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Department of Biological Sciences, ‘Neuroscience Solutions to Cancer’ Research Group, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2AZ, UK
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(Received 7 May 2004;
accepted after revision 9 July 2004;
first published online 14 July 2004)
Corresponding author M. B. A. Djamgoz: Department of Biological Sciences, Imperial College London, Sir Alexander Fleming Building, South Kensington Campus, London SW7 2AZ, UK. Email: m.djamgoz{at}imperial.ac.uk
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Introduction |
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7 mM Zn2+ and high levels of polyamines and myo-inositol (Lynch & Nicholson, 1997). On the other hand, blood contains 135 µM citrate, but this is one of the highest among the related intermediates of the tricarboxylic acid cycle (Inoue et al. 2002b). Intracellular citrate is important for generation of energy as well as synthesis of fatty acids, isoprenoids and cholesterol (Inoue et al. 2002a). A variety of cells have transporters that enable absorption of citrate from body fluids, as in intestine, kidney (Pajor, 1999), placenta, liver and brain (Inoue et al. 2002a). These are Na+-coupled dicarboxylate (NaDC) transporters which exist in two main isoforms: NaDC1, which has a low affinity, and NaDC3, which has a high affinity for succinate and other dicarboxylates (Bai & Pajor, 1997). Another Na+-dependent transporter, Na+-coupled citrate transporter (NaCT), isolated from rat brain, enables citrate uptake with high affinity (Inoue et al. 2002b). At present, the ionic mechanism(s) involved in the transport of citrate out of prostatic epithelial cells into the lumen is not known. In the present study, we have used the prostatic epithelial cell line PNT2-C2, derived originally from normal human prostate and immortalized by TSV40 transfection (Cussenot et al. 1991). These cells are thought to represent highly differentiated luminal epithelia (e.g. Lang et al. 2000, 2001; Usmani et al. 2002). Whole-cell patch clamp techniques were used both to introduce citrate into the cells and to record electrophysiologically the membrane current resulting from its transmembrane transport.
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Methods |
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The PNT2-C2 cells were grown in Roswell Park Memorial Institute (RPMI)-1640 medium, supplemented with 10% fetal calf serum and 2 mML-glutamine, in 10 cm Petri dishes maintained in a humidified incubator with 5% CO2 at 37°C. Three days before the patch-clamp recordings, the cells were re-plated into 2 cm Petri dishes at a density of 5 x 104 per dish. Three other normal human epithelial cell lines, as follows, were also studied for comparison: MCF-10A (breast), 16HBE-140 (lung) and HEK-293 (kidney), cultured as described before (Soule et al. 1990; Mulier et al. 1998; Benton et al. 2003).
Electrophysiology
Prior to patch-clamp recording, the growth medium was replaced with an external bath (EB) solution containing (mM): NaCl (118), NaHCO3 (26), KCl (5.4), MgCl2 (1), CaCl2 (2.5), D-glucose (5.6) and Hepes (5), pH 7.2, adjusted with 1 M NaOH or 1 M HCl. Patch pipettes were prepared and recordings were performed as described earlier (Grimes et al. 1995). The holding potential was –45 mV unless stated otherwise. Citrate was dialysed into the cells by adding 0.5 or 10 mM sodium or potassium citrate salt to the patch pipette solution. The same procedure was used for intracellular application of other reagents, such as Zn2+, NaCl, glutamate and other Krebs cycle intermediates. All chemicals were purchased from Sigma (Poole, UK) except 9-AC, lonidamine and SCH28080 which were purchased from Tocris Cookson (Bristol, UK), TTX, from Alomone (Towcester, UK), and RIPA from Upstate (Milton Keynes, UK).
