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Am J Physiol Renal Physiol 273: F674-F679, 1997;
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Vol. 273, Issue 5, F674-F679, November 1997

Inhibition of PAH transport by parathyroid hormone in OK cells: involvement of protein kinase C pathway

Junya Nagai, Ikuko Yano, Yukiya Hashimoto, Mikihisa Takano, and Ken-Ichi Inui

Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto 606-01, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously shown that the p-aminohippurate (PAH) transport system in OK kidney epithelial cell line is under the regulatory control of protein kinase C. Parathyroid hormone (PTH) could activate protein kinase C, as well as protein kinase A, in OK cells. In the present study, the effect of PTH on PAH transport was studied in OK cells. PTH inhibited the transcellular transport of PAH from the basal to the apical side, as well as the accumulation of PAH in OK cells. Basolateral PAH uptake was inhibited by PTH in a dose- and time-dependent manner. Protein kinase A activators did not affect the transcellular transport or the accumulation of PAH. The PTH-induced inhibition of the accumulation of PAH was blocked by a protein kinase C inhibitor staurosporine. These results suggest that PTH inhibits the PAH transport in OK cells and that the messenger system mediated by protein kinase C, not protein kinase A, plays an important role in the regulation of PAH transport by PTH.

organic anion transport; hormonal control; renal secretion; opossum kidney cell

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE ORGANIC ANION TRANSPORT system of the renal proximal tubule has physiological roles in the excretion of a wide variety of anionic compounds, including endogenous metabolites and xenobiotics, into the urine (19, 20). This function is important because many organic anions are toxic and need to be removed as promptly as possible. The organic anion transport system has been studied using different tissue preparations, including renal cortical slices, isolated proximal tubules, and purified brush-border and basolateral membrane vesicles (17, 20, 25). These studies have revealed the kinetics, driving forces, and substrate specificities of the organic anion transport system. However, little information is available concerning the regulation of organic anion transport, especially by hormone via the receptor-mediated generation of intracellular second messengers. This might be partly due to the lack of a good model system with which to study the regulatory factors for organic anion transport under well-defined conditions. We reported that OK cells, which were established from the American opossum kidney (11), are a useful in vitro model system with which to study the organic anion transport across intact epithelial cells (8, 16, 23). Moreover, we showed that the transport of p-aminohippurate (PAH), a typical organic anion, in OK cells is regulated by protein kinase C (24).

Parathyroid hormone (PTH) has multiple effects on the kidney. In addition to the stimulation of gluconeogenesis and 25-hydroxyvitamin D3 1alpha -hydroxylase activity, the major physiological effect of PTH is on the regulation of tubular transport processes (14). In renal proximal tubule, PTH regulates the activities of apically located Na+-phosphate cotransport and Na+/H+ exchange by activating intracellular regulatory pathways (protein kinase A, protein kinase C) via PTH receptor-mediated production of intracellular messengers [adenosine 3',5'-cyclic monophosphate (cAMP), diacylglycerol] (3, 15). OK cells have specific PTH receptors coupled to the protein kinase A and protein kinase C pathways as well as various transport systems similar to those in renal proximal tubules (3, 15, 21). In addition, PTH/PTH-related peptide receptor has been cloned by COS-7 expression, using an OK cell cDNA library (9). Thus OK cells could be useful in studying the regulation of the organic anion transport system by PTH.

The purpose of this study is to examine whether PAH transport in OK cells is regulated by PTH. The results show that PTH decreases the activity of the PAH transport in OK cells via the messenger system mediated by protein kinase C, rather than by protein kinase A.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. OK cells were cultured in plastic dishes (Corning Glass Works, Corning, NY) in medium 199 (Flow Laboratories, Rockville, MD) containing 10% fetal bovine serum (Whittaker Bioproducts, Walkersville, MD) without antibiotics, in an atmosphere of 5% CO2-95% air at 37°C, and subcultured every 5-7 days using 0.02% EDTA and 0.05% trypsin (8). OK cells were used between passages 73 and 97.

