTS
), these data suggest that the 5-CH3-H4PteGlu that crossed the BL membrane was delivered through the cell into the AP membrane and appeared as AP binding. These studies were conducted with 5-CH3-H4PteGlu at a 10 nmol/L concentration since human plasma 5-CH3-H4PteGlu concentrations range form 5-20 nmol/L (McMartin et al. 1986). Similar binding results were observed at 25 and 50 nmol/L (data not shown).
DISCUSSION ACKNOWLEDGMENT 
Fig. 2.
5-Methyltetrahydrofolate (5-CH3-H4PteGlu) transport in human proximal tubule (HPT) cells. HPT cell monolayers were incubated for the indicated time period with 10, 25, or 50 nmol [3H]-5-CH3-H4PteGlu/L without (total) or with a 1000-fold excess of unlabeled 5-CH3-H4PteGlu (nonspecific control). Experiments were conducted in quadruplicate inserts to determine the total transport and in duplicate inserts for the nonspecific control. Incubations were initiated from the apical (AP)-to-basolateral (BL) (panel A) or BL-to-AP (panel B) directions by adding the substrate to the AP or BL chambers, respectively. After the incubations, inserts were washed, and AP and BL transport were measured as described in Materials and Methods. Data, expressed as fmol 5-CH3-H4PteGlu transported per mg of cellular protein, represent the specific transport calculated by subtracting the nonspecific transport from the total transport. Nonspecific transport constituted less than 10% of the total transport in all cases. Values are means ± SEM of three different experiments with separate isolates.
[View Larger Version of this Image (12K GIF file)]
, Rothberg et al. 1990). As such, the possible role of a folate receptor-mediated transport pathway in the bidirectional uptake of 5-CH3-H4PteGlu by HPT cells was assessed initially by co-incubating with colchicine, a microtubule depolymerizer that leads to malfunctions in vesicular processing (Brown et al. 1991). A number of studies have shown that such effects of colchicine block the endocytic accumulation of ricin (Sandvig and van Deurs 1990) and ferritin (Thatte et al. 1994). Figure 3 shows the effects of colchicine on the AP binding and bidirectional intracellular transport of 5-CH3-H4PteGlu. Inhibition of 5-CH3-H4PteGlu binding and transport from the A-B direction occurred in a concentration-dependent manner. Inhibition of both the binding and transport approached about 75-80% at a 1 mmol/L concentration of colchicine, suggesting that the majority of the 5-CH3-H4PteGlu uptake from the A-B direction occurred by a microtubule-sensitive endocytic process. On the other hand, colchicine did not affect 5-CH3-H4PteGlu transport from the B-A direction, suggesting that movement of 5-CH3-H4PteGlu across the BL membrane occurred by a microtubule-insensitive mechanism.
Fig. 3.
5-Methyltetrahydrofolate (5-CH3-H4PteGlu) binding and transport in colchicine-treated human proximal tubule (HPT) cells. HPT cell monolayers were preincubated for 1 h with 0.01, 0.10, and 1.0 mmol colchicine/L as detailed in Materials and Methods. 5-CH3-H4PteGlu uptake studies were conducted for 4 h by adding [3H]-5-CH3-H4PteGlu (final concentration, 10 nmol/L) in the apical (AP) or basolateral (BL) chamber without (total) or with a 1000-fold excess of unlabeled 5-CH3-H4PteGlu (nonspecific control). After the incubations, inserts were washed, then AP binding and AP and BL transport were measured as described in Materials and Methods. Data, expressed as fmol 5-CH3-H4PteGlu bound or transported per mg of cellular protein, represent the specific binding or transport calculated by subtracting the respective nonspecific values from the total values. Nonspecific binding and transport represented less than 10% of the total values in all cases. Values are means ± SEM of three different experiments with separate isolates. Asterisks indicate significant difference from control experiments that represent 100% binding or transport activities in the absence of colchicine (P < 0.05).