Enzyme spectrophotometry
Cellular uptake and release of citrate were measured by the spectrophotometric ‘citrate lyase method’ described earlier (Petrarulo et al. 1995). For the uptake experiments, PNT2-C2 cells were plated in Petri dishes at a density of 3 x 104 cm–2, grown for 3 days, washed carefully and then incubated with 10 mM sodium citrate for 30 min. After thorough washing with EB solution, the cells were digested in lysis (RIPA) buffer (100 µl dish–1). The citrate content of the medium, containing the cellular uptake, was determined as before (Petrarulo et al. 1995). Absorption of 330 nm in the medium was checked before citrate lyase was added to the cuvettes and this reading was subtracted from the final value read after addition of the enzyme. For the release experiments, the cells were plated in 96-wells dishes at a density of 3 x 104 cm–2 and grown for 3 days. They were washed and incubated in EB solution containing 10 mM sodium citrate for 30 min. The cells were washed carefully and incubated in 50 µl well–1 EB solution (normal and modified ionic content) for 5–60 min. Absorbances in all EB solutions used (not exposed to cells) were read and found to be insignificant. Supernatant was collected and citrate content determined as before (Petrarulo et al. 1995).
Ionic substitutions
Effects of different ions on citrate transport were studied by applying extracellular solutions with modified ionic content, whilst (i) dialysing the cells with intracellular citrate in steady-state or (ii) pulsing the cells with extracellular citrate (10 mM). Any effect was compared with control data obtained from experiments performed on cells patched with normal intracellular pipette solution for (i), or without extracellular citrate for (ii), respectively. Details of the extracellular solutions with modified ionic content used in the electrophysiological and the spectrophotometric experiments are given in Table 1 and respective figure legends.
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The stoichiometry of the K+ dependence of inward citrate transport was determined as described earlier (Chen et al. 1998), based upon the following equation:
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Data analysis
Data were analysed as means ± standard error of the mean. Each measurement was made on a minimum of seven cells. Change in a parameter of interest is indicated as ‘
’ expressed as either a percentage change, defined as follows:
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Results |
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Cells had a normal resting potential of –36 ± 1.9 mV. At the holding potential of –45 mV, membrane currents recorded with normal patch pipettes were very steady (Fig. 1A). In contrast, cells patched with pipettes containing citrate revealed a prominent outward current (Icit) that developed slowly, reached a steady-state value after about 1 min and remained stable for several minutes (Fig. 1A). Citrate applied similarly to normal human breast (MCF-10A), lung (16HBE-140) and kidney (HEK-293) epithelial cell lines produced no current (Fig. 1A). Spectrophotometric analysis showed that PNT2-C2 cells increased their citrate content by some 15% (P < 0.05) when incubated in 10 mM sodium citrate for 30 min (Fig. 1B), whilst the citrate content of the incubation solution decreased (data not shown). Citrate was released from the cells preloaded with sodium citrate, the amount of the efflux increasing with time (Fig. 1C). Readings from the EB solution not exposed to cells were insignificant.
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The dose dependence of Icit was tested in the concentration range 0.01–10 mM intracellular citrate (Fig. 1G). The current was dose dependent up to 1 mM. Further increase in the citrate concentration to 10 mM caused no further statistically significant change.
Inhibitor profile of Icit
The effects of a range of membrane transport inhibitors on Icit were tested for the two working concentrations of intracellular citrate: 0.5 and 10 mM (Fig. 2A). Phloretin increased Icit whilst diethyl pyrocarbonate (DEPC) and LiCl decreased it; none had any effect on the control membrane current. Following application of ouabain for 1 min, there was a decrease in response to citrate but this was statistically the same as in control recordings. Similar application of dinitrophenol (DNP), amiloride, tetrodotoxin (TTX) or SCH28080did not affect the membrane currents of cells under control conditions or with intracellular citrate present.
