PTH stimulates a Clminus -dependent and EIPA-sensitive current in chick proximal tubule cells in culture

Gary Laverty1, Colleen McWilliams1, Amanda Sheldon1, and Sighvatur S. Árnason2

1 Department of Biological Sciences, University of Delaware, Newark, Delaware 19716; and 2 Department of Physiology, University of Iceland, IS-101 Reykjavík, Iceland


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The electrophysiological effects of parathyroid hormone (PTH) were studied in a primary cell culture model of the chick (Gallus domesticus) proximal tubule. In this model, confluent monolayers are grown on permeable filters and exhibit vectorial transport, including glucose-stimulated current. Under short-circuit conditions, PTH, at 10-9 M, induced a positive current [short-circuit current (Isc)] response, with an average 2-min peak response of 14.30 ± 1.58 µA/cm2 over the baseline Isc, followed by a slow decay. The PTH response was dose dependent, with a half-maximal response at 5 × 10-9 M and maximal response at 5 × 10-8 M. Forskolin and dibutyryl-cAMP also stimulated Isc, as did the phosphodiesterase inhibitor IBMX. In contrast, the phorbol ester PMA inhibited baseline Isc. The PTH response was nearly abolished by apical addition of 100 µM EIPA, an inhibitor of Na+/H+ exchangers, and partially blocked by the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 100 µM) and glibenclamide (300 µM). Higher doses of EIPA or NPPB alone (500 µM) were almost fully effective, with no or slight additional effects of NPPB or EIPA, respectively. The anion exchange inhibitor DIDS (100 µM) and the Na+ channel blocker amiloride (10 µM) had no effect. Bilateral reduction of Cl- in the buffer, from 137 to 2.6 mM, abolished the PTH response; increasing Cl- concentration restored the Isc response, with a half-maximal effect at 50 mM. These data suggest that, in the chick proximal tubule, PTH activates both an Na+/H+ exchanger and a Cl- channel that may be functionally linked.

avian kidney; short-circuit current; chloride channels; cystic fibrosis transmembrane regulator; glibenclamide


    INTRODUCTION
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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PARATHYROID HORMONE (PTH) is known to affect a number of proximal tubule (PT) transport systems. In rats, rabbits, and other mammalian species, PTH inhibits Na+, fluid, and bicarbonate reabsorption in vivo and in vitro (2, 4, 16, 29; for a review, see Ref. 15). A major part of this effect is linked to inhibition of an apical Na+/H+ exchanger (NHE) isoform, identified as NHE3 (10, 14, 16, 54). On the basis of recovery of intracellular pH from an imposed ammonium chloride acid load, PTH treatment inhibits an amiloride- and EIPA-sensitive exchanger in both native tissues and in the proximal-like opossum kidney (OK) cell line (10, 17, 40). Furthermore, studies with brush-border membrane vesicles (BBMV) have shown that PTH treatment reduces the activity of pH gradient-driven 22Na uptake (14, 16, 20). PTH was also shown to decrease the Vmax for 22Na uptake by OK cells (30). These findings are suggestive of a PTH-induced decrease in transporter number. In support of this, more recent immunoblotting studies and subcellular fractionation experiments have shown a PTH-induced internalization of NHE3 from the apical membrane to a subapical, intracellular compartment (10, 14, 16, 18, 54). PTH also reduces NHE3 activity via phosphorylation of cytoplasmic domains of the transporter, thus indicating a dual mechanism of regulation (10, 14). These effects of PTH appear to be mediated largely via the cAMP-dependent protein kinase (PKA) signaling pathway (1, 10, 17, 20, 29, 40). Similarly, PTH also inhibits an apical sodium phosphate (Pi) cotransport system, resulting in increased urinary excretion of Pi (17, 54).

There is much less known about PTH function in the avian PT. There is some evidence for an apical NHE in chickens (35), and PTH in vivo leads to increased whole animal urinary flow rates, Na+ excretion, and urinary pH, suggesting PTH inhibition of this system (26). However, this has not been demonstrated directly. Moreover, there are a number of known differences in proximal transport characteristics between birds and mammals. For example, we previously demonstrated that superficial PTs of the European starling do not acidify the urine (24); i.e., there was no measurable pH gradient between the tubule lumen and peritubular blood and therefore no preferential bicarbonate reabsorption, as seen in mammalian PTs (15). We and others were also unable to detect, by histochemical methods, PT carbonic anhydrase activity, whereas distal tubule and collecting duct segments were clearly positive (25, 37). Martinez et al. (28) found that superficial, nonlooped nephrons of chickens seem to possess none of the known mammalian basolateral acid-base transporters.