[View Larger Version of this Image (19K GIF file)]
, Kamen et al. 1991). The effects of probenecid (10 mmol/L) on the AP binding and bidirectional intracellular transport of 5-CH3-H4PteGlu are shown in Figure 4. Probenecid did not significantly alter 5-CH3-H4PteGlu binding, although it markedly inhibited intracellular transport of 5-CH3-H4PteGlu, both from the A-B and B-A directions. The inhibition of transport appeared to be greater from the B-A direction (about 70% inhibition after 1 h) compared with that from the A-B direction (about 50% inhibition).
Fig. 4.
5-Methyltetrahydrofolate (5-CH3-H4PteGlu) binding and transport in probenecid-treated human proximal tubule (HPT) cells. HPT cellular 5-CH3-H4PteGlu uptake studies were conducted for 15 and 60 min with 10 mmol probenecid and 10 nmol [3H]-5-CH3-H4PteGlu/L (final incubation concentrations) added to the apical (AP) or basolateral (BL) chambers without (total) or with a 1000-fold excess of unlabeled 5-CH3-H4PteGlu (nonspecific control). After the incubations, inserts were washed, then AP binding and AP and BL transport were measured as described in Materials and Methods. Data, expressed as fmol 5-CH3-H4PteGlu bound or transported per mg of cellular protein, represent the specific binding or transport calculated by subtracting the respective nonspecific values from the total values. Nonspecific binding and transport represented less than 10% of the total values in all cases. Values are means ± SEM of three different experiments with separate isolates. Asterisks indicate significant difference from control experiments that represent 100% binding or transport activities in the absence of probenecid (P < 0.05).
[View Larger Version of this Image (13K GIF file)]
Fig. 5.
5-Methyltetrahydrofolate (5-CH3-H4PteGlu) binding and transport in colchicine + probenecid-treated human proximal tubule (HPT) cells. HPT cell monolayers were preincubated for 1 h with 1.0 mmol colchicine/L or 1.0 mmol colchicine + 10.0 mmol probenecid/L as detailed in Materials and Methods. 5-CH3-H4PteGlu uptake studies were conducted for 4 h by adding [3H]-5-CH3-H4PteGlu (final concentration, 10 nmol/L) in the apical (AP) or basolateral (BL) chambers without (total) or with a 1000-fold excess of unlabeled 5-CH3-H4PteGlu (nonspecific control). After the incubations, inserts were washed, then AP binding and AP and BL transport were measured as described in Materials and Methods. Data, expressed as fmol 5-CH3-H4PteGlu bound or transported per mg of cellular protein, represent the specific binding or transport calculated by subtracting the respective nonspecific values from the total values. Nonspecific binding and transport represented less than 10% of the total values in all cases. Values are means ± SEM of three different experiments with separate isolates. Asterisks indicate significant difference from control experiments that represent 100% binding or transport activities in the absence of colchicine and probenecid (P < 0.05).
[View Larger Version of this Image (12K GIF file)]
, Wang et al. 1992, Westerhof et al. 1991). Indeed, PteGlu inhibited about 80-90% of the 5-CH3-H4PteGlu binding to the AP membrane. As shown in the inset of Fig. 6, probenecid did not inhibit the remaining, PteGlu-insensitive 5-CH3-H4PteGlu binding. Figure 6 also shows that PteGlu inhibited about 40-50% of the transport of 5-CH3-H4PteGlu from the AP direction. That the A-B 5-CH3-H4PteGlu transport was inhibited by PteGlu suggests that about half of the 5-CH3-H4PteGlu may be transported via folate receptor-mediated endocytosis. The addition of probenecid (Fig. 6, inset) almost completely inhibited the remaining, PteGlu-insensitive, 5-CH3-H4PteGlu transport.
Fig. 6.