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In order to elucidate the ion(s) coupled to outward citrate transport, solutions with altered ionic content were applied extracellularly and their effects on Icit and control membrane current (i.e. no intracellular citrate present) were compared. Changing the concentrations of Na+ and Mg2+ had no influence on the membrane currents recorded under control conditions or with 0.5 mM intracellular citrate present. On the other hand, the cells were sensitive to changes in extracellular Cl–, Ca2+ and K+. Importantly, however, it was only for K+ that there was a significant difference in the membrane currents recorded under control conditions and with 0.5 or 10 mM intracellular citrate. Thus, with 0.5 or 10 mM intracellular citrate present, increasing the extracellular K+ concentration ([K+]o) reduced Icit significantly in a concentration-dependent log-linear manner, the slopes of the changes being similar, 34.4 and 39.8 pA per 10-fold change in [K+]o for 0.5 and 10 mM intracellular citrate, respectively (Fig. 2C and D). The dependence of Icit on the K+ gradient was seen also by applying the K+ channel blocker 4-aminopyridine (4-AP), which reduced Icit (Fig. 2A). As already noted (Fig. 1F), the role of the K+ gradient in citrate transport was apparent also when comparing the membrane currents produced by K+versus Na+ salts of citrate. The K+ dependence of citrate uptake and release was also demonstrated in spectrophotometric measurements. Thus, in high-K+ medium, uptake of citrate was increased (Fig. 1B) whilst release was reduced (Fig. 1C). In contrast, Na+ had no effect on the uptake or release of citrate (Fig. 1B and C).
Thus, the various, independent lines of evidence were highly consistent in showing that Icit, as well as citrate uptake and release, depended upon the transmembrane K+ gradient.
pH sensitivity of Icit
The pH sensitivity of Icit was studied by three different experimental approaches, as follows (Fig. 2E and F).
From these experiments, it was concluded that Icit was sensitive particularly to acidification of pHi and not affected by changes in pHo.
Membrane current induced by extracellular citrate
Application of extracellular sodium citrate (10 mM) caused an inward current (Icit/in) of –40 ± 8 pA (Fig. 3A). This current reversed at 1.7 ± 1.3 mV (Fig. 1E); there was no difference between this and the reversal potential of Icit. No desensitization was seen when extracellular citrate was applied repeatedly to the same cell (tested up to 5 times) or its application was maintained (up to 5 min). The voltage dependences of currents induced by pulses of 5 or 10 mM extracellular citrate (with equimolar intracellular citrate present) were linear but with significantly different reversal potentials: –7.9 ± 1.3 and 8.4 ± 2.1 mV, respectively (P < 0.001) (Fig. 3C). Icit/in had the same ionic dependence as Icit. First, potassium citrate was more effective than equimolar sodium citrate (Fig. 3D). Second, Na+, Cl–, Ca2+ and Mg2+ had no involvement (Fig. 3Ba and c and E) whilst increasing the extracellular K+ concentration had a strong, dose-dependent enhancement effect on Icit/in (Fig. 3Ba and b and E). Acidification of the extracellular bath (pHo 6.5) reduced Icit/in significantly whilst alkalinization (pHo 8) had no effect on the citrate current (Fig. 3F). The inhibitor profile of Icit/in was also very similar to that of Icit: (i) phloretin slightly increased the citrate uptake, (ii) DEPC and Li+ were inhibitory, and (iii) ouabain, DNP and amiloride had no effect and 4-AP blocked Icit/in to the same extent as Icit (Fig. 2A).
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Effects of anion channel and transporter inhibitors
The effects of several anion transporter inhibitors on Icit/in and resting Cl– conductance, monitored by measuring the Cl– current (ICl) induced by lowering the extracellular Cl– concentration 10-fold, were also tested (Fig. 2B). 4,4'-Diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and diphenylamine-2-carboxylate (DPC) had no effect on Icit/in or ICl. Importantly, however, (i) 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) significantly reduced Icit/in but had no effect on ICl, whilst (ii) ICl was significantly reduced by anthracene-9-carboxylic acid (9-AC) and lonidamine but these inhibitors had no effect on Icit/in (Fig. 2B). These results suggested that the bulk of citrate transport did not involve a Cl– channel.
