Introduction

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Examination of renal transporters of organic cations has been carried out almost exclusively with radioactive substrates (Boom et al., 1992; Hohage et al., 1994b, 1996). Besides the difficulties in the handling of radioactive substances in general, a further disadvantage is given by the limitation of online monitoring of transport processes in living cells with this approach.

Ullrich et al. established a new technique for the analysis of the renal transport of organic cations with the fluorescent cation (ASP⁺) (Rohlicek and Ullrich, 1993; Pietruck and Ullrich, 1995). The study of dynamics of both the transport rate and potential effects of other substances on the cation transporter is possible with this dye. Thus, an increase in cellular fluorescence after addition of ASP⁺ to the incubation medium can be used to study the dynamics of transport via the renal organic cation transporter.

Our previous work demonstrated that PKC stimulates the transport of organic anions and organic cations across basolateral membranes of freshly isolated S2 segments of rabbit kidney proximal tubules (Hohage et al., 1994a, b). In this study, LLC-PK₁ and IHKE-1 cells were used as experimental models. LLC-PK₁ cells from the pig are of renal proximal origin (Hull et al., 1976) and are accepted widely as a model to study transport processes in the proximal tubule. LLC-PK₁ cells absorb and transport tetraethylammonium and cimetidine via transport systems for organic cations (Saito et al., 1992; Fouda et al., 1990; McKinney et al., 1988; Bendayan et al., 1994). No information on regulation of this transport is available. In addition, human IHKE-1 cells recently have become available to investigators and show several characteristics of proximal tubules (Tveito et al., 1989; Jessen et al., 1994; Hirsch et al., 1998).

In the present study, the effects of protein kinase activation on transport of ASP⁺ in LLC-PK₁ and IHKE-1 cells were investigated by use of dynamic fluorescence microscopy.

	Methods

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Cell culture. LLC-PK₁ cells (kindly provided by Dr. M. Mohrmann, Kinderklinik Universität Freiburg, Germany) were grown by serial passages (187-200) in 50-ml tissue culture flasks (Greiner, Frickenhausen, Germany). The cells were fed with Dulbecco's modified Eagle's medium (Biochrom, Berlin, Germany) supplemented with 0.5 mM L-glutamine (Gibco BRL/Life Technologies, Eggenstein, Germany), 100,000 U/l penicillin and 100 mg/l streptomycin (Biochrom). In addition, the medium contained 10% fetal calf serum (Biochrom), as published before (Kleta et al., 1995).

ASP⁺ fluorescence measurements. Fluorescence microscopy experiments were performed with an inverted microscope (Axiovert 135, Zeiss, Oberkochen, Germany) equipped with an oil immersion objective (100×/1.3). The excitation light was generated by a xenon-quartz lamp (XBO 75W, Zeiss). A filter wheel (Physiologisches Institut, Universität Freiburg, Germany) rotating at 10 Hz and loaded with a 450- to 490-nm filter provided a pulsating excitation light. The excitation light was reduced by a grey filter to lower possible quenching of the dye. An iris diaphragm allowed the reduction of the area of measurement to ~40 µm² covering about five cells. The excitation light was reflected by a dichroic mirror (560 nm) to the perfusion chamber in which the cells grown on cover slips formed the bottom. The perfusion rate was held at 10 to 12 ml/min corresponding to an exchange rate of about 20/min with a HCO₃-free Ringer's-like solution (see below, 37°C; pH 7.4). The fluorescence signals of ten pulses every second were averaged and plotted as a function of time. Fluorescence emission, passing through a band pass filter of 575 to 640 nm, was measured with a photo multiplier tube (Hamamatsu H 3460-04, Herrsching, Germany). Experiments were controlled and data analyzed with an AT-486 computer system and specific software (U. Fröbe, Universität Freiburg, Germany). The ASP⁺ concentration in the superfusate was 1 µmol/l. At this concentration the uptake of ASP⁺ was linear for at least 10 min (Stachon et al., 1996). Because ASP⁺ fluorescence is bleached readily by light in aqueous solutions, the whole device was protected from light. Under these conditions, no significant bleaching was observed (Stachon et al., 1996). The uptake of ASP⁺ was measured as an increase in cellular fluorescence with time. Experiments generally were performed for 10 min, where the first 5 min served as the control and the last 5 min as the experimental period. The uptake rate during 100 to 200 s (control) was compared with that during 500 to 600 s (experimental). Background fluorescence of about 80 counts/s was mostly caused by ASP⁺ in solution and thus stayed constant throughout the experiments. The background fluorescence was subtracted from every experiment. Effects of agonists are given in percent changes, as absolute uptake rates varied considerably between monolayers.

