Acute application of TNF stimulates apical 70-pS K+ channels in the thick ascending limb of rat kidney
Yuan Wei,
Elisa Babilonia,
Paulina L. Pedraza,
Nicholas R. Ferreri, and
Wen-Hui Wang
Department of Pharmacology, New York Medical College, Valhalla, New York
10595
Submitted 17 March 2003
; accepted in final form 19 May 2003
 |
ABSTRACT
|
---|
TNF has been shown to be synthesized by the medullary thick ascending limb
(mTAL) (21). In the present
study, we used the patch-clamp technique to study the acute effect of TNF on
the apical 70-pS K+ channel in the mTAL. Addition of TNF (10 nM)
significantly stimulated activity of the 70-pS K+ channel and
increased NPo [a product of channel open probability
(Po) and channel number (N)] from 0.20 to 0.97.
The stimulatory effect of TNF was observed only in cell-attached patches but
not in excised patches. Moreover, addition of TNF had no effect on the
ROMK-like small-conductance K+ channels in the TAL. The
dose-response curve of the TNF effect yielded a Km value
of 1 nM, a concentration that increased channel activity to 50% maximal
stimulatory effect of TNF. The concentrations required for reaching the
plateau of the TNF effect were between 5 and 10 nM. The stimulatory effect of
TNF on the 70-pS K+ channel was observed in the presence of
N
-nitro-L-arginine methyl ester. This
indicated that the effect of TNF was not mediated by a nitric oxide-dependent
pathway. Also, inhibition of PKA did not affect the stimulatory effect of TNF.
In contrast, inhibition of protein tyrosine kinase not only increased activity
of the 70-pS K+ channel but also abolished the effect of TNF.
Moreover, inhibition of protein tyrosine phosphatase (PTP) blocked the
stimulatory effect of TNF on the 70-pS K+ channel. The notion that
the TNF effect results from stimulation of PTP activity is supported by PTP
activity assay in which treatment of mTAL cells with TNF significantly
increased the activity of PTP. We conclude that TNF stimulates the 70-pS
K+ channel via stimulation of PTP in the mTAL.
ROMK channel; protein tyrosine kinase; protein tyrosine phosphatase; NG-nitro-L-arginine methyl ester
THE THICK ASCENDING LIMB (TAL) is an important nephron segment
responsible for reabsorption of 20-25% of the filtered Na+ load
(11). Recent studies indicate
that the TAL produces TNF in response to bacterial LPS, ANG II, and elevated
extracellular calcium (15,
21,
28). Incubation of the TAL
with TNF in vitro inhibited ouabain-sensitive Rb+ uptake
(10), suggesting that TNF may
inhibit transcellular sodium transport in the TAL. However, the sites at which
TNF may affect membrane transport in the TAL have not been determined.
The apical K+ channels play an important role in K+
recycling, which is essential for maintaining the normal function of Na-K-Cl
cotransporter in the TAL (11).
There are at least three types of K+ channels:
Ca2+ activated, large conductance, and 30 and 70 pS
(3,
13,
29). It is generally accepted
that the 70- and 30-pS K+ channels are major K+ channels
responsible for K+ recycling
(30). In addition, it is
possible that ROMK is an important component of both 70- and 30-pS
K+ channels because neither K+ channel can be found in
ROMK knockout mice (18). We
showed that apical K channels are regulated by a variety of signaling
pathways, including nitric oxide (NO), protein kinase C, and protein tyrosine
phospatase (PTP) (12,
19,
32). TNF has been shown to
activate these same pathways
(1,
5,
16,
20,
35). Thus the main goal of the
present study is to investigate the acute effect of TNF on apical
K+ channels in the mTAL and delineate the mechanism by which TNF
regulates the apical K+ channels.