Transport measurements. PAH transport was measured in OK cell monolayers cultured in Transwell chambers (Costar, Cambridge, MA). To prepare cell monolayers, cells were seeded at a density of 4 × 105 cells/cm2 on polycarbonate membranes (3 µm pore size) in Transwell cell chambers (4.71 cm2 surface area), which were placed in six-well cluster plates. The volume of medium inside and outside the chambers was 1.5 and 2.6 ml, respectively. Fresh medium was replaced every 2 days, and the cells were used between the 5th and 7th days after seeding. Transport was measured at 37°C in Dulbecco's phosphate-buffered saline, containing (in mM) 137 NaCl, 3 KCl, 8 Na2HPO4, 1.5 KH2PO4, 1 CaCl2, and 0.5 MgCl2 (for PAH transport), or in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline, containing (in mM) 137 NaCl (or for Na+-independent transport, 137 choline chloride), 5.4 KCl, 1 CaCl2, 0.5 MgCl2, and 14 HEPES-tris(hydroxymethyl)aminomethane (for phosphate transport) supplemented with 5 mM D-glucose.

The transcellular transport of PAH across OK cell monolayers was measured as described (8). To measure the cellular uptake of [14C]PAH (15 µM) and [32P]phosphate (100 µM), the reaction was initiated by adding each buffer containing 5 mM D-glucose and the substrate to either the basal or the apical side of the monolayers. D-[3H]mannitol (15 µM) was added simultaneously to correct for extracellular trapping and nonspecific uptake of the substrate in the PAH uptake experiments. After an incubation for a specified period, the uptake medium was aspirated and discarded, and the membrane was rapidly washed three times with ice-cold buffer containing 5 mM D-glucose. The cell monolayers on the membrane were solubilized in 0.5 ml of 0.1 M sodium hydroxide, and the amount of substrate taken up by the cells was measured by counting the radioactivity.

Cell treatment. Stock solutions of dibutyryladenosine 3',5'-cyclic monophosphate (dibutyryl-cAMP), 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), forskolin, and staurosporine were prepared in dimethyl sulfoxide (DMSO). The final concentration of DMSO during exposure was 0.25-0.5%. The compounds were applied to both the basolateral and apical sides for a specified period. The control cells were incubated with the same concentration of DMSO in each experiment. Finally, the cell monolayers were washed three times with the uptake buffer before measuring the uptake.

Analytical methods. The radioactivity was determined in 5 ml of ACS II (Amersham International, Buckinghamshire, UK) by liquid scintillation counting using an external standard to correct for quenching. The appropriate crossover correction was given to separate the radioactivities of 3H and 14C. Protein was determined by the method of Bradford (2) with bovine gamma -globulin as the standard.

Statistical analysis was performed by Student's t-test, or by the one-way analysis of variance with the Dunnett's test for post hoc analysis (P < 0.05 for significance).

Materials. p-[Glycyl-1-14C]aminohippurate (1.53 GBq/mmol), D-[3H]mannitol (728.9-828.8 GBq/mmol), and KH232PO4 (37 GBq/mmol) were obtained from Du Pont-New England Nuclear (Boston, MA). Synthetic bovine (1---34)-PTH, phorbol 12-myristate 13-acetate (PMA), and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma Chemical (St. Louis, MO). Dibutyryl-cAMP, forskolin, and staurosporine were purchased from Wako Pure Chemicals (Osaka, Japan). 8-Br-cAMP was purchased from Nacalai Tesque (Kyoto, Japan). All other chemicals used were of the highest purity available.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of PTH and dibutyryl-cAMP on Na+-phosphate cotransport. Figure 1 shows the effect of PTH and dibutyryl-cAMP on phosphate uptake from the apical side of OK cells. Phosphate was transported sodium dependently. PTH produced a time-dependent decrease in Na+-phosphate cotransport. In addition, the uptake of phosphate was inhibited by pretreatment for 3 h with dibutyryl-cAMP (10-5 M). These findings showed the regulation coupled to PTH receptor by PTH and the activation of protein kinase A by dibutyryl-cAMP. Therefore, OK cells are useful in studying the regulation of PAH transport by PTH.


