INTRODUCTION

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RENAL ELIMINATION of a wide variety of organic anions (OA) from the body is an essential process for human and animal health. Transport of OA into proximal cells across the basolateral membrane is the active step in transtubular secretion (11, 17). There is a single transport process at the basolateral membrane, the "classic" OA transporter (OAT), that accepts a broad range of chemical structures and for which p-aminohippurate (PAH) and fluorescein (FL) are prototypical substrates (17, 18). Only recently has this basolateral transporter been cloned (13, 14, 19). In accordance with the sequence data, there are consensus sites for phosphorylation of the transporter protein by protein kinase C (PKC), thereby making it a potential target for regulation via pathways involving the activation of PKC. Indeed, some years before the sequence data became available, studies on the possible regulation of OAT suggesting that phosphorylation might be important were performed. In 1994, Hohage et al. (6) observed the stimulation of PAH accumulation in isolated S2 segments of rabbit kidney by phorbol esters. In contrast to these data, Miller (9) as well as Takano et al. (15) reported an inhibition of OA accumulation by PKC in killifish renal proximal tubules (PT) and opossum kidney OK cells, respectively. In addition, Halpin and Renfro (4) showed that OA secretion in proximal tubular cells of winter flounder is inhibited by phorbol esters and may underlie dopaminergic and adrenergic regulation. The reason(s) for these apparent discrepancies is not known at present and might be species specific or due to differences in the actual experimental conditions, as, for example, the buffer composition.

We took advantage of an experimental protocol described recently by Welborn et al. (18) to determine the initial transport rate (1st 25 s) of OAT in isolated S2 segments of rabbit kidney in real time as well as in paired experiments. As has been shown in previous studies (18), the data obtained with this system and FL as a substrate reflect the activity of OAT, as do measurements of PAH uptake. Our data show that in rabbit PT, activation of PKC, either directly with 4--phorbol 12-myristate 13-acetate (PMA) or indirectly with ligands of receptors coupled to the PKC pathway, inhibits the activity of OAT. This effect is not due to changes in counterion availability but rather to a direct inhibition of OAT.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

Chemicals. Spectral-grade FL was purchased from Molecular Probes (Eugene, OR). Cell-Tak was obtained from Collaborative-Biomedical Products (Bedford, MA). All other chemicals were purchased from commercial sources and were of the highest available purity. All compounds added to the superfusion solution other than FL were checked for interference with the photon counts obtained from FL under our experimental conditions. No significant interference could be observed.

Solutions. A modified rabbit Ringer solution, used in the experiments as a dissection buffer, a superfusion buffer, and an uptake medium, consisted of the following (in mmol/l): 110 NaCl, 25 NaHCO₃, 5 KCl, 2 Na₂HPO₄, 1.8 CaCl₂, 1 MgSO₄, 10 sodium acetate, 8.3 glucose, 5 alanine, 4 lactate, and 0.9 glycine, with an osmolality of ~290 mosmol/kgH₂O. Before use, buffer solutions were filtered (0.4-µm pore size) and aerated for 20 min with the use of 95% O₂-5% CO₂, and the pH was adjusted to 7.4 with NaOH or HCl.

Animals and PT dissection. Adult male New Zealand White rabbits were killed by intravenous injection with pentobarbital sodium. A kidney was immediately removed, perfused with a HEPES-sucrose buffer (250 mmol/l sucrose, 10 mmol/l HEPES, pH 7.4 with Tris base), and transversely sliced with the use of a single-edge razor. A kidney slice was placed in a plastic petri dish containing ice-cold dissection buffer and aerated with 95% O₂-5% CO₂. Segments of PT were individually dissected from the cortical zone, and a segment was transferred to an aluminum superfusion chamber containing superfusion buffer. The chamber floor consisted of a no. 1 glass coverslip coated with 1 µl of Cell-Tak. The chamber was transferred to the stage of an Olympus IMT microscope and superfused with buffer at 5 ml/min. The chamber was fitted with a water jacket, and its temperature, as well as that of the incoming superfusion buffers, was maintained at 37°C. With the use of two-way switching valves, superfusion buffers could be changed in a few seconds while a constant flow rate and temperature were maintained. A small vacuum line on the side of the chamber removed overflow.