 |
METHODS
|
---|
Preparation of medullary TAL. Pathogen-free Sprague-Dawley rats
(5-6 wk; Taconic Farms, Germantown, NY) were used in the experiments. Animals
were kept on normal rat chow and had free access to water for 7 days before
use. The rats were killed by cervical dislocation, and the kidneys were
rapidly removed. Several thin (0.5-1 mm) slices were cut from the kidney and
placed in ice-cold NaCl Ringer. The medullary TAL (mTAL) tubules were isolated
with two watch-making forceps and placed on a 5 x 5-mm cover glass
coated with polylysine (Collaborative Research, Bedford, MA). The cover glass
was transferred to a chamber mounted on an inverted microscope (Nikon,
Melville, NY), and the tubules were superfused with a bath solution containing
(in mM) 140 NaCl, 5 KCl, 1.8 MgCl2, 1.8 CaCl2, 5
glucose, and 10 HEPES (pH = 7.4). We used a sharpened pipette to open the mTAL
to gain access to the apical membranes. The tubule was superfused with NaCl
Ringer, and the temperature was maintained at 37°C.
Patch-clamp technique. Electrodes were pulled with a Narishige
model PP83 vertical pipette puller and had resistances of 4-6 M
when
filled with 140 mM NaCl. The channel current recorded by an Axon200A
patch-clamp amplifier was low-pass filtered at 1 kHz using an eight-pole
Bessel filter (902LPF, Frequency Devices, Haverhill, MA). The current was
digitized by an Axon interface (Digitada1200), collected by an IBM-compatible
Pentium computer (Gateway 2000) at a rate of 4 kHz, and analyzed using the
pClamp software system 6.04 (Axon Instruments, Burlingame, CA). Channel
activity was defined as NPo, a product of channel open
probability (Po) and channel number (N). The
NPo was calculated from data samples of 60-s duration in
the steady state as follows
 | (1) |
where ti is the fractional open time spent at
each of the observed current levels. Because three types of K+
channels have been identified in the mTAL
(3,
6,
13,
29), we measured the channel
current at three different membrane potentials in each patch to estimate the
conductance of the K+ channel in the patch.
Cell cultures and measurement of protein tyrosine kinase and PTP
activity. Cultured mTAL cells were used to measure the activity of
protein tyrosine kinase (PTK) and PTP before and after treatment with TNF. The
method for isolation and primary culture of mTAL cells has been previously
described (28). The TAL cells
were incubated in the presence or absence of TNF (10 nM) for 10 and 15 min. We
followed the method described previously to measure PTK
(31). Cells were lysed in 100
µl of RIPA buffer [150 mM NaCl, 50 mM Tris·HCl (pH 7.4), 50 mM
-glycerophosphate, 50 mM NaF, 1 mM sodium orthovanadate, 2.5 mM EDTA, 5
mM EGTA, 0.5 mM dithiothreitol, 1% Triton X-100, 0.5% sodium deoxycholate,
0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5
µg/ml leupeptin, and 2 µg/ml pepstatin]. For activity assay of PTK, 10
µl of the sample were incubated in a total volume of 30 µl containing
200 µM[32P]ATP (1 cpm/fmol), 12 mM magnesium acetate, 2 mM
MnCl2, 0.3 mM dithiothreitol, 0.5 mM sodium orthovanadate, 0.5 mM
ammonium molyb-date, and 2 mM R-R-Src peptide
(Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Gly). Reactions were quenched by
adding 200 ml of 75 mM phosphoric acid, and 15 µl of cocktail were spotted
on phosphocellulose paper. After several washes, the amount of 32P
incorporated into R-R-Src peptide was assessed using a liquid scintillation
counter.