The avian PT also possesses specific systems for secretory transport (basolateral to apical) of both urate and Pi, although there is little known about these systems (3, 7, 13, 36, 52). Such secretory processes may have evolved in part to compensate for the lower filtration rates found in nonmammalian nephrons (7, 36). Thus PTH in birds is thought to increase Pi excretion by both inhibition of a reabsorptive flux, as in mammals, and by stimulation of a secretory flux (13, 36, 52).

We have recently developed a primary cell culture model of the avian (chick) PT using methods similar to those developed for rabbit and rat (45). Cells grown as confluent monolayers on permeable membrane filters become highly polarized and exhibit transepithelial transport, measurable by classic electrophysiological methods. Using this approach, we undertook these studies to investigate PTH effects on the avian PT. The results demonstrate a novel effect of PTH on this system involving stimulation of a Cl--dependent and EIPA-sensitive short-circuit current (Isc).


    MATERIALS AND METHODS
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Reagents and supplies. The growth media and supplements, collagen, Percoll, PTH [bovine PTH-(1-34) fragment], and all other agonists and antagonists used in this study were obtained from Sigma (St. Louis, MO). Dispase and collagenase were from Roche Molecular Biochemicals (Indianapolis, IN). The membrane filters used were Nunc (Naperville, IL) 10-mm tissue culture inserts with an 0.02-µm Anopore membrane. Inhibitors and agonists were prepared as 1,000× stock solutions in DMSO or water.

Cell culture. Chick PT cultures were prepared as described previously (45), using methods similar to those for mammalian primary cultures (12, 19, 48). Briefly, 4- to 7-day-old White Leghorn chicks were killed by cervical dislocation (approved by the International Animal Care and Use Committee), and kidney tissue was removed asceptically. Pieces of tissue were pooled from five to seven chicks in ice-cold Hanks' balanced salt solution (HBSS) with penicillin and streptomycin. The pooled tissue was then minced and enzymatically disaggregated in a solution containing 1 mg/ml collagenase A and 0.6 U/ml Dispase for 30 min at 37°C. This digested material was then triturated with a 10-ml pipette and sieved through a stainless steel screen (30 mesh, 0.52-mm openings). The filtrate, at this point, consisted of short, intact tubule fragments of ~100-200 µm in length. Following the techniques described for isolation of rat PTs (48), the tubule suspension was washed multiple times in HBSS by low-speed centrifugation and then placed in a 1:1 mixture of Percoll and 2× Krebs-Henseleit buffer containing (in mM) 240 NaCl, 8 KCl, 2 KH2PO4, 30 NaHCO3, 2.4 CaCl2 · 2H2O, 2.4 MgSO4 · 7H2O, 10 glucose, and 20 HEPES.

The suspension was centrifuged through the Percoll density gradient at 15,000 g for 30 min (4°C). For chick kidney, this process resulted in two major tissue bands at low and high densities. The high-density band, designated as the "PT band," consisted almost entirely of short PT fragments, as assessed by microscopic appearance and marker enzyme enrichment (45). The PT band was removed from the Percoll and washed several times in HBSS and one time in growth media. These washing steps and all subsequent work was done in the absence of antibiotics. A final suspension of tubules was prepared in 3-4 ml of growth media and used for seeding culture inserts.

Before each preparation (1 day), 12 Nunc tissue culture inserts were collagen coated by soaking the filters in a 20:1 dilution of type I calfskin collagen, removing excess solution and allowing the filters to completely air-dry. The inserts were prewetted with growth medium several hours before seeding. The growth media used was serum-free and antibiotic-free DME/F-12 (1:1) supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenite, 5 × 10-8 M hydrocortisone, and 20 µM ethanolamine.