Measuring FL uptake into rabbit PT. Initial rates of FL uptake were calculated from measurements of epifluorescence intensity as described previously (18). In brief, a monochromater (Photon Technology International, Brunswick, NJ) equipped with a 75-W xenon lamp was used to generate excitation light at 490 nm (±1-2 nm). A 490-nm dichroic mirror (model 490DCLP; Omega Optical, Brattleboro, VT) directed excitation light to the PT segment through a ×40 oil-immersion fluor objective (1.3 NA, Nikon). Emitted light passed through a 520-nm long-pass filter (Omega Optical) before reaching a photomultiplier tube (model HC120; Hamamatsu, Bridgewater, NJ). Photomultiplier output was recorded at 1-s intervals with the use of LabView software (National Instruments, Austin, TX) installed in a personal computer. Because the halftime for solution exchange in the chamber was 1.5-2 s, the first 5-10 s of the fluorescence record (i.e., ~5 halftimes) was discarded after the switch to a buffer containing FL. The next 25 s of the record was linear. The slope of this line was calculated and represents the initial rate of FL uptake. The interval between two measurements was 10 min to allow washout of FL (18). Routinely, uptake was determined under control conditions at least three times, and an experimental maneuver was only performed if those three control uptakes were stable (5% difference). The last control uptake before starting an experimental maneuver was normalized to 100%, and all the other uptake rates are expressed as a percentage of this control uptake rate. Because FL shows only weak pH dependence at physiological pH levels (a change in pH from 8.0 to 7.2 caused a drop in FL fluorescence of only 5%), possible changes in intracellular pH would not have affected our measurements, as we have determined with the setup described here. Furthermore, stimulation of PKC is expected to activate the basolateral Na⁺/H⁺ exchange (10), thereby leading to a rise in pH. Yet, a rise in pH would, if anything at all, enhance fluorescence and would thereby mimic an increase in FL uptake rather than a decrease, as observed in this study.

Statistical analysis. Unless indicated otherwise, data are means ± SE. Sample size (n) refers to n separate tubules. For each series, tubules from at least four rabbits were used. Comparisons of observed differences to determine their statistical significance at the 0.05 level were performed with the use of either Student's t-test or analysis of variance and a posttest, employing the Student-Newman-Keuls method.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

In the present study we used bicarbonate-buffered, nutrient-rich media and physiological incubation temperatures (18). These conditions are likely to keep the rates of transport and/or cellular metabolism under conditions closely resembling the in vivo situation and therefore allow the investigation of physiological regulatory processes. We tested four different maneuvers, all known to lead to an activation of PKC in PT (1, 3, 7): application of 1) PMA, 2) 1,2-dioctanoyl-sn-glycerol (DOG), 3) the ₁-receptor agonist phenylephrine (PE), and 4) bradykinin (BK). To show that the observed effects were indeed predominantly due to PKC activation, we used the PKC inhibitor bisindolylmaleimide I (BIM) (2, 16) in a concentration at which no other effects have been described thus far (10⁷ mol/l).

As already mentioned above, the setup used in this study allows us to determine the initial uptake rate of FL in real time and in paired experiments (18). Figure 1 shows a typical experiment: after three runs under control conditions (Fig. 1, A-C) in which the uptake rate stayed virtually constant (Fig. 1I), application of 10⁶ mol/l PE induced a time-dependent (Fig. 1, D-F) and reversible inhibition of FL (Fig. 1, G-H). During the 60 s when FL uptake was determined, PE was not present, in order to use the same transport substrate solution during the whole experiment. This also held true for all the other modulators of FL uptake (PMA, DOG, BIM, and BK). Addition of the modulators to the FL solution did not alter the effects of the modulators on FL uptake (data not shown). Previously, it has been shown that FL uptake in the system used here is virtually completely mediated by OAT (18). Thus the method applied here is indeed suitable to detect rapid regulatory changes in the activity of OAT in paired experiments.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Original recordings of a typical fluorescein (FL) uptake experiment (see METHODS for details). Control uptake of FL was determined 3 consecutive times with intervals of 10 min (A-C). Uptake remained virtually constant during control period (see I). Addition of 106 mol/l phenylephrine (PE) led to a time-dependent (D-F) and reversible (G-H) inhibition of FL uptake. I: slopes for different uptake measurements (A-H) are provided; bar indicates presence of PE. s, Seconds; p, photons.