We used 32P-labeled myelin basic protein described originally by
Tonks et al. (27) as a
substrate for the measurement of PTP activity. The release rate of
32P from the basic protein is used as an index of PTP activity, and
the value obtained in the presence of vanadate is used as background. The
activity of PTP was measured with a BioLabs PTP assay kit following the
instruction provided by the vender (Bio-Rad, Hercules, CA). Briefly, cultured
mTAL cells were incubated in the presence or absence of TNF (10 nM) for 10
min. After treatment, cells were lysed in 100-µl buffer solution containing
50 mM NaCl, 50 mM Tris·HCl (pH 7.4), 2.5 mM EDTA, 2.0 mM
dithiothreitol, 0.01% Brij 35, 1.0 µg/ml aprotinin, 1.0 µg/ml leupeptin,
1.0 µg/ml pepstatin, and 2 mM phenylmethyl sylfonyl fluoride. The mixture
(10 µl) was added to a tube containing 30-µl assay buffer composed of 1
mM NaCl, 50 mM Tris·HCl (pH 7.4), 1 mM EDTA, 5 mM dithiothreitol, 0.01%
Brij 35, and 1 mg/ml BSA at 30°C for an additional 5 min. Ten microliters
of the 32P-labeled myelin basic protein were added to the sample
followed by incubation for 10 min at 30°C. Reactions were terminated by
adding 200 µl of ice-cold 20% trichloride acetate into the tube. The sample
was centrifuged for 5 min at 12,000 g, the resultant supernatant was
carefully taken, and the amount of 32P released from the peptide
was measured using a liquid scintillation counter.
Solution and statistics. The pipette solution was composed of (in
mM) 140 KCl, 1.8 Mg2Cl, and 5 HEPES (pH = 7.4).
NG-nitro-L-arginine methyl ester (L-NAME) and
phenylarsine oxide were purchased from Sigma (St. Louis, MO), whereas H8 and
herbimycin A were obtained from Biomol (Plymouth Meeting, PA). Herbimycin A
was dissolved in DMSO and the final concentration of DMSO was <0.1% that
had no effect on channel activity. TNF was obtained from PeproTech (Princeton,
NJ) and diluted in sterile water at a concentration of 29 µM as a stock
solution. The data are means ± SE. We used paired and unpaired
Student's t-tests to determine the statistical significance. If the
P value was <0.05, the difference was considered to be
significant.
 |
RESULTS
|
---|
We first examined the effect of TNF on the 70-pS K+ channel
activity in a cell-attached patch. Figure
1 is a representative recording showing that addition of 10 nM TNF
increased NPo from 0.20 ± 0.02 to 0.97 ± 0.1
(n = 7). The effect of TNF on channel activity is concentration
dependent: addition of 50 and 500 pM TNF increased NPo
from 0.20 ± 0.02 to 0.33 ± 0.04 (n = 6) and 0.45
± 0.05 (n = 10), respectively. The stimulatory effect of TNF
was maximal at concentrations between 5 and 10 nM
(Fig. 2). Therefore, the
calculated Km value of TNF, a concentration that increased
channel activity to 50% the maximal value, was
1 nM
(Fig. 2). In addition to the
70-pS K+ channel, a ROMK-like 30-pS K+ channel was also
expressed in the mTAL (3,
18,
29). We examined the effect of
10 nM TNF on the ROMK-like 30-pS K+ channel. To determine the
effect of TNF on the 30-pS K+ channel, patches containing the 30-pS
K+ channels with a relatively low Po were
selected. Figure 3 is a
recording made in a cell-attached patch demonstrating that TNF did not affect
the activity of the 30-pS K+ channel (n = 3) because
NPo = 0.51 ± 0.05 (TNF) was not different from 0.50
± 0.05 (control).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1. Recording demonstrating the effect of 10 nM TNF on the 70-pS K+
channel activity. The experiment was performed in a cell-attached patch and
the pipette holding potential was 0 mV. Top: time course of the
experiment and the 2 parts of data indicated by numbers are extended to show
the fast time resolution. C-, channel closed.