Six to seven drops of the final tubule suspension were seeded in each prepared filter insert; they were placed in individual wells of a 24-well culture dish with 0.5 ml growth media added to both the outer wells and inner cup. Cells were grown in a 37°C incubator with an atmosphere containing 5% CO2 and were fed every second day. Under these growth conditions, monolayers typically reached confluence within 7-10 days after seeding, as determined with a "dipstick"-style resistance meter (EVOM meter; WPI, Sarasota, FL). Monolayers were previously shown to be highly polarized, with apical microvilli and proximal-like electrophysiological characteristics, including glucose-stimulated Isc (45).

Electrophysiology. Filter inserts with intact monolayers were mounted in modified Ussing chambers with adapters fitted for the Nunc 10-mm cups (Warner Instrument, Hamden, CT). An "O" ring sealed the outside of the cup within the adapter. Thus the epithelial monolayer formed an intact barrier between circulating apical and basolateral Ringer solutions, with no edge damage. A transport buffer containing (in mM) 130 NaCl, 4 KCl, 1.3 CaCl2, 1 MgSO4, 5 HEPES, and 25 NaHCO3 was circulated on both sides and gassed with 5% CO2-95% O2 (pH 7.5). For Cl- substitution experiments, NaCl and KCl were partially or completely replaced with gluconate salts on both sides. Heated reservoirs kept the buffers (16 ml on each side) at 37°C. The monolayers were short-circuited with an automatic two-channel voltage clamp (DVC 1000; WPI) with correction for fluid resistance compensation. Ringer-agar bridges were used to electrically couple the apical and basolateral solutions to a matched pair of calomel half-cells for measurement of the potential difference (PD). A second set of bridges was connected to a pair of Ag/AgCl wires for passing current. Isc was measured continuously and displayed on a strip-chart recorder, with intermittent measurement of the open-circuit PD. Transepithelial resistance (TER) was also monitored continuously by current deflections in response to 2-s changes in the clamping voltage (to 1 mV) every 5 min.

Experiments were always run in pairs on monolayers selected for similar resistances from the same culture. For each experimental group (e.g., EIPA inhibition), data were collected from at least four different cultures. We have observed that most of the variation in responses occurs between cultures, with a high degree of consistency between monolayers from the same culture. Once a stable baseline Isc was obtained, glucose was added to both apical and basolateral solutions to a final concentration of 5 mM. The resulting increase in Isc was regarded as a check on the proximal-like behavior of these cultures (45). All other agents were added after the Isc had stabilized again, after glucose addition (postglucose baseline). PTH, agonists, and inhibitors were added from concentrated stocks, with a minimum of 15 min between additions (20 min after PTH addition). Changes in the Isc from the previous, extrapolated current values were calculated in units of microamperes per square centimeter (0.5 cm2 growth surface on the Nunc 10-mm inserts). For the antagonist studies, one monolayer of a pair was chosen at random to serve as a control. After postglucose stabilization, the antagonist was added to one monolayer and an equal volume (16 µl) of appropriate vehicle to the other. This was followed 15-20 min later by PTH addition (10-9 M) to the basolateral side of both the control and antagonist-treated monolayers.

The following agents were used at the indicated final concentrations, derived from various published studies (see DISCUSSION): channel/transporter blockers: EIPA, 100 or 500 µM, apical; glibenclamide, 300 µM, apical; 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 100 or 500 µM, apical; amiloride, 10 µM, apical; and DIDS, 100 µM, apical; and agonists: forskolin, 0.2 or 10 µM, basolateral; dibutyryl-cAMP (DBcAMP), 500 µM, both sides; and PMA, 100 nM, both sides. In some experiments, the phosphodiesterase inhibitor IBMX was added to both sides at 100 µM. The dose response to PTH was tested in the range of 10-10 to 1.4 × 10-7 M, with a "standard" concentration of 1.0 × 10-9 M used for inhibitor and Cl- substitution studies. According to our dose-response studies, this concentration gave ~40% of the maximal response.

Immunoblotting. Standard Western blotting methods were used to investigate the possible presence of a CFTR-like protein in PT culture extracts. Monolayers were extracted in 1% Nonidet P-40 containing a protease inhibitor cocktail (Complete Mini; Roche Molecular Biochemicals). Total protein (30 µg) was loaded on 8% SDS-polyacrylamide gels and probed with the following two separate commercial anti-human CFTR antibodies: a COOH-terminal monoclonal MAB 25031 (R & D Systems, Minneapolis, MN) and monoclonal MA1-935 (Affinity Bioreagents, Golden, CO). Neither antibody detected specific CFTR antigen in these extracts.

