Vol. 284, Issue 5, F987-F995, May 2003
PTH stimulates a Cl
-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
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ABSTRACT |
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 × 109 M and maximal response at 5 × 108
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
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INTRODUCTION |
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).
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MATERIALS AND METHODS |
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 × 108 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 (109 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
1010 to 1.4 × 107 M, with a
"standard" concentration of 1.0 × 109 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.
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RESULTS |
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 
· 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).
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 × 109 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 109 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 109 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).
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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
1010 to 1.37 × 107 M. Figure
3 shows a clear dose-dependent response
over a 100-fold range, with a threshold at 5 × 1010
M, an apparent half-maximal activation of 14 µA/cm2 at
5 × 109 M, and a maximal response of 28 µA/cm2 at 5 × 108 M PTH. The avian
antidiuretic hormone arginine vasotocin had no effect on
Isc in these cells, even at 106 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 × 109 M PTH.
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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.
The effects of these inhibitors on monolayer responses to 1 × 109 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).
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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
(109 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.
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DISCUSSION |
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 109 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 × 109 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 
-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.
| |
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ABSTRACT
INTRODUCTION