|
|

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 3. Recording showing the effect of TNF (10 nM) on the 30-pS K+
channel in the medullary thick ascending limb (mTAL). Top: 2 traces
are 1-min channel activity recorded in the absence (control) or presence of
TNF. Two parts indicated by numbers are extended to show the fast time
resolution. The experiment was conducted in a cell-attached patch and the
holding potential was 0 mV.
|
|
As the dose-response curve of TNF was established, 5 nM TNF was used in
experiments designed to study the mechanism by which TNF stimulates the 70-pS
K+ channel. Several factors that have been shown to stimulate the
70-pS K+ channel include protein kinase A (PKA), the
NO-cGMP-dependent pathway, and PTP
(12,
19,
29). Therefore, we examined
the effect of TNF on the 70-pS K+ channel in the presence of PKA
inhibitors (Fig. 4). Inhibition
of PKA did not significantly affect channel activity. Moreover, inhibition of
PKA did not alter the TNF effect because application of TNF further increased
channel activity from 0.3 ± 0.03 to 1.10 ± 0.1 (n = 5)
in the presence of H8. This suggested that the stimulatory effect of TNF does
not result from stimulation of PKA.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 4. Recording demonstrating the effect of 5 nM TNF on the 70-pS K+
channel in the presence of H8 (5 µM). The experiment was carried out in a
cell-attached patch and the holding potential was 0 mV. Top: channel
activity in the presence of H8 (control) and TNF + H8 with slow time
resolution. Two parts of the data are demonstrated with extended time
course.
|
|
Stimulation of TNF receptors has been shown to increase NO production
(5,
16). Because the NO-dependent
cGMP pathway can stimulate activity of the 70-pS K+ channel, we
investigated the role of NO in mediating the effect of TNF on the 70-pS
K+ channel in the presence of L-NAME, an inhibitor of NO
synthase (NOS). Inhibition of NOS decreased the NPo of the
70-pS K+ channel to 0.13 ± 0.02 (n = 5). This is
consistent with the previous observation that L-NAME inhibited
activity of the 70-pS K+ channel
(19). However,
L-NAME did not block the effect of TNF because it increased
NPo from 0.13 ± 0.02 to 0.9 ± 0.1
(n = 5) in the presence of 0.5 mM L-NAME
(Fig. 5).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 5. Effect of 5 nM TNF on the 70-pS K+ channels in the presence of
0.2 mM NG-nitro-L-arginine methyl ester
(L-NAME). The experiment was carried out in a cell-attached patch
and the holding potential was 0 mV. Top: channel activity in the
presence of L-NAME and TNF + L-NAME with slow time
resolution. Two parts of the data are demonstrated with extended time
course.
|
|
Several studies demonstrated that stimulation of the TNF receptor increases
PTP (1,
26). We previously observed
that the 70-pS K+ channel is regulated by PTK and PTP
(12). If the effect of TNF was
the result of stimulation of PTP, which leads to enhancement of tyrosine
dephosphorylation, inhibition of PTK should mimic the effect of TNF such that
the effect of TNF on the 70-pS K+ channel activity should be absent
in the presence of a PTK inhibitor such as herbimycin A. This possibility has
been tested by experiments in which the effect of TNF was examined in the
tubule treated with herbimycin A. Figure
6 is a recording showing the effect of TNF on channel activity in
the presence of herbimycin A. We confirmed the previous finding that
inhibition of PTK significantly stimulates the 70-pS K+ channel
(12) and increases
NPo from 0.24 ± 0.02 to 1.22 ± 0.1
(n = 5). Furthermore, in the presence of herbimycin A, the
stimulatory effect of TNF on the 70-pS K+ channel is absent because
TNF did not significantly increase channel activity (NPo =
1.25 ± 0.1). This indicates that the effects of TNF and herbimycin A
are not additive.