K+ stoichiometry
Pulses of 10 and 5 mM citrate salt solutions were applied extracellularly whilst the cell membrane potential was held at different values in the range –90 to 10 mV (Fig. 4A). From these data, the stoichiometry equation was solved, giving the value of s as 3.9 ± 0.2. A similar protocol was applied to study the stoichiometry in reverse, i.e. to determine the number of citrate ion(s) transported for 1 K+. In this case, the intracellular patch pipette contained 0.5 mM citrate whilst 0.05 mM citrate was added to the extracellular solution. High concentrations of K+ (54 and 108 mM) were then applied extracellularly and current–voltage relationships were determined as before (Fig. 4B). This analysis gave a value of 0.23 ± 0.1 for the number of citrate ions transported together with 1 K+.
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Effect of intracellular EDTA on citrate transport
The possible involvement intracellular cationic (e.g. Ca2+, Mg2+ and Zn2+) chelation in the membrane currents elicited by citrate (intracellular or extracellular) was studied by applying intracellular EDTA (1 mM). First, application of intracellular EDTA by itself induced an inward current (–59 ± 19 pA), opposite of the effect of intracellular citrate (Fig. 5A and C). Second, the amplitude of Icit did not change significantly: 180 ± 20 (normal) versus 141 ± 25 pA (EDTA) (P > 0.05) (Fig. 5C); and the time course remained the same (Fig. 5B). Finally, the amplitude of Icit/in did not change: –44 ± 1.7 (normal) versus–41 ± 1.9 pA (EDTA) (P > 0.05) (Fig. 5D).
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The possibility of there being basal m-aconitase activity was studied by applying intracellular Zn2+, which would inhibit the enzyme and thus block citrate oxidation. Inclusion of Zn2+ in the patch pipette induced a delayed outward current of 50 ± 9 pA. This current was reduced when [K+]o was increased in log-linear function, the slope being 31.4 pA per 10-fold change in [K+]o. The opposite effect was obtained when N,N,N',N'-tetrakis(2-pyridylmethyl) etylenediaminepentaethylene (TPEN; 50 µM), a membrane-permeant Zn2+ chelator, was applied extracellularly. This generated an inward current of –16 ± 11 pA. There was no effect of the control solvent DMSO (0.08%). These effects were consistent with PNT2-C2 cells having basal Zn2+-sensitive m-aconitase activity controlling cellular production of citrate and its transport outward.
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Discussion |
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We have used a prostatic epithelial cell line derived originally from normal human prostate gland and immortalized by TSV40 antigen transfection (Cussenot et al. 1991). PNT2-C2 cells are thought to be highly differentiated luminal epithelia (Lang et al. 2001) that would be a good model for the physiology of citrate, which is released from epithelial cells into the lumen in the prostate gland. Although as an immortalized cell line, PNT2-C2 cells may not be wholly normal, this cell line has been used extensively as a model of normal prostate epithelia, and reacted to the application of intracellular and extracellular citrate in a highly consistent manner. This was confirmed by independent, complementary spectrophotometric experiments in which citrate uptake and release were measured inside cells and supernatant, respectively. These data, taken together, suggest that PNT2-C2 cells are able to transport citrate in either direction, outward transport being some 4 times more efficient than inward.
In response to depolarizing voltage pulses, an outward (K+) current was generated. Other studies carried out on normal rat prostatic cells also have shown the presence of K+ channels (Ouadid-Ahidouch et al. 1999; Kim et al. 2002). In contrast, a voltage-activated Na+ current, generated by rat and human prostate cancer cells (Grimes et al. 1995; Laniado et al. 1997), was absent. Of the extracellular ions tested under control conditions, only K+, Cl– and Ca2+ produced a membrane current, whilst reduction in Na+ or Mg2+ had no effect. Similar results were obtained from epithelial cells of normal rat prostate (Mycielska et al. 2003). These data are consistent with the cells having significant K+ permeability at rest.
Characteristics of Icit
Time course. Application of intracellular citrate resulted in a slowly developing outward current which reached a plateau within about 1 min. This delay is likely mainly to be due to the time necessary for complete intracellular dialysis, estimated to take around 1 min on average (e.g. Higure et al. 2003). However, we cannot exclude the possibility that there might be an intermediate step needed to activate the citrate transporter to its full extent.