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  		Fig. 1.   Effect of parathyroid hormone (PTH) and dibutyryl-cAMP on phosphate uptake from the apical side of OK cell monolayers. Confluent monolayers were incubated with PTH (10-7 M) for 30 min or 3 h and with dibutyryl-cAMP (10-5 M) for 3 h. After the cells were washed, [32P]phosphate (100 µM) was added to the apical side of the monolayers, and [32P]phosphate uptake for 5 min at 37°C was measured. Each column is the mean ± SE of 3 monolayers of a typical experiment. * P < 0.05, significant difference from control.

Effect of PTH on the basal-to-apical transport and accumulation of PAH. We examined the effect of PTH on the transcellular transport from the basal to apical side and accumulation of [14C]PAH in OK cells. PTH (final concentration 10-7 M) or its vehicle was added at 25 min after initiation of transport measurement. The transcellular transport activities of PAH at 30, 45, and 60 min from start of the PAH transport (5, 20, and 35 min after the addition of PTH or its vehicle, respectively) were measured. At 30 and 45 min, the transcellular transport of PAH slightly decreased by PTH addition compared with its vehicle (data not shown), and PTH significantly inhibited the transcellular transport of PAH from the basal to apical side at 60 min (P < 0.05) (Fig. 2A). The simultaneously measured accumulation of PAH in OK cells at 60 min was also significantly inhibited by PTH (Fig. 2B). The inhibition of transcellular transport and intracellular accumulation of PAH by PTH suggested that PTH affects, at least in part, the basolateral transport of PAH in OK cells. Therefore, the effects of PTH on PAH uptake across basolateral membrane of OK cells were further studied.


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  		Fig. 2.   Effect of PTH on basal-to-apical transport (A) and accumulation (B) of p-aminohippurate (PAH) by OK cell monolayers. [14C]PAH (15 µM) and D-[3H]mannitol (15 µM) were added to the basal side of monolayers, and distilled water (open bars, final concentration 0.25% vol/vol) or PTH (solid bars, final concentration 10-7 M) was added at 25 min after start of the transport measurement. A: at 60 min, medium on apical side was collected (100 µl), and radioactivity levels were counted to determine transcellular transport of [14C]PAH. D-[3H]mannitol was used to correct for paracellular flux. PAH transport in absence of PTH (control) was 180.0 ± 15.1 pmol · cm-2 · 60 min-1. B: after a 60-min transport measurement, accumulation of [14C]PAH in OK cells was determined. D-[3H]mannitol was used to correct for extracellular trapping and nonspecific uptake. PAH accumulation in absence of PTH (control) was 53.7 ± 4.5 pmol · mg protein-1 · 60 min-1. Each column is the mean ± SE of 9 monolayers of 3 separate experiments. * P < 0.05, significant difference from each control.

Effect of PTH pretreatment concentration and time on basolateral PAH uptake. We examined the effect of various concentrations of PTH on the PAH uptake for 1 min from the basal side of OK cells. The OK cells were treated with various concentrations (10-11-10-7 M) of PTH for 15 min, then the basolateral PAH uptake for 1 min was measured. As shown in Fig. 3, PTH inhibited the PAH uptake in a dose-dependent manner, and it was significant at 10-7 M (P < 0.05). We further examined the dose response curve after 3-h pretreatment with PTH. The dose response curve after 3-h pretreatment slightly shifted to the left compared with that after 15-min pretreatment, but there was no significant difference between two experimental groups at each PTH concentration (10-11-10-7 M) (data not shown).


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  		Fig. 3.   Dose-dependent effect of PTH on PAH uptake from the basal side of OK cell monolayers. Confluent monolayers were incubated for 15 min with various concentrations of PTH (10-11-10-7 M). After the cells were washed, [14C]PAH (15 µM) and D-[3H]mannitol (15 µM) were added to the basal side of monolayers, and [14C]PAH uptake for 1 min at 37°C was measured. Each point is the mean ± SE of 6-7 monolayers of 3 separate experiments. * P < 0.05, significant difference from control.