Data analysis and statistics. Data are expressed as means ± SE. The responses to PTH, forskolin, DBcAMP, and PMA were analyzed by measuring the changes in current at 2, 10, and 20 min after addition. Effects of PTH in the presence or absence of inhibitors were analyzed at 2 and 10 min after hormone addition. Two-minute peak responses to PTH were used for the PTH dose-response and Cl- substitution series. Significant differences between groups (P < 0.05) were assessed with ANOVA followed by a Tukey test or with paired and unpaired Student's t-tests.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Baseline electrophysiological characteristics. Table 1 presents baseline electrophysiological measurements and Isc responses to glucose addition for all groups used in this study. Baseline Isc was somewhat variable, ranging from mean values of 7 to 15 µA/cm2, but with no statistically significant differences among groups. All monolayers grown under these conditions and tested with normal Cl- transport buffer consistently displayed low PDs and a modest TER, ranging from 0.6 to 1.9 mV and 63 to 150 Omega  · cm2, respectively. There were some significant differences in TER among groups in Table 1, mostly compared with the higher TER seen in the monolayers tested at the lowest Cl- concentrations ([Cl-]; 2.6 mM). All monolayers also consistently displayed a glucose-stimulated increment in Isc, attributable to a Na+-glucose luminal cotransporter and characteristic of the vertebrate PT (13, 15, 45).

                              
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Table 1.   Baseline electrophysiological characteristics

PTH-induced current response. Figure 1 presents tracings from two examples of experiments on paired PT monolayers. As seen in Fig. 1A, top trace, exposure of the chick monolayers to 1.0 × 10-9 M PTH resulted in a positive current response that peaked at 2 min and slowly decayed thereafter. Data from 43 PTH-treated monolayers (taken from the control monolayers from all antagonist groups combined) are summarized in Fig. 2. The peak response at 2 min averaged 14.30 ± 1.58 µA/cm2, falling thereafter to 6.78 ± 0.63 and 3.38 ± 0.44 µA/cm2 at 10 and 20 min, respectively, still significantly different from the baseline current (P < 0.01).


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Fig. 1.   Short-circuit current (Isc) recordings from two sample experiments (A and B), each showing tracings (top and bottom) from a matched pair of proximal cell monolayers. All monolayers were initially tested for glucose responsiveness (a). Spikes represent command voltage steps for measurement of transepithelial resistance (TER). A: experiment showing positive Isc responses to 10-9 M parathyroid hormone (PTH; c) and 10 µM forskolin (d) in a control (top trace) and EIPA-treated (bottom trace) monolayer pair. EIPA (100 µM, apical) reduced the postglucose baseline current (b) and nearly abolished both PTH and forskolin-activated Isc responses. B: experiment showing opposite effects of 500 µM dibutyryl (DB)-cAMP (top trace) and 100 nM PMA (bottom trace), activators of PKA and PKC signaling pathways, respectively. Whereas DBcAMP stimulated a slow increase in Isc, PMA reduced the baseline current. EIPA (d) abolished both the cAMP-induced current and part of the residual current in both monolayers.



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Fig. 2.   Summary of agonist effects on Isc responses. Isc values (2, 10, and 20 min) are plotted for 10-9 M PTH (n = 43), 10 µM forskolin (n = 8), 500 µM DBcAMP (n = 8), and 100 nM PMA (n = 9). PMA and DBcAMP were added to both apical and basolateral bathing solutions; PTH and forskolin were added to the basolateral side only. Note marked overshoot (2-min response) to forskolin and decrease (negative change in Isc) in response to PMA. Values are means ± SE. * Values significantly different from baseline, P < 0.01 (paired t-test).

Positive Isc responses were also produced by the adenylate cyclase activator forskolin, as demonstrated in Fig. 1A, top trace, and by the membrane permeable DBcAMP (Fig. 1B, top trace). An early, rapid peak response to forskolin is clearly evident, followed by a sustained late response, whereas the response to DBcAMP exhibits a slower, monotonic rise in Isc. In contrast, 100 nM of the phorbol ester PMA, an activator of protein kinase C (PKC), slowly inhibits the current in these cells over the 20-min time course (Fig. 1B, bottom trace). The time courses for these experimental groups are summarized in Fig. 2. The marked 2-min overshoot after forskolin is clearly seen. The sustained responses at 10 and 20 min were similar for forskolin and DBcAMP, averaging between 9 and 12.5 µA/cm2. In contrast, the slow decrease in Isc with PMA reached an average 20-min value of -7.28 ± 1.83 µA/cm2.