5-Methyltetrahydrofolate (5-CH3-H4PteGlu) binding and transport in human proximal tubule (HPT) cells in the presence of folic acid (PteGlu) alone or in combination with probenecid. HPT cellular 5-CH3-H4PteGlu uptake studies were conducted for 4 h with 10 nmol [3H]-5-CH3-H4PteGlu/L in the presence of the indicated concentrations of PteGlu alone or of PteGlu (10 nmol/L) + 10 mmol probenecid/L (inset). Substrate and effectors were added together in the apical (AP) chambers without (total) or with a 1000-fold excess of unlabeled 5-CH3-H4PteGlu (nonspecific control). After the incubations, inserts were washed, then AP binding and transport were measured as described in Materials and Methods. Data, expressed as fmol 5-CH3-H4PteGlu bound or transported per mg of cellular protein, represent the specific binding calculated by subtracting the nonspecific values from the total values. Values are means ± SEM of three different experiments with separate isolates. All values were significantly different from control experiments that represent 100% binding or transport activity in the absence of PteGlu (P < 0.05).
[View Larger Version of this Image (16K GIF file)]
, Wang et al. 1992, Westerhof et al. 1991). Accordingly, the apical 5-CH3-H4PteGlu binding due to the folate receptor remained largely unaffected by 5-HCO-H4PteGlu. The A-B transport of 5-CH3-H4PteGlu, which, according to the above studies, appears to occur mostly via a folate receptor-mediated process, was also not inhibited except at 1000 nmol/L. In contrast, 5-HCO-H4PteGlu significantly inhibited the B-A transport (P < 0.05).
Fig. 7.
5-Methyltetrahydrofolate (5-CH3-H4PteGlu) binding and transport in human proximal tubule (HPT) cells in the presence of 5-formyltetrahydrofolate (5-HCO-H4PteGlu). HPT cellular 5-CH3-H4PteGlu uptake studies were conducted for 4 h by adding 10 nmol [3H]-5-CH3-H4PteGlu/L and the indicated concentrations of 5-HCO-H4PteGlu to the apical (AP) or basolateral (BL) chambers without (total) or with a 1000-fold excess of unlabeled 5-CH3-H4PteGlu (nonspecific control). After the incubations, inserts were washed, then AP binding and AP and BL transport were measured as described in Materials and Methods. Data, expressed as fmol 5-CH3-H4PteGlu bound or transported per mg of cellular protein, represent the specific 5-CH3-H4PteGlu binding and transport calculated by subtracting the nonspecific values from the total values. Values are means ± SEM of three different experiments with separate isolates. Asterisks indicate significant difference from control experiments that represent 100% binding or transport activities in the absence of 5-HCO-H4PteGlu (P < 0.05).
[View Larger Version of this Image (12K GIF file)]
). This reabsorptive process has been shown in several different types of renal systems to involve primarily a folate receptor-mediated process (Birn et al. 1993, Rothberg et al. 1990). However, secretion of folate by the kidney has also been observed in certain circumstances (Eisenga et al. 1987, Williams and Huang 1982), but the physiological importance of secretion is not known. Our previous studies in cultured HPT cells have established that both reabsorptive and secretory transport pathways for folates exist in cultured HPT cells (Morshed and McMartin 1996a and 1996b). In fact, intracellular transport of 5-CH3-H4PteGlu occurs to about the same extent across the AP and BL membranes (Fig. 2), suggesting that the secretory transport may be quantitatively important. The present studies have shown that the AP and the BL transport processes are mediated to a different extent by the two folate transport proteins, the folate receptor and the reduced folate carrier.