Figure 4 shows the effect of PTH pretreatment periods (5 to 60 min) on PAH uptake from the basal side of OK cells. Exposure to PTH caused a time-dependent decrease in PAH uptake, which was significant at a period of 15 min or more (P < 0.05).


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  		Fig. 4.   Effect of time on PTH-induced inhibition of PAH uptake from the basal side of OK cell monolayers. Confluent monolayers were incubated for various periods with PTH (10-7 M), and [14C]PAH uptake from the basal side of monolayers was measured as described in Fig. 3. Each point is the mean ± SE of 8-9 monolayers of 3 separate experiments. * P < 0.05, significant difference from control at time 0.

Effect of protein kinase A activators on PAH transport. OK cells have specific PTH receptors coupled to not only the protein kinase C pathway but also the protein kinase A pathway. We have already reported that protein kinase C activators, such as active phorbol esters and diacylglycerols, inhibit PAH transport in OK cells, and protein kinase C may have an important role in the regulation of PAH transport (24). Therefore, we further analyzed the effect of protein kinase A activators on PAH transport in OK cells. Figure 5 shows the effect of protein kinase A activators, which are cAMP analogs, dibutyryl-cAMP and 8-Br-cAMP (10-5 M), an adenylate cyclase activator forskolin (10-5 M), and a phosphodiesterase inhibitor IBMX (10-3 M). However, exposure to these protein kinase A activators for 3 h had no effect on the initial rate of PAH uptake from the basal side of OK cells. Moreover, neither the transcellular transport from the basal to the apical side nor the steady-state accumulation of PAH in OK cells was affected by the incubation of dibutyryl-cAMP and forskolin (10-5 M) for 3 h before transport measurements (Fig. 6).


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  		Fig. 5.   Effect of protein kinase A activators on PAH uptake from the basal side of OK cell monolayers. Confluent monolayers were incubated for 3 h with cAMP analogs, forskolin (10-5 M) and 3-isobutyl-1-methylxanthine (IBMX) (10-3 M). After the cells were washed, [14C]PAH uptake from the basal side of monolayers was measured as described in Fig. 3. Each column is the mean ± SE of 6-7 monolayers of 3 separate experiments.


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  		Fig. 6.   Effect of protein kinase A activators on basal-to-apical transport (A) and accumulation (B) of PAH by OK cells. Confluent monolayers were incubated for 3 h without (open circle ) or with 10-5 M dibutyryl-cAMP (black-triangle) or 10-5 M forskolin (black-square). After the cells were washed, transcellular transport at 15, 30, 45, and 60 min (A) and accumulation at 60 min (B) of [14C]PAH were measured as described in Fig. 2. Each point or column is the mean ± SE of 6-8 monolayers of 3 separate experiments.

To examine whether protein kinase C and protein kinase A systems mutually interact on the inhibition of PAH transport in OK cells by PTH, the effects of a protein kinase C activator PMA (10-8 M) alone and in combination with dibutyryl-cAMP (10-5 M) were examined. PMA inhibited PAH uptake from the basal side of OK cells as described previously (24). However, its inhibitory effect was not affected by coincubation with dibutyryl-cAMP (Fig. 7).


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  		Fig. 7.   Effect of pretreatment with phorbol 12-myristate 13-acetate (PMA) alone or in combination with dibutyryl-cAMP on PAH uptake from the basal side of OK cell monolayers. Confluent monolayers were incubated for 3 h in absence or presence of PMA (10-8 M) and/or dibutyryl-cAMP (10-5 M). After the cells were washed, [14C]PAH uptake from the basal side of monolayers was measured as described in Fig. 3. Each column is the mean ± SE of 5 or 6 monolayers from 2 separate experiments. * P < 0.05, significant difference from control.