The forskolin overshoot was most likely because of a high rate of cAMP production, followed by secondary regulation by phosphodiesterase and possibly other regulatory controls. This interpretation was supported by a separate series of experiments performed with the phosphodiesterase inhibitor IBMX, combined with lower doses of forskolin. In these experiments, 100 µM IBMX alone raised the baseline postglucose Isc by 7.53 ± 1.09 µA/cm2 (n = 15). Subsequent addition of 0.2 µM forskolin further raised Isc by 9.10 ± 1.62 µA/cm2 without an overshoot. Raising the forskolin concentration to the standard level of 10 µM had no further significant effect on Isc (0.20 ± 0.11 µA/cm2), indicating that IBMX and low-dose forskolin maximally stimulated this transport system. These data, combined with the observation that DBcAMP also stimulates Isc in a monotonic fashion, suggest that cAMP activates a single, coupled transport process in these cells.

The PTH stimulation of Isc was tested over a range of cumulative concentrations of the hormone, from 10-10 to 1.37 × 10-7 M. Figure 3 shows a clear dose-dependent response over a 100-fold range, with a threshold at 5 × 10-10 M, an apparent half-maximal activation of 14 µA/cm2 at 5 × 10-9 M, and a maximal response of 28 µA/cm2 at 5 × 10-8 M PTH. The avian antidiuretic hormone arginine vasotocin had no effect on Isc in these cells, even at 10-6 M (data not shown).


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Fig. 3.   PTH dose-response curve for hormone-induced Isc. Changes in Isc are plotted as a function of sequentially increasing doses of PTH for 14 proximal monolayers (means ± SE). Half-maximal stimulation was seen at 5 × 10-9 M PTH.

Effects of transporter and channel blockers on the PTH-stimulated Isc. A number of transport inhibitors were found to partially block the postglucose Isc, as exemplified by EIPA, an inhibitor of NHEs (Fig. 1A, bottom trace). Table 2 summarizes the initial effects of these inhibitors on post-glucose Isc and TER values. The NHE inhibitor EIPA, the Cl- channel blockers glibenclamide and NPPB, and the Na+ channel blocker amiloride all significantly reduced Isc, with EIPA causing the greatest effect. DIDS, a blocker of Cl-/base exchange, had no overall effect on Isc, although some of the individual monolayers showed either increases or decreases in current. EIPA, glibenclamide, and NPPB also significantly increased TER, as did DIDS. Amiloride, however, had no significant effect on this parameter.

                              
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Table 2.   Effects of inhibitors on postglucose Isc and TER

The effects of these inhibitors on monolayer responses to 1 × 10-9 M PTH are shown in Fig. 4. To preserve information about the PTH time course, both 2- and 10-min responses are plotted. The open bars show significant Isc responses to PTH in the control monolayers of these five groups, with means ranging from 13.2 to 21.7 µA/cm2 at the 2-min time points. The filled bars show significant inhibition of this response in paired monolayers with apical addition of EIPA, glibenclamide, and NPPB (added 15-20 min before PTH addition). EIPA at 100 µM significantly reduced the 2-min PTH response from 14.81 ± 3.41 to 2.63 ± 0.92 µA/cm2 and the 10-min response from 6.56 ± 1.24 to 1.94 ± 0.64 µA/cm2 (P < 0.05). The Cl- channel blockers glibenclamide and NPPB also significantly attenuated the PTH response, although not as fully as EIPA. Average inhibition ranged from 45 to 65% at the 2- and 10-min time points. Ten micromolar amiloride, a dose normally used to effectively block electrogenic epithelial Na+ channels, was completely ineffective against the PTH response as was also the case with 100 µM DIDS.