Fluorescence measurements of [Ca⁺⁺]_i. Cellular Ca⁺⁺ concentrations ([Ca⁺⁺]_i) of IHKE-1 cells were measured with the Ca⁺⁺-sensitive dye fura-2 as described previously (Schlatter et al., 1995; Ankorina et al., 1997). Cells were incubated with the acetomethyl ester of fura-2 (fura-2-AM, 5 µmol/l, Sigma) dissolved with 0.1 g/l pluronic F-127 (Calbiochem) in standard solution for 40 min at 37°C in the dark. The incubation was followed by an equilibration period of 15 min while superfusing the cells with control solution at 37°C. Loaded cells were excited at 340, 360 and 380 nm with a filter wheel (Physiologisches Institut, Universität Freiburg, Germany) rotating at 10 Hz and a xenon-quartz lamp (XBO 75 W, Zeiss) as light source. Fura-2 emission was recorded at 500 to 530 nm with a single photon counting tube (H3460-04, Hamamatsu, Herrsching, Germany). Measurements were taken from approximately five cells with the aid of an iris diaphragm. The ratio of the emissions after excitations at 340 and 380 nm at 10 Hz was calculated and 10 consecutive datapoints were averaged yielding a time resolution of 1 Hz. Signal noise and autofluorescence were measured before loading the cells with fura-2-AM and subtracted from the measured signals for each experiment. Experiments were controlled and data analyzed with an AT-486 computer system and specific software (U. Fröbe, Universität Freiburg, Germany). Calibration of [Ca⁺⁺]_i was done at the end of each experiment by incubating the cells with the Ca⁺⁺ ionophore ionomycin (1 µmol/l, Sigma) in the presence (1.3 mmol/l) and nominal absence of Ca⁺⁺ [with 5 mmol/l ethyleneglycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid present] according to standard methods (Grynkiewicz et al., 1985).

Materials. ASP⁺ was obtained from Molecular Probes (Eugene, OR). ASP⁺ is positively charged at the physiological pH of 7.4, because of a pK_a of 13.6 (Pietruck and Ullrich, 1995). ASP⁺ has a low lipophilicity (n-octanol/H₂0 ratio, 0.14) (Irion et al., 1993) and does not permeate membranes significantly. As standard superfusate solution a HCO₃-free Ringer's-like solution containing (in mM): NaCl, 145; K₂HPO₄, 1.6; KH₂PO₄, 0.4; D-glucose, 5; MgCl₂, 1; and Ca-gluconate, 1.3 was used. All chemicals were obtained from Merck or Sigma. Human ANP (hANP) was a kind gift of the Niedersächsische Institut für Peptidforschung, Hannover, Germany. ATP, bradykinin, oxytocin, angiotensin II, 1,2-dioctanoylglycerol, 8-Br-cAMP and 8-Br-cGMP were obtained from Sigma.

Statistics. Data are represented as mean values ± S.E., N denotes the number of monolayers used in each series. Statistical analysis was done with a two-sided paired t test, and a P value of < .05 was considered to be statistically significant.

	Results

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ASP⁺ uptake under control conditions was similar in IHKE-1 cells (0.52 ± 0.02 count, n = 212) and in LLC-PK1 cells (0.77 ± 0.05 count, n = 114). Stimulation of PKC previously was found to activate organic anion and cation transport in proximal tubules (Hohage et al., 1994a, b). In our study, 1,2-dioctanoylglycerol, a membrane-permeable diacylglycerol analog, modulated ASP⁺ accumulation in both cell lines at a concentration of 1 µmol/l. In IHKE-1 cells uptake was increased significantly by 35 ± 9% (figs. 1 and 2), whereas in LLC-PK₁ cells ASP⁺ accumulation significantly decreased by 20 ± 7% (fig. 2). Figure 1 shows the linearity of ASP⁺ uptake under control conditions and after stimulation with forskolin and diacylglycerol.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Original recordings of ASP+ uptake and the effects of forskolin and diacylglycerol on uptake rate.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent changes in ASP+ accumulation by DOG in IHKE-1 (circle) and LLC-PK1 cells (square). Data are presented as changes in fluorescence increase expressed in percentages of the paired controls. Mean value ± S.E.M. Numbers in parentheses represent the number of individual experiments. (*) indicates statistical significance of the effect (P < .05).