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 6. Effect of 5 nM TNF on the 70-pS K+ channels in the presence of
herbimycin A (Herb; 2 µM). The experiment was carried out in a
cell-attached patch and the holding potential was 0 mV. Top: channel
activity under control conditions, in the presence of herbimycin A, and in the
presence of TNF + herbimycin A with slow time resolution. Three parts of the
data are demonstrated with extended time course.
|
|
Although the observation that the effects of TNF and herbimycin A are not
additive supports the notion that the effect of TNF on the 70-pS K+
channel is mediated by stimulation of PTP activity, it is possible that TNF
cannot further increase channel activity because it is already at peak
following inhibition of PTK. Therefore, we examined whether inhibition of PTP
can reverse the stimulatory effect of TNF. Because addition of phenylarsine
oxide (PAO), an inhibitor of PTP, decreased channel activity, we selected the
patches with a high NPo to study the effect of TNF in the
presence of PAO. Inhibition of PTP not only decreased NPo
from 0.44 ± 0.04 to 0.21 ± 0.01 (n = 5) but also
abolished the effect of TNF on channel activity because
NPo did not significantly alter and was 0.18 ± 0.01
(Fig. 7). Moreover, we examined
the effect of PAO on channel activity in the mTAL, which was challenged with
TNF. Application of TNF increased channel activity from 0.24 ± 0.02 to
0.92 ± 0.1 (Fig. 8).
However, in the presence of TNF, addition of PAO reduced channel activity from
0.92 ± 0.1 to 0.1 ± 0.01 (n = 5)
(Fig. 8). This further suggests
that the effect of TNF on the 70-pS K+ channel is mediated by
stimulation of PTP.

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 7. Effect of 1 µM phenylarsine oxide (PAO) and 5 nM TNF + PAO on the
activity of the 70-pS K+ channels. Data are summarized from 5
cell-attached patches. *Data are significantly different from the
control value (P < 0.05).
|
|

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 8. Recording illustrating the effect of PAO in the presence of TNF. The
experiment was carried out in a cell-attached patch and the holding potential
was 0 mV. Top: channel activity in the presence of TNF (5 nM), PAO (1
µM) + TNF, and wash-out with slow time resolution. Three representative
parts indicated by numbers are extended at fast time scale.
|
|
To examine whether TNF treatment can stimulate the activity of PTP, we used
cultured mTAL cells (28) to
measure the activity of PTP in the presence or absence of 10 nM TNF for 10
min. Results summarized in Fig.
9 show that treatment of mTAL cells with 10 nM TNF increased the
release of 32P from the substrate from 2,050 ± 100 pmol/mg
protein (control) to 4,200 ± 300 pmol/mg protein (n = 4). The
effect of TNF on PTP was specific because TNF did not alter the activity of
PTK (control 35,777 ± 2,000 pmol/mg protein, TNF 35,250 ± 2,100
pmol/mg protein, n = 5) in mTAL cells.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 9. Effect of TNF (10 nM) on the activity of protein tyrosine phosphatase (PTP)
in the cultured mTAL cells. The cells were treated with vehicle (control) or
TNF for 10 min and the release rate of 32P from the substrate, as
an index of activity of PTP, was measured with the synthesized peptide.
*Data are significantly different from the control value (without
TNF), P < 0.05.
|
|
 |
DISCUSSION
|
---|
The main findings of the present study are that acute application of TNF
stimulates the apical 70-pS K+ channel in the mTAL and that the
effect of TNF on channel activity is blocked by inhibition of PTP and PTK.
Similar observations that TNF acutely regulates K+ channel activity
have been reported in retinal ganglion cells
(8) and microglia cells
(22). In the present study, we
suggest that the effect of acute application of TNF on the 70-pS K+
channel is possibly mediated by stimulation of PTP activity. This conclusion
is supported by four lines of evidence: 1) inhibition of PTK
abolished the effect of TNF on channel activity; 2) the effect of TNF
was absent in the presence of PTP inhibitor; 3) application of PAO
reversed the stimulatory effect of TNF; and 4) treatment of mTAL
cells with TNF increased PTP activity. We hypothesize that acute application
of TNF stimulates PTP activity and leads to enhancement of tyrosine
dephosphorylation, which increases the activity of the apical 70-pS
K+ channel in the mTAL. However, when PTK has been inhibited by
herbimycin A, the effect of TNF is absent because inhibition of PTK also leads
to increased tyrosine dephosphorylation.