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Fig. 4.   Summary of inhibitor effects on PTH-induced Isc responses. Isc responses (2 and 10 min) are shown for paired monolayers in the absence (open bars) or presence (filled bars) of various antagonists. All inhibitors were added to the apical bathing solution at the following concentrations (no. of pairs in parentheses): EIPA, 100 µM (n = 8); glibenclamide (Glib), 300 µM (n = 9); 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 100 µM (n = 9); DIDS, 100 µM (n = 9); amiloride (Amil), 10 µM (n = 8). Values are means ± SE. All open bars represent values significantly elevated over baseline Isc (P < 0.01, paired t-test). * Significant decreases in Isc responses with inhibitor compared with matched control monolayers (P < 0.05, unpaired t-test).

To evaluate the inhibitor efficacy and additivity of the Cl- dependence and EIPA sensitivity of this system, a separate series of experiments was performed with the NHE inhibitor EIPA and the Cl- channel blocker NPPB. Isc was first maximally stimulated with IBMX and 0.2 µM forskolin as described above. Addition of 100 µM EIPA reduced the Isc by 19.80 ± 3.68 µA/cm2 from a stimulated baseline of 35.20 ± 4.44 µA/cm2. Increasing the EIPA concentration in this series (n = 5) to 500 µM reduced Isc by an additional 11.70 ± 1.29 µA/cm2. The combined inhibition was 90% of the total stimulated current. When these monolayers were further treated with 500 µM NPPB, the Isc declined nonsignificantly by 0.20 ± 0.20 µA/cm2. In a complementary set of experiments, monolayers were sequentially exposed to 100 µM NPPB, 500 µM NPPB, and 500 µM EIPA. From a stimulated Isc of 34.10 ± 4.22 µA/cm2, these treatments decreased Isc by 9.70 ± 1.55, 15.00 ± 1.35, and 3.80 ± 1.35 µA/cm2, respectively (n = 5). The combined inhibition was 84% of the total stimulated current. Thus these data demonstrate that higher doses of each inhibitor alone were almost fully effective on the transport currents in these monolayers and that the Cl--dependent and EIPA-sensitive components do not appear to be additive.

Dependence of the PTH response on [Cl-]. The PTH-induced Isc response was found to be completely dependent on the presence of Cl- (Fig. 5). Symmetrical reduction of [Cl-] in the bathing solutions to 2.6 mM essentially abolished the PTH response. Increments in [Cl-] between 25 and 137 mM restored the PTH response in a dose-dependent manner. Half-maximal stimulation was obtained at [Cl-] levels of 50 mM and maximal stimulation at levels >65 mM.


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Fig. 5.   Cl- dependence of the PTH-induced Isc response. Peak responses (at 2 min) to PTH (10-9 M) are shown from separate groups of monolayers bathed on both sides with buffers containing 2.6 (n = 8), 25 (n = 5), 50 (n = 4), 65 (n = 5), 80 (n = 5), 110 (n = 5), or 137 (n = 20) mM Cl-. Cl- substitution was made with sodium and potassium gluconate salts. Half-maximal stimulation occurred at 50 mM Cl- concentration. Data are means ± SE.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study describes a novel effect of PTH on a chick PT culture system. This primary culture model has previously been shown to exhibit properties characteristic of vertebrate PTs, including apical microvilli and vectorial transport, exemplified by glucose-stimulated Isc (45). In the present study, PTH at 10-9 M consistently induced a positive increase in Isc that was EIPA sensitive. The Isc response peaked at 2 min and then decayed over a 10- to 20-min time course, possibly reflecting receptor desensitization (Figs. 1A and 2). The Isc response was dose dependent over a 100-fold range, with a half-maximal effect at 5 × 10-9 M PTH. This dose-response relationship is similar to that seen for PTH inhibition of fluid reabsorption in vivo (4) and for stimulation of cAMP production (9) and inhibition of NHE activity (17) in the OK PT cell line. The lack of response to even high doses of the avian antidiuretic hormone arginine vasotocin indicates specificity of this PTH response in these chick PT cells.

Forskolin resulted in a similar Isc response to PTH. The large 2-min overshoot appeared to be because of secondary phosphodiesterase activation, since separate experiments with IBMX and 10-fold lower forskolin maximally stimulated Isc without an overshoot (see RESULTS). The sustained current after forskolin showed little decay with time. The membrane-permeant cAMP analog DBcAMP also increased Isc, but with a slower onset and lack of overshoot (Figs. 1B and 2). These observations suggest that PTH is acting on this transport system via the adenlylate cyclase/PKA signaling pathway. Interestingly, the phorbol ester PMA, an activator of PKC, caused a decrease in baseline Isc. Thus these two signaling pathways, both of which are activated by PTH in proximal cells (1, 9), appear to have opposite effects on this Isc response. In the OK cell model, it has generally been observed that activation of either the PKA or PKC signaling pathways inhibits NHE activity (1, 17). On the other hand, PKC stimulation resulted in increased Na+/H+ activity in both native tissues (rabbit BBMV) and primary cultures of rabbit PTs (19, 51). Thus the potential role of PKC regulation of apical NHE activity is uncertain but may be related to different PKC isoforms present in various cell types or under different assay conditions (15).