, Rothberg et al. 1990, Selhub et al. 1987). The present results confirm that the folate receptor plays a major role in the AP-directed transport of 5-CH3-H4PteGlu in the HPT cell. PteGlu, which has a significantly greater affinity for the folate receptor than does 5-CH3-H4PteGlu (Antony 1992, Wang et al. 1992) and markedly less affinity for the reduced folate carrier (Westerhof 1991), almost completely blocked the binding of 5-CH3-H4PteGlu to the AP membrane (Fig. 6). At the same time, PteGlu decreased the transport of 5-CH3-H4PteGlu via the AP membrane by about 50%, presumably because of its effects on folate receptor-mediated binding. Similarly, 5-HCO-H4PteGlu, which has poor affinity for the folate receptor, did not affect either AP binding or AP-directed transport of 5-CH3-H4PteGlu (Fig. 7). Finally, colchicine, which disrupts the vesicular trafficking that takes place during endocytic processes (Brown et al. 1991), significantly inhibited both AP binding and AP-mediated transport (Fig. 3). Since the folate receptor-mediated transport is thought to occur via an endocytic uptake into vesicles, with recycling of the folate receptor to the AP membrane via dense apical tubules (Birn et al. 1993), these results suggest that the AP-mediated transport of 5-CH3-H4PteGlu occurs to a major extent by a folate receptor-mediated endocytosis.
, Wang et al. 1992), these data indicate the involvement of additional 5-CH3-H4PteGlu transport pathways that were not saturated at such low concentrations. Saturation of 5-CH3-H4PteGlu transport by the reduced folate carrier is generally reported in the 100-1000 nmol/L range (Antony 1992, Henderson et al. 1980), which is consistent with these results. Furthermore, although PteGlu blocked the binding of 5-CH3-H4PteGlu completely, it inhibited the AP-mediated transport by only 50%, suggesting that another pathway was responsible for the remaining 50% of transport. The studies with probenecid, a known inhibitor of renal anion exchange processes (Pritchard and Miller 1993), suggest that the additional pathway for AP-mediated 5-CH3-H4PteGlu transport would be the reduced folate carrier, which is an anion exchange mechanism known to be inhibited by probenecid (Fort et al. 1993, Kamen et al. 1991). In the HPT cells, probenecid significantly decreased the AP-mediated transport of 5-CH3-H4PteGlu by about 50%, without altering AP binding (Fig. 4).
). The present studies showed no inhibition of AP binding at 10 mmol probenecid/L, which significantly decreased AP transport. More importantly, the studies of the combination of probenecid with PteGlu or with colchicine demonstrated that the inhibition of AP transport by probenecid occurred because of an effect on the reduced folate carrier rather than the folate receptor. In the presence of PteGlu which would completely block the folate receptor, probenecid increased the inhibition of AP transport from about 50% to 90% (Fig. 6). Similarly, probenecid exacerbated the effects of colchicine on transport (Fig. 5), presumably through a pathway independent of the folate receptor-mediated endocytosis. In both of these cases, probenecid did not further inhibit the binding of 5-CH3-H4PteGlu. These results suggest that both the folate receptor and the reduced folate carrier participate in the AP-directed transport of 5-CH3-H4PteGlu into HPT cells.
, Middleton et al. 1989). In contrast, most previous cell culture studies of folate transport have been carried out in cells such as the KB, MA104 or L1210 cell, which are tumor or transformed cell lines (Antony 1992, Henderson et al. 1980, Kamen et al. 1991) and whose transport systems may be different from normal tissue. The folate receptor activity in KB and MA104 cells is overexpressed (Antony 1985, Rothberg et al. 1990), similar to the reported overexpression of the folate receptor in numerous carcinomas (compared to the corresponding normal tissue) (Ross et al. 1994). The folate receptor seems to be lacking in L1210 cells, although clonal adaptation can produce sublines that express higher levels of the folate receptor (Henderson et al. 1988). The high folate receptor activity in cell lines could possibly obfuscate evidence for transport by the reduced folate carrier. For example, a study in normal human lymphocytes by Fort et al. (1993) suggested primarily a reduced folate carrier-mediated uptake. The HPT cells may utilize both the folate receptor and the reduced folate carrier for folate uptake because the folate receptor is not overexpressed in normal cells in culture.