Figure 3 shows the effects of various agonists on ASP⁺ accumulation. In IHKE-1 cells a significant stimulation of ASP⁺ accumulation was observed upon addition of each of three stimulators of the phospholipase C [ATP 0.1 mmol/l (65 ± 30%), oxytocin 0.1 µmol/l (70 ± 20%) and bradykinin 1 µmol/l (66 ± 14%)]. Angiotensin II (0.1 nmol/l) did not modulate ASP⁺ accumulation (ASP⁺ accumulation, 6 ± 8%, n = 6) nor cellular Ca⁺⁺ activity significantly (see table 2). However, in LLC-PK₁ cells addition of angiotensin II (0.1 nmol/l) to the superfusate solution decreased accumulation of ASP⁺ significantly by 20 ± 5%. In IHKE-1 cells, the effect of oxytocin (0.1 µmol/l, n = 7) on ASP⁺ accumulation was reduced to a stimulation of only 23 ± 11% in the absence of extracellular Ca⁺⁺.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Changes in ASP+ accumulation in IHKE-1 cells after addition of ATP (0.1 mmol/l), oxytocin (Oxy; 0.1 µmol/l) in the presence and absence of extracellular Ca++ and bradykinin (BK; 1 µmol/l) to the bath solution. The effect of angiotensin II (AII; 0.1 nmol/l) is shown in LLC-PK1 cells. Data are presented as changes in fluorescence increase expressed in percentages of the paired controls. The columns represent mean values ± S.E.M. The numbers in parentheses represent the number of individual experiments. (*) indicates statistical significance of the effects (P < .05).

Figure 4 summarizes the effects of the adenylate cyclase activator forskolin on ASP⁺ accumulation. Addition of 0.5 and 1 µmol/l forskolin resulted in a significant increase in ASP⁺ accumulation in IHKE-1 cells by 20 ± 8% and 35 ± 13%, respectively. An increase in the forskolin concentration to 10 µmol/l led to an increase in ASP⁺ accumulation similar to control. ASP⁺ accumulation in LLC PK₁ cells was only slightly inhibited with high concentrations of forskolin (10 µmol/l), without any stimulation at low concentrations.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Concentration-dependent effects in ASP+ accumulation by forskolin in IHKE-1 (circle) and LLC-PK1 (square) cells. Data are presented as changes in fluorescence increase expressed in percentages of the paired controls. Mean value ± S.E.M. The numbers in parentheses represent the number of individual experiments. (*) indicates statistical significance of the effect (P < .05).

To further support the involvement of PKA in the regulation of cellular ASP⁺ accumulation in both cell lines, 8-Br-cAMP, a membrane-permeable analog of the second messenger cAMP, was added to the bath solution. IHKE-1 cells showed a significant increase in ASP⁺ accumulation by 125 ± 25%, whereas no significant effect was found in LLC-PK₁ cells (fig. 5).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effects of 8-Br-cAMP on ASP+ accumulation in IHKE-1 and LLC-PK1 cells. Data are presented as changes in fluorescence increase expressed in percentages of the paired controls. The bars represent the mean value ± S.E.M. The numbers in parentheses represent the number of individual experiments. (*) indicates statistical significance of the effect (P < .05).