K+ and lack of Na+ sensitivity. The release and uptake of citrate were found to be sensitive particularly to the transmembrane K+ gradient. First, increasing the extracellular K+ concentration significantly affected citrate transport in either direction (in both electrophysiological and spectrophotometric experiments). None of the other common inorganic ions had any effect (except H+, but see section below). Second, potassium citrate (intracellular or extracellular) was more effective than sodium citrate. Furthermore, the K+ channel blocker 4-AP slightly but significantly decreased the citrate current. The effect of phloretin was also interesting, since this agent increasedIcit (and Icit/in). This can be explained by assuming that phloretin enhanced K+ activity by activating Ca2+-activated K+ (BK) channels, as in Aplysia neurones (Zhang et al. 2002). Similar channels were found in rat ventral prostate epithelial cells (Kim et al. 2003). In all other citrate and dicarboxylate transporters described to date, Na+ was cotransported into cells (e.g. Inoue et al. 2002a,b; Hering-Smith et al. 2000). In the case of the prostatic citrate transporter, however, Na+ had no direct role and amiloride, which is known to block epithelial Na+ channels, had no effect. The inhibitory effect of 4-AP on the citrate current would suggest that a K+ channel may be a part of a regulatory pathway involved in recycling the citrate-coupled K+ efflux. In addition, H+–K+-ATPase has been localized to the basal membrane and could pump K+ into the cells (Mobasheri et al. 2003).
These data emphasize a key role for K+, consistent with the direction of citrate transport in the prostate, i.e. out of epithelial cells into the lumen. In turn, this could explain why the concentration of K+ in prostatic fluid (65 mM; Kavanagh, 1985), is some 10-fold higher than in blood (4 mM, e.g. Overgaard et al. 2002).
Zn2+ sensitivity. Application of intracellular Zn2+ caused an outward current that developed over 2 min; the opposite was seen by chelating intracellular Zn2+. The outward current could be due to Zn2+ efflux through a membrane channel or transporter. Such mechanisms have been described in many systems (Borovansky et al. 1997; McMahon & Cousins, 1998) and it was also found that prostate cancer cells (LNCaP and PC-3) express ZIP1, a Zn2+ transporter (Franklin et al. 2003). In the case of PNT2-C2 cells, however, the Zn2+-induced current was delayed and was sensitive to [K+]o, similar to Icit/Icit/in. This is consistent with (i) the Zn2+-induced outward current being due to the activity of the citrate transporter, including being K+ sensitive, and (ii) there being a significant level of basal citrate production due to Zn2+-induced suppression of m-aconitase (Costello & Franklin, 2000).
pH sensitivity. PNT2-C2 cells were sensitive to changes in pHo and pHi. The biggest effects were seen by alkalization of pHo and acidification of pHi. These effects were consistent with the effect of pH on the valency of citrate (96–98% trivalent at pH > 7.1 but mainly divalent at pH < 7; Hering-Smith et al. 2000) and the prostatic transporter preferring the trivalent form of citrate that would be dominant at the prevailing physiological pHi (Hering-Smith et al. 2000). A citrate transporter (NaCT) has recently been characterized in brain cells and shown to have much higher affinity for the trivalent form (Inoue et al. 2002a,b). In PNT2-C2 cells, change in pHo (in either direction) did not affect citrate extrusion from cells. On the other hand, acidifying pHo significantly reduced citrate uptake (Icit/in) whilst alkalization had no effect. Application of weak acid (propionic acid), which has been shown to acidify pHi (Szatkowski & Thomas, 1989), and hence increase the tendency for citrate to be divalent, resulted in net inward current, consistent with a decrease in Icit. A similar result was obtained by acidifying pHi with extracellular NH4Cl. These effects are consistent with the prostatic citrate transporter strongly favouring the trivalent form of citrate. Prostate epithelial cells produce, accumulate and release very high levels of citrate, so it seems necessary for the transporter to have high affinity for the most common, trivalent form of citrate at physiological pHi.