, Middleton et al. 1989) to be the minimal medium necessary for proper growth and differentiation of HPT cells. This media contained micromolar concentrations of folate (McMartin et al. 1992), while most of the 5-CH3-H4PteGlu transport studies in cell lines have been carried out after adapting the cells to media of folate concentration in the 10 nmol/L range [these concentrations approximate those in normal human plasma (McMartin et al. 1986) so are considered physiological]. The ideal system would be to examine folate transport in HPT cells adapted to media with lower folate concentrations, but whether the cells would survive in such media and differentiate properly has not been determined. However, previous studies have shown that exposure of HPT cells to normal growth media does not increase cell folate content, which was about 300 pmol/g cell protein in freshly isolated cells and about 150 pmol/g protein in cells cultured through two passages (Morshed and McMartin 1996b). Also before uptake studies, cells are removed from the growth media and washed thrice so that the experiments are conducted with physiological amounts of 5-CH3-H4PteGlu externally. Although cellular folate accumulation does not occur with growth in high folate-containing media, such pre-exposure may result in a down-regulation of the folate receptor and an increased reliance on the reduced folate carrier for folate transport in these HPT cells.
). In contrast, recent studies using isolated perfused rat kidneys in vitro suggested that reabsorption occurred primarily via nonspecific pathways, with little physiological role for the folate receptor (Muldoon et al. 1996). For example, in microinfused rat proximal tubules (Selhub et al. 1987), uptake of PteGlu could be inhibited by 5-CH3-H4PteGlu, whereas in the perfused rat kidney, 5-CH3-H4PteGlu reabsorption was not inhibited by PteGlu. In cultured HPT cells, 5-CH3-H4PteGlu transport was inhibited by about 50% by PteGlu, suggesting a major, but not exclusive role for the folate receptor (similar results have been observed in preliminary studies in cultured rat proximal tubule cells). Thus, the results in cultured cells and in vivo suggest the importance of the folate receptor, while those in perfused kidneys do not. The reasons for these differences are not known, but may be related to the high flow rates in closed perfusion systems (Henderson et al. 1995).
), a colon carcinoma cell line. Previously Corrocher et al. (1985) have reported the presence of specific folate binding proteins in isolated AP and BL membranes from rat kidneys. In their studies, the AP binding was also about three times the magnitude of the BL binding. The functional significance of the BL binding is not yet known. As noted above, the binding does not seem to play much of a role in the BL-directed transport of 5-CH3-H4PteGlu into the HPT cell. Another interesting aspect of the binding studies was the observation that significant amounts of 5-CH3-H4PteGlu were relatively rapidly bound to the AP folate receptor, even when the 5-CH3-H4PteGlu was added to the BL compartment. Since the HPT cells formed an exceptionally tight monolayer when grown on these membrane inserts (Morshed and McMartin 1995), there was minimal leakage of 5-CH3-H4PteGlu paracellularly (Morshed and McMartin 1996b). Hence, the 5-CH3-H4PteGlu that appeared bound to the AP membrane was taken up across the BL membrane and transferred through the cell to the AP membrane. One explanation for this phenomena would be bidirectional transport across the AP membrane by the reduced folate carrier, followed by binding of the secreted 5-CH3-H4PteGlu to the AP membrane. Alternatively, the 5-CH3-H4PteGlu-folate receptor complex could be created intracellularly after BL uptake, then recycled to the AP membrane as in the usual endocytic recycling. The opposite process (binding of AP-directed 5-CH3-H4PteGlu to the BL membrane) also occurred but to a much smaller extent. The importance and the mechanisms of these "reverse" bindings are not clear.
FOOTNOTES
Manuscript received 9 September 1996. Initial reviews completed 4 October 1996. Revision accepted 30 January 1997.
LITERATURE CITED
-mediated transport and reduced folate carrier-mediated transport in L1210 leukemia cells.
J. Biol. Chem.
1995;
270:7842-7849
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