To investigate an additional potential regulatory mechanism, PKG was stimulated via both human ANP (10 nmol/l) and the second messenger 8-Br-cGMP (0.1 nmol/l). In IHKE-1 cells, addition of both activators resulted in an increase in ASP⁺ accumulation by 35 ± 5% and 28 ± 6%, respectively. However, in LLC-PK₁ cells no significant effects were observed with both substances (fig. 6). To test whether stimulation of ASP⁺ accumulation involves a phosphorylation/dephosphorylation process the effect of an inhibitor of phosphatases I and IIa was examined. In the presence of calyculin A (10 nmol/l) ASP⁺ accumulation was stimulated significantly by 39 ± 6% (n = 5).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Changes in ASP+ accumulation in IHKE-1 and LLC-PK1 cells by hANP and 8-Br-cGMP. Data are presented as changes in fluorescence increase expressed in percentages of the paired controls. Mean value ± S.E.M. The numbers in parentheses represent the number of individual experiments. (*) indicates statistical significance of the effect (P < .05).

These results are summarized in table 1. In IHKE-1 cells, stimulation of all three investigated transduction pathways increased ASP⁺ accumulation via the luminal membrane. However, in LLC-PK₁ cells ASP⁺ accumulation decreased upon addition of PKC activators. Neither of the two other systems investigated, i.e., cAMP- and cGMP-dependent protein kinases, had any significant effect on ASP⁺ accumulation in LLC-PK₁ cells.

Intracellular Ca⁺⁺ activity [Ca⁺⁺]_i. to examine which of the receptors for ATP, oxytocin, angiotensin II or hANP were coupled to changes in [Ca⁺⁺]_i in IHKE-1 cells [Ca⁺⁺]_i was measured. Increases in [Ca⁺⁺]_i were observed only for ATP (100 µmol/l), bradykinin (1 µmol/l) and oxytocin (100 nmol/l). ANP (10 nmol/l) and angiotensin II (100 pmol/l) revealed no changes in [Ca⁺⁺]_i. These data are summarized in table 2.

	Discussion

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An important function of the proximal tubule in the kidney is the secretion and reabsorption of different xenobiotics and drugs. Most of these substances are organic anions and organic cations. The transport of organic cations has been investigated in radioactive uptake studies (Boom et al., 1992; Brandle and Greven, 1992; Hohage et al., 1996) and three different transport systems have been described so far (Wright, 1996; Roch-Ramel et al., 1992; Ullrich, 1994). With the help of the fluorescent dye ASP⁺ a method was established to monitor organic cation transport in living cells without radioactive substrates (Rohlicek and Ullrich, 1993; Pietruck and Ullrich, 1995; Stachon et al., 1996, 1997). With this fluorometric method we investigated the regulation of organic cation transport in cell lines derived from the proximal tubule of pig (LLC-PK₁) and humans (IHKE-1).

PKC. Regulation of organic cation transport by kinases first was reported by us for proximal tubules of rabbit (Hohage et al., 1994b). Activators of PKC, like phorbol esters and diacylglycerol analogs, stimulated organic cation transport time- and concentration-dependently. This stimulation was inhibited by the PKC inhibitor staurosporin (Hohage et al., 1994b). In IHKE-1 cells activation of PKC by DOG led to an increase in ASP⁺ fluorescence. ATP, bradykinin and oxytocin, which increase [Ca⁺⁺]_i in IHKE1 cells and probably also activate PKC, showed a similar effect on ASP⁺ accumulation. The fact that oxytocin stimulated ASP⁺ accumulation to a much lower degree when extracellular Ca⁺⁺ was omitted indicates that the PKC involved is Ca⁺⁺ dependent. In contrast to the situation in IHKE1 cells, in LLC-PK₁ cells ASP⁺ fluorescence was decreased after application of angiotensin II which is known to stimulate PKC in these cells (Karim et al., 1995). Unfortunately, the use of PKC inhibitors like bisindolylmaleimid or calphostin C was not feasible because these substances show high autofluorescence. The involvement of PKC isoforms in the regulation of several transport processes of the proximal tubule was described previously. The organic anion transporter of rabbit proximal tubule was stimulated by PKC (Hohage et al., 1994a), whereas the para-aminohippuric acid transporter of OK-cells was inhibited by this kinase (Takano et al., 1996). Na⁺/H⁺ exchange of rat proximal tubules also was modulated by PKC (Wang and Chan, 1990; Rebouças and Malnic, 1996). The Na⁺/HCO₃ exchanger of the rabbit proximal tubule was stimulated (Yamada et al., 1996), whereas the Na⁺/phosphate transporter when expressed in Xenopus laevis oocytes was inhibited by PKC. Such regulation by PKC can involve direct phosphorylation of the transporter or regulation of trafficking of the transporter as it had been shown for the Na⁺/glucose transporter where the rabbit isoform is down-regulated whereas the human isoform is up-regulated by PKC (Hirsch et al., 1996).