Lack of effect of intracellular ion chelation
To test the possibility that the observed citrate-induced currents might involve intracellular ionic chelation (Westergaard et al. 1994), cells were dialysed with EDTA, known to chelate several divalent ions including Ca2+, Mg2+ and Zn2+ (Sakabe et al. 1994). Application of intracellular EDTA caused an inward current, which could be due to chelation of intracellular Zn2+, since TPEN had the same effect. This is opposite of Icit and hence could not explain the main outward current produced by application of intracellular citrate. Furthermore, Icit and Icit/in were not affected by intracellular EDTA (Fig. 5). It seemed highly unlikely therefore that chelation of the main intracellular cations could explain the membrane currents produced by intracellular or extracellular citrate.
Ion channel versus transporter?
Some studies have suggested that citrate can be transported through Cl– channels (e.g. Miyamoto et al. 1998). Although PNT2-C2 cells were found to have some resting Cl– conductance, its partial blockage by 9-AC and lonidamine had no effect on Icit/in, and retrospectively, suppressing Icit/in by NPPB did not affect ICl (Fig. 2B). These results would suggest that a Cl– channel could not solely be responsible for the citrate-induced currents. Another evidence that supports this comes form the shift in the reversal potential of the currents induced by different concentrations of extracellular citrate (at fixed transmembrane concentration gradients). It was concluded therefore that an ion channel could not be the primary mechanism mediating the transport of citrate.
Outward versus inward transport of citrate, and K+ stoichiometry
Applying extracellular citrate elucidated an inward current, Icit/in. The amplitude of this current was only some 25% of the outward current, Icit, generated by equimolar intracellular citrate. Icit/in could be due to (i) decreased basal citrate release caused by the excess external citrate; (ii) the outward citrate transporter being forced to work in reverse; or (iii) activity of a totally different, inward citrate transporter. Importantly, all characteristics of Icit and Icit/in tested (ionic, including pH dependence, inhibitor profile, reversal potential) were essentially the same for the two currents. In particular, both currents were highly sensitive to change in the transmembrane K+ concentration gradient. Such close similarity would make (iii) seem unlikely. The possibilities (i) and (ii) could not readily be distinguished with the available data. Since the reduction in Icit/in caused by TPEN was only 25%, the basal production and outward transport of citrate could be much smaller than the amount of citrate which can be taken up when introduced extracellularly, thereby favouring hypothesis (ii), i.e. that the transporter can work in reverse. Many other transmembrane transporters, such as the Na+–Ca2+ exchanger, even the Na+–K+-ATPase and the 
-aminobutyric acid transporter are known to function bidirectionally (Wu et al. 2003). Interestingly, this ability may relate to prostate cancer where the epithelial cells release much less citrate and may even revert to transporting citrate mainly inward (Costello et al. 1999). Nevertheless (i) and (ii) would both represent the activity of the same citrate–K+ cotransporter. Our analyses of stochiometry, taking either K+ or citrate as the ‘driving’ ion, suggested consistently that the transporter carries 1 citrate anion together with 4 K+. We cannot rule out the possibility of there being a minor Na+-dependent uptake mechanism removing citrate from blood.
Concluding remarks
We conclude that normal prostatic epithelial cells possess a citrate transporter which mediates the outward cotransfer of one trivalent citrate anion alongside 4 K+ at physiological pH and thus generates a net current outward. This citrate transporter is novel by virtue of its K+ dependence and association with prostate epithelia. The transporter would work more efficiently in the outward direction, as normal, releasing citrate into extracellular space/prostatic fluid, where its concentration may reach 180 mM (Kavanagh, 1994). Consequently, the transporter current can be recorded by introducing excess citrate into the cell from the whole-cell patch pipette. Our previous recordings from intact pieces of rat prostate showed that prostate possessed a negative lumen potential that could facilitate the activity of the transporter (Mycielska et al. 2003). It is likely that the K+ ions transported alongside citrate are regulated by a voltage-gated K+ channel (Fig. 3A) and/or other counter-balancing ion transporters (e.g. Na+–K+-ATPase, H+–K+-ATPase) also known to be expressed in prostatic epithelia (Mobasheri et al. 2001, 2003).
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