In the mammalian PT, PTH is known to inhibit Na+, fluid, and bicarbonate reabsorption, largely through its inhibition of the NHE3 isoform of the NHE family (10, 14, 16, 54). However, in this chick primary culture model, PTH appears to stimulate an NHE activity that is either linked to or dependent on a Cl- transport process. Several observations support this conclusion. First, the Isc response to PTH was nearly abolished by apical addition of 100 µM EIPA (Figs. 1A and 4). This amiloride analog is generally considered to be a selective inhibitor of NHE transporters, although various isoforms may have different inhibitory constants (18, 34). In contrast, amiloride itself, at a dose that inhibits electrogenic epithelial Na+ channel activity (32, 53), had no effect on the PTH-induced current response, although it did slightly decrease the baseline Isc (Table 2 and Fig. 4). Thus it seems unlikely that PTH upregulates Na+ channels in this system, as has been proposed for mammalian PTs (15).

Second, regarding Cl- dependency, both Cl- replacement and two different Cl- channel blockers significantly inhibited the PTH-induced response. Glibenclamide, a sulfonylurea receptor inhibitor used to stimulate beta -cell insulin release, has also been widely used as a blocker of CFTR Cl- channels (41, 43). Similarly, the arylaminobenzoate NPPB is known as a potent inhibitor of a variety of Cl- channels (33, 38, 41). Both of these blockers, when added to the apical side, significantly reduced the PTH-induced Isc response (Fig. 4). In contrast, apical addition of 100 µM DIDS has no significant effect on this PTH response. DIDS is primarily used at these concentrations as a blocker of Cl-/base or Cl-/formate/oxalate exchangers of the PT (49). This compound also blocks some types of Cl- channels, although typically at higher concentrations (32-34, 38, 41). However, it is ineffective against CFTR channels from the extracellular side (41). Thus this result also rules out such anion exchangers as a possible mechanism behind the PTH-induced current response. It should be noted that, when used at higher concentrations, both NPPB and EIPA eliminated ~80-90% of the total stimulated current, and there was almost no additivity of these two blockers (see RESULTS).

Bilateral reduction of [Cl-] in the bathing solutions, from 137 to 2.6 mM, essentially abolished the Isc response (Fig. 5). Increasing [Cl-] over the range of 25-137 mM restored the full hormone response, with a half-maximal response at 50 mM. These data suggest that a low-affinity Cl- transport process is somehow linked to the PTH response. Because, in the present study, we did not measure isotope fluxes, it is not possible to identify the ion(s) responsible for the Isc response. Given the simple composition of the transport buffer used in these studies, positive currents would most likely be mediated by Cl- secretion (basolateral-to-apical flux), Na+ reabsorption, or some combination of these. A number of studies have suggested that intracellular Cl- levels in PT cells lie above electrochemical equilibrium; i.e., increased apical Cl- conductance would result in Cl- exit from the cell, rather than entry (44). It should also be noted that, under these short-circuit conditions, passive (i.e., electrically driven) coupling of Cl- fluxes is eliminated. Thus, taken together, these data suggest that PTH activates both NHE activity and a Cl- channel, one that is possibly CFTR related. These activities appear to be functionally linked, either at the transport level or via a common regulatory pathway.

Previous clearance studies in chickens have shown that, as in mammals, PTH administration results in whole animal diuresis, natriuresis, and urinary alkalinization (26), suggesting a conventional inhibitory action of PTH on NHE3 activity of the PT, in addition to the stimulated system seen in this study. This leads to the question of whether a PTH-induced Isc response may also be present in mammalian PTs, masked by the much larger inhibitory effect on Na+ reabsorption. To our knowledge, there are no other reports of PTH-stimulated current in PT cells. However, several observations suggest that a similar system may be present in mammals. First, in primary cultures of human PT cells, forskolin caused a similar positive Isc response to that seen in the chick cultures (47). PTH was not tested in these human cells, however, and possible implications of the forskolin response were not addressed.