Figure 2 shows the effects of DOG (10⁵ mol/l) or PMA (at concentrations of 10⁸ and 10⁷ mol/l) on OAT activity. Under control conditions (i.e., addition of the vehicle only after the three control runs), the activity of OAT remained virtually constant (there was a slight decrease to 95% of control at 60 min). However, the addition of PMA or DOG induced a rapid and dramatic decrease in OAT activity. At 10⁷ mol/l PMA there was already a significant reduction after 5 min, as shown in Fig. 2. PMA was added at time (t) = 25 min, and the next uptake determination was performed at t = 30 min. In the presence of 10⁷ mol/l PMA, uptake was reduced significantly, even after this short period of exposure (5 min). These data show that PKC activation inhibits OAT in rabbit PT as well. In four additional experiments, we determined the effect of 10⁷ mol/l PMA after 45 and 55 min of exposure. Uptake was reduced to 31 ± 6% of control after 55 min, and there was no significant difference between uptake at 45 and 55 min (Fig. 2, inset). Thus the maximum degree of inhibition by PMA is to 31% of control, a value comparable with the human kidney PAH transporter (8). To confirm that the effects of PMA were indeed due to PKC activation, we used the inhibitor BIM. Although the inhibition constant of BIM for inhibition of PKC in vitro is very low (14 nmol/l, see Ref. 16), the 50% inhibitory concentration of BIM for the inhibition of PKC-induced effects in intact cells is ~200-250 nmol/l (2). This can be explained, at least in part, by the fact that BIM has to compete with high intracellular ATP concentrations for binding to PKC. Because BIM looses its specificity for PKC at higher concentrations, we used it at a concentration of 100 nmol/l; even so, one cannot expect a complete inhibition of PKC under these conditions. However, the specificity of BIM is retained. Application of BIM alone had no significant effect on the activity of OAT under our conditions (Fig. 3), suggesting that there is no constitutive suppression of OAT activity via PKC. As shown in Fig. 4, BIM reduced the inhibitory effect of PMA to less than one-half of the original effect. Therefore, we can conclude that the effect of PMA was indeed mediated by PKC stimulation. Of course, we cannot completely rule out the possibility that part of the action of PMA was not due to PKC activation. PKC-independent modulation of transport activity by PMA has been proposed, for example, for the Na⁺/H⁺ exchanger NHE3 (5). However, the underlying mechanisms are still unknown and possibly may involve the action of other kinases.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effect of protein kinase C (PKC) stimulation with phorbol 12-myristate 13-acetate (PMA) or 1,2-dioctanoyl-sn-glycerol (DOG) on initial uptake rate of FL. All values are normalized to last control measurement. Control, addition of vehicle in which PMA or DOG was dissolved only (dimethyl sulfoxide; final concentration was always 1:10,000). Sample size (n) = 6 for each value plotted. * P < 0.05 vs. last control measurement. Inset: effect of 107 mol/l PMA in 4 experiments in which time of exposure was extended to 55 min; symbols correspond to those as defined in main.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   PKC inhibitor bisindolylmaleimide I (BIM; 107 mol/l) did not affect FL uptake significantly during time of exposure used in this study; n = 6 for each value plotted.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Change of FL uptake after 25 min vs. last control value under different conditions. Time control shows that FL uptake was virtually constant during period of experiments (n = 6). PKC inhibitor BIM (107 mol/l) reduced effect of PMA (108 mol/l, n = 6). PMA (108 mol/l) induced more change in presence than in absence of exogenous -ketoglutarate (KG; 105 mol/l, n = 6). Methyl succinate (MS; 103 mol/l, n = 4) did not alter effect of PMA (108 mol/l). resp, Respective.

Because the initial transport rate of OAT is affected by the availability of -ketoglutarate as a counterion, changes in OAT activity could be due to changes in metabolism that provide -ketoglutarate (18). To test this possibility, we determined the effect of PMA in the continuous presence of -ketoglutarate (10⁵ mol/l) in the superfusion solution under the assumption that in this case, changes in metabolism would not lead to significant changes in the availability of -ketoglutarate for OAT. As shown in Fig. 4, PMA exerted an even greater effect on OAT in the presence than in the absence of exogenous -ketoglutarate. This exaggeration of the PMA effect is most probably due to an enhanced OAT activity in the presence of exogenous -ketoglutarate, as shown previously (18). Thus we conclude that the effect of PKC stimulation is not due to changes in cellular metabolism but rather to a more direct interaction with OAT. Furthermore, the inhibitory action of PKC activation is not due to an interaction with the Na⁺--ketoglutarate cotransporter because 1) this transport has been shown to be unaffected by PKC stimulation (12) and 2) PMA still inhibited OAT activity when the Na⁺--ketoglutarate cotransporter was functionally eliminated (18) by the addition of 1 mmol/l methyl succinate. In the presence of 1 mmol/l methyl succinate, 10⁸ mol/l PMA reduced OAT activity by 52 ± 5% after 25 min (n = 4) compared with 43 ± 5% in the absence of methyl succinate (no significant difference). Thus OAT itself is regulated by PKC. The effect of BIM, -ketoglutarate, and methyl succinate on FL uptake modulation after 5, 15, and 35 min of exposure to the different drugs was also tested. The effects were qualitatively the same: BIM prevented inhibition, methyl succinate had no effect on transport modulation, and -ketoglutarate increased the inhibitory effect of PMA slightly. For reasons of clarity, we present only the 25-min data in Fig. 4 (see also Fig. 7).