PKA. Similar to the activation of PKC activation of PKA by forskolin or the membrane-permeable analog of cAMP, 8-Br-cAMP, also led to a stimulation of ASP⁺ accumulation in IHKE-1 cells. In LLC-PK₁ cells forskolin reduced organic cation transport, however, only in very high concentrations, which are no more specific for PKA activation. Such effects of high concentrations of forskolin have been described previously (Ruiz et al., 1995, 1996). In IHKE-1 cells 10 µmol/l forskolin did not increase ASP⁺ accumulation further, but rather tended to decrease the stimulation again, which also might indicate a similar nonspecific effect of forskolin in these cells. Regulation by PKA was reported for several different transport systems in the proximal tubule of various species. Among those is the Na⁺/H⁺ exchanger from LLC-PK₁ cells which is activated by PKA (Azuma et al., 1996), the Na⁺/HCO₃ exchanger and the Na⁺+K⁺-ATPase from isolated proximal tubules of rabbit and rat, respectively, which are both inhibited by PKA (Ruiz et al., 1996; Aperia et al., 1994). Furthermore, it was shown that the Na⁺/glucose cotransporter isoforms of rabbit and human are up-regulated by PKA when expressed in X. laevis oocytes (Hirsch et al., 1996).

PKG. We also tested for a third regulatory pathway, PKG, with human ANP and 8-Br-cGMP, the membrane-permeable analog of cGMP. ANP is known to increase intracellular cGMP in IHKE-1 (Hirsch et al., 1997) and LLC-PK₁ (Leitman et al., 1988) cells. Whereas in IHKE-1 cells an increase of ASP⁺ fluorescence was seen with either hANP or the second messenger cGMP, there was no significant effect in LLC-PK₁ cells with both substances. Thus, in the latter cells either the PKG is absent or the regulation of the organic cation transport differs from that in IHKE-1 cells. In proximal tubules an inhibition of the Na⁺ + K⁺-ATPase caused by intracellular cGMP was described previously as well (Aperia et al., 1994).

Phosphatases. ASP⁺ accumulation in IHKE1 cells apparently is controlled by phosphorylation and dephosphorylation even under basal, nonstimulated conditions, because an inhibition of phosphatases I and IIa stimulated ASP⁺ accumulation without prior activation of kinases. A similar basal phosphorylation/dephosphorylation activity has been described for the Na+/glucose cotransporter before (Hirsch et al., 1996).

Possible involvement of Na⁺/H⁺ exchanger. Organic cation transport in proximal tubular cells also critically depends on intracellular pH (pH_i). pH_i mainly is regulated by the activity of Na⁺/H⁺ exchanger which is highly expressed in the proximal tubule. One can speculate that the observed changes in intracellular organic cation accumulation after stimulation of the second messenger systems are not direct effects on the cation transporters but secondary effects caused by stimulation of Na⁺/H⁺ exchanger and consecutive changes in pH_i. There are, however, strong facts against this assumption. In our study, ASP⁺ accumulation was stimulated by all three second messengers. Na⁺/H⁺ exchange, however, was regulated in a different way by the second messenger systems investigated in this study (Kandasamy et al., 1995). Furthermore, we demonstrated recently that ASP⁺ accumulation in LLC-PK₁ cells is reduced by 20% by extracellular alkalinization (pH = 8.5) and stimulated by acidification (pH = 6.0) by 60% (Stachon et al., 1997). ASP⁺ accumulation in the present study was, however, not or only slightly modified in LLC-PK₁ cells by PKA or PKC stimulation, which modulate Na⁺/H⁺ exchange in these cell lines (Azuma et al., 1996; Casavola et al., 1992). Thus, modulation of Na⁺/H⁺ exchanger activity does not seem to contribute to organic cation accumulation.