Effect of staurosporine on PTH-induced inhibition of PAH accumulation. To clarify whether protein kinase C activation is linked directly to the inhibition of PAH transport by PTH, we examined the effect of staurosporine, a potent inhibitor of protein kinase C, on the PTH-induced inhibition of PAH accumulation. When cells were pretreated with staurosporine before adding PTH, the inhibitory effect of PTH on the PAH accumulation was almost completely blocked (Fig. 8).


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  		Fig. 8.   Effect of staurosporine on PTH-induced inhibition of PAH accumulation of OK cell monolayers. [14C]PAH (15 µM) and D-[3H]mannitol (15 µM) were added to the basal side of monolayers with staurosporine (10-6 M) or its vehicle, and distilled water (open bars, final concentration 1% vol/vol) or PTH (solid bars, final concentration 10-6 M) was added at 25 min after starting of the transport measurement. At 60 min, accumulation of [14C]PAH in OK cells was determined as described in Fig. 2. PAH accumulations in absence of PTH (control) for vehicle and staurosporine were 101.5 ± 6.8 and 73.3 ± 8.5 pmol · mg protein-1 · 60 min-1, respectively. Each column is the mean ± SE of 7-8 monolayers of 3 separate experiments. * P < 0.05, significant difference from each control.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

OK cells have various transport systems similar to those in renal proximal tubules, including Na+-coupled transport systems for amino acids, hexoses, proton, and inorganic phosphate (12), in addition to the PAH transport system we reported (8, 16, 23). OK cells also have specific PTH receptors coupled to the protein kinase A and protein kinase C pathways (3, 15). In addition, PTH/PTH-related peptide receptor has been cloned by COS-7 expression, using an OK cell cDNA library (9). Thus OK cells are useful in studying the effect of PTH on transport systems in renal proximal tubules. Furthermore, several studies have shown that PTH regulates the activities of apically located Na+-phosphate cotransport, as well as Na+/H+ exchange, via the activation of protein kinase A and/or protein kinase C pathways (7, 21, 22). In this study, we evaluated whether PTH regulates the PAH transport system expressed in OK cells via the protein kinase A and/or protein kinase C pathways.

Miyauchi et al. (13) have shown that the OKH, a clonal cell line of OK cells, is resistant to inhibition of Na+-phosphate cotransport by PTH, suggesting that the resistance is associated with the lack of stimulated phospholipase C. To assess the OK cells we have used, the effect of PTH and dibutyryl-cAMP on Na+-phosphate cotransport was examined. Na+-dependent phosphate uptake from the apical side of OK cells was inhibited by dibutyryl-cAMP and PTH, suggesting that the OK cells are useful for examining the effect of protein kinase A activation and PTH on the PAH transport system.

PTH inhibited both the transcellular transport and intracellular accumulation of PAH in OK cell monolayers. These findings indicated that the inhibitory effect of PTH is related, at least in part, to the inhibition of PAH uptake from the basal side of OK cells. Therefore, we further studied the effect of PTH on the basolateral uptake of PAH in OK cells. The basolateral uptake of PAH in OK cells was inhibited by PTH in a dose- and time-dependent manner. However, the dose-response curve given in Fig. 3 for PTH inhibition of PAH uptake in OK cells showed a rather low sensitivity to PTH. Quamme et al. (21) demonstrated that half the maximally inhibitory effect of PTH on Na+-phosphate was 10-12-10-11 M. The difference between these sensitivities to PTH remains unclear.