To investigate whether PKC-mediated regulation also takes place after physiological stimulation of receptors that are coupled to the PKC pathway, we applied PE or BK, both of which are known to bind to receptors in the plasma membrane (₁-receptor and BK receptor, respectively) that couple to the PKC pathway (1, 3, 7). As shown in Figs. 5 and 6, both substances led to a reversible inhibition of OAT activity. Furthermore, the action of both substances was prevented by the PKC inhibitor BIM (Fig. 7, A and B), showing that they indeed acted via stimulation of PKC. The effects of PE and BK in the presence of 10⁷ mol/l BIM were not significantly different from time control in the presence of BIM (Fig. 3), indicating that these two substances acted exclusively via PKC. We also tested the effect of PE in the presence of 2 × 10⁷ mol/l BIM: there was no significant effect of PE (95 ± 3% of control, n = 4) or BK (96 ± 4% of control, n = 3) on FL uptake compared with time control. The reversibility of the effect also showed that PKC-mediated inhibition of OAT was not due to a unspecific toxic action on the cells. The rapid time course of the reversibility may be the result of a rapid dephosphorylation of the transporter itself by endogenous phosphatases. Yet, a direct phosphorylation of OAT has not been demonstrated thus far. Alternatively, OAT activity may be decreased indirectly by PKC (e.g., retrieval from the plasma membrane or phosphorylation of regulatory proteins), and dephosphorylation of regulatory factors accounts for the reversibility. De novo synthesis of transport proteins can be excluded from the time course of recovery. Of course, our data do not allow us to draw a final conclusion with respect to the mechanisms underlying reversibility, and detailed future studies will have to address this question. Nevertheless, OAT seems to be under the physiological control of two regulatory systems: circulating hormones, like BK, and the autonomic nervous system, represented by the ₁-receptor ligand phenylephrine. The in vivo importance of these effects remains to be determined. However, the existence of such a regulation in freshly isolated PT argues strongly in favor of the physiological relevance of the observed regulation. Additional work is needed to determine whether OAT is phosphorylated directly by PKC or whether there are additional events involved.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Bradykinin (BK; 106 mol/l) induced a time-dependent and reversible inhibition of FL uptake; n = 6 for each value plotted. * P < 0.05 vs. last control value; # P < 0.05 vs. last value in presence of BK.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   PE (106 mol/l) induced a time-dependent and reversible inhibition of FL uptake; n = 6 for each value plotted. * P < 0.05 vs. last control value. # P < 0.05 vs. last value in presence of PE.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   PKC inhibitor BIM (107 mol/l) abolished effect of BK (A) and PE (B). Effect of both substances was not significantly different from time control (see Fig. 3 in presence of BIM).

The data of the present study are in good agreement with those obtained by Miller (9), Takano et al. (15), Halpin and Renfro (4), and Lu et al. (8), who also reported an inhibitory action of PKC on OA transport, albeit in other species. The reasons for the apparent discrepancy with the data of Hohage et al. (6) are not clear at the moment. Two possible explanations are 1) the difference in the composition of the buffer solution, because Hohage et al. did not use nutrient-rich media, and 2) determination of steady-state accumulation of PAH in the tubules, not initial transport rates, by Hohage et al. The different regulation of OA transport by oxymetazoline (4) and PE is most probably due to the fact that oxymetazoline interacts primarily with ₂-receptors, whereas PE is an ₁-agonist (7).

In summary, our data show that the initial transport rate of OAT in rabbit PT is under the negative control of physiological stimuli (e.g., hormones or the autonomic nervous system) that act via the activation of PKC. This action is not mediated by changes in metabolism or counterion availability but by a direct action on the transporter itself.