Second, patch-clamp studies in primary cultures of rabbit PTs revealed a PTH-activated Cl- channel (46). This Cl- conductance could also be activated by forskolin, by a catalytic subunit of PKA, and, interestingly, also by PKC exposure. cAMP- and PKA-activated Cl- channels were also observed in primary cultures of rat PTs (12). Furthermore, PTH and cAMP have been shown to increase Cl- membrane permeability in rat kidney BBMV (27).

Regarding the possibility of CFTR localization to the PT, several studies have demonstrated CFTR mRNA in PT segments or in cultured cells (21, 31, 38), whereas expression at the protein level has been observed by some (6, 11), but not other, investigators (38). In the present study, attempts were made to detect CFTR protein in monolayer extracts using two different anti-human CFTR antibodies. Although these attempts were unsuccessful, this could indicate that the antibodies lacked sensitivity or that the transporter activity is species specific or distinct from the human CFTR protein, despite pharmacological similarities. Nevertheless, the recent realization that CFTR controls a wide variety of membrane channels and transporters (22, 42) and that a family of intracellular signal complex proteins, known as Na+-H+ exchanger regulatory factors (NHERFs), bind both NHE and CFTR via PDZ domains (50) provides a possible model for linkage of NHE and CFTR function.

Although the transport mechanism behind the PTH-induced Isc response is unknown, an intriguing possibility is suggested by recent studies of a novel Cl--dependent NHE found in rat distal colon crypt cells (33, 34, 39). As determined by pH gradient-stimulated 22Na uptake by apical membrane vesicles, this "Cl-NHE" requires Cl- and is sensitive to both NPPB and EIPA. High, channel-blocking concentrations of DIDS also inhibited activity, but lower concentrations, used to inhibit anion exchangers, had no effect (33, 34). Moreover, an antibody to CFTR also partially blocked Cl-NHE activity (33). Recently, this transporter was cloned from rat distal colon crypt cells and shown to have homology with NHE1, but with a markedly shortened and novel COOH-terminal domain (39). Of particular interest, cDNA probes for this transporter revealed widespread distribution of specific mRNA in several rat tissues, including kidney. This again raises the possibility of a system similar to that described in the present study in mammalian PTs. In this regard, Choi et al. (8) have shown that 50% of EIPA-sensitive proximal NHE activity remains intact in NHE2 plus NHE3 double-knockout mice, suggesting the presence of an as yet undefined NHE activity.

It is unclear what physiological function this system might have in the avian and/or mammalian PT or how its activation by PTH fits in with the other known effects of this hormone. Because these studies were performed in a tissue culture environment, in vivo implications need to be considered with caution. Among other factors, hormone release patterns (pulsatile vs. continuous), stability, and concentration in the whole animal will be different. Furthermore, it is possible that the observed current response represents only one manifestation of a multistep process in vivo. Nevertheless, it is interesting to consider several avian PT transport systems that are affected by PTH. In birds, PTH is known to stimulate a secretory component of Pi transport (52) in addition to its inhibition of reabsorptive transport (13, 36). PTH was also shown to increase urate clearance in birds, although the mechanism of this response was not determined (23). It is also of interest that Cl- secretion has been correlated with net fluid secretion in vertebrate PTs, clearly demonstrated in teleosts (5) and hypothesized to exist in other vertebrates (44). Potential interactions among these transport systems deserve further study.


    ACKNOWLEDGEMENTS

This study was funded by National Science Foundation Grant IBN-9870810 (G. Laverty). Additional support was from The Icelandic Research Council and the University of Iceland's Sattmalasjodur (S. S. Árnason). Funding was also provided (A. Sheldon) by a grant from the Howard Hughes Medical Institute to the University of Delaware (Improving Undergraduate Biology Education).


    FOOTNOTES

Address for reprint requests and other correspondence: G. Laverty, Dept. of Biological Sciences, Univ. of Delaware, Newark, DE 19716 (E-mail: Laverty{at}udel.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published December 27, 2002;10.1152/ajprenal.00281.2002

Received 6 August 2002; accepted in final form 15 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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