OK cells have PTH-responsive dual signal pathways that activate both protein kinase A and protein kinase C. We previously demonstrated that protein kinase C activation by phorbol esters and diacylglycerols inhibits the PAH transport in OK cells (24). Therefore, it is likely that the inhibition of PAH transport by PTH is due, at least in part, to the activation of protein kinase C. The present study with staurosporine, a protein kinase C inhibitor, has demonstrated the involvement of protein kinase C on the inhibitory effect of PAH transport by PTH. In contrast, various protein kinase A activators, such as cAMP analogs (dibutyryl-cAMP and 8-Br-cAMP), an adenylate cyclase activator (forskolin), and a phosphodiesterase inhibitor (IBMX), had no effect on PAH transport in OK cells. Moreover, neither a dose-dependent (10-7-10-3 M) nor time-dependent (5-180 min) effect by dibutyryl-cAMP was observed (data not shown). Friedman et al. (4) have shown that activation of both protein kinase A and protein kinase C pathways is necessary for PTH stimulation of calcium uptake in mouse distal convoluted tubule cells. However, the inhibitory effect on the basolateral PAH uptake by a protein kinase C activator PMA was not affected by dibutyryl-cAMP (Fig. 7). These results suggested that protein kinase A activation is not involved in the regulation of PAH transport in OK cells.

Several studies suggested that the protein kinase C-mediated pathway is more important than the protein kinase A pathway in regulating Na+-phosphate cotransport. Quamme et al. (21) demonstrated that Na+-phosphate cotransport in OK cells is inhibited by PTH at the concentrations with which activation of phospholipase C occurs without increasing cAMP. In a clonal OK cell line lacking PTH-dependent phospholipase C activation, PTH was not able to reduce Na+-phosphate cotransport despite a stimulation of adenylate cyclase (13). Moreover, Hayes et al. (6) showed that the rat renal brush-border membrane Na+-phosphate cotransporter (NaPi-2) expressed in Xenopus laevis oocytes is inhibited by pharmacological activation of protein kinase C but not by protein kinase A. Not only in OK cells but also in a rat osteosarcoma cell line UMR-106, which possesses PTH-responsive dual signal transduction systems, the protein kinase C system is involved exclusively in the stimulation of Na+-phosphate cotransport by PTH without any contribution of the protein kinase A system (1).

The inhibition of PAH transport by PTH may be related to PTH-dependent stimulation of renal gluconeogenesis. Wang and Kurokawa (26) reported that PTH stimulated glucose production from alpha -ketoglutarate in proximal tubules. The regulation of gluconeogenesis by PTH may affect the intracellular concentration of dicarboxylates such as alpha -ketoglutarate. In addition, a recent report of Pritchard (18) with rat renal cortical slices showed that PAH transport was modulated by changes in intracellular alpha -ketoglutarate concentration. Therefore, the inhibitory effect of the basolateral PAH transport by PTH in OK cells may result from alteration of the intracellular dicarboxylate (alpha -ketoglutarate) concentration.

Kippen et al. (10) studied the effect of PTH and cAMP on PAH transport in rabbit proximal tubule suspension. In contrast to this study with OK cells, they showed the stimulatory effect of PTH and cAMP on PAH uptake. The reasons for this discrepancy are unclear but may be related to methodological differences (culture cell and tubule suspension) or species differences (opossum and rabbit). Recently, Halpin and Renfro (5) studied the regulation of active net secretion of a xenobiotic organic anion, 2,4-dichlorophenoxyacetic acid (2,4-D), by flounder proximal tubule primary cultures. Activation of protein kinase C but not protein kinase A caused a decrease in active net secretion of 2,4-D. Moreover, they demonstrated dopaminergic inhibition and alpha -adrenergic stimulation of 2,4-D net secretion. On the basis of these findings, various compounds may be regulating the renal secretion of organic anions.

In conclusion, we demonstrated that PTH inhibits PAH transport in OK cells. In addition, we showed that the pathway via protein kinase C, rather than protein kinase A, plays a crucial role in the regulation of PAH transport by PTH in OK cells. The mechanism by which protein kinase C activation induces the inhibition of PAH transport activity as well as the physiological role of the regulation by PTH need further studying.

    ACKNOWLEDGEMENTS

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, and by Grants-in-Aid from the Japan Health Sciences Foundation.

    FOOTNOTES

Address for reprint requests: K. Inui, Dept. of Pharmacy, Kyoto Univ. Hospital, Sakyo-ku, Kyoto 606-01, Japan.

Received 11 March 1997; accepted in final form 18 June 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Renal Physiol 273(5):F674-F679
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