PTK and PTP have been shown to play important roles in the regulation of
the 70-pS K+ channel: stimulation of PTK decreases, whereas an
increase in PTP activity augments activity of the 70-pS K+ channel
(12). The effect of PTK on the
70-pS K+ channel is possibly the result of the stimulation of
tyrosine phosphorylation of the 70-pS K+ channel or its closely
associated proteins because addition of exogenous c-Src PTK could inhibit the
70-pS K+ channel in inside-out patches
(12). We also observed that
TNF did not inhibit the ROMK-like 30-pS K+ channel in the TAL. This
suggests that the 30-pS K+ channel is not directly regulated by
PTP. The finding is consistent with previous findings that stimulation of PTK
activity inhibits only the 70-pS K+ channel but not the 30-pS
K+ channel (12).
Therefore, the mechanism by which PTP or PTK regulates the apical 30-pS
K+ channel is different from that of the 70-pS K+
channel. In addition, the observation that inhibition of PTP decreased the
ROMK-like 35-pS K+ channel in the cortical collecting duct (CCD)
(34) indicates further that
ROMK-like small-conductance K+ channels in the mTAL and CCD are
also differentially modulated by PTK and PTP. Because ROMK1 is in the CCD
whereas ROMK2/3 is in the TAL
(4), this strongly suggests
that the mechanism by which PTK regulates ROMK1 or ROMK2/3 is different. The
physiological significance of the finding that only the 70-pS but not the
30-pS K+ channel is regulated by TNF is not clear. It is speculated
that the 30-pS K+ channel may be responsible for providing a basal
level of apical K+ conductance, whereas the 70-pS K+
channel activity is subjected to modulation by a variety of factors such as
TNF.
TNF is a cytokine produced by a variety of cells including the epithelial
cells in the mTAL (21). TNF
production increases in response to inflammation, injury, and infection. It
has been reported that IL-1 and -2 increase TNF production
(20). TNF plays an important
role in inflammation, cell differentiation, and cell death
(20). The role of TNF in renal
pathophysiological mechanisms has been well established. For instance, TNF may
contribute to the chronic tubule injury associated with hypercalcemia.
Moreover, TNF can be produced by physiologically relevant stimulations. It has
been shown that stimulation of the Ca2+-sensing receptor
increases TNF generation (15,
28).
Stimulation of the Ca2+-sensing receptor is expected
to decrease Na+ reabsorption in the mTAL
(14). One mechanism by which
stimulation of the Ca2+-sensing receptor inhibits
Na+ transport is to block K+ recycling across the apical
membrane in the mTAL. We previously demonstrated that raising extracellular
Ca2+ inhibits the apical 70-pS K+ channel by
increasing 20-HETE generation
(33). Because K+
recycling is important, inhibition of K+ channels should lead to
decreased Na+ absorption in the mTAL
(11). However, this
20-HETE-dependent mechanism may be responsible for acute stimulation of the
Ca2+-sensing receptor, whereas the TNF-cyclooxygenase
(COX)-dependent pathway is involved in mediating the effect of sustained
stimulation of the Ca2+-sensing receptor in the mTAL. It
has been shown that a prolonged stimulation of the
Ca2+-sensing receptor by incubation of mTAL cells in a
media containing high Ca2+ for more than 3 h increases
COX-2 expression and PGE2 generation. TNF has been shown to play a
key role in inducing COX-2 following stimulation of the
Ca2+-sensing receptor
(28). Because PGE2
has been shown to inhibit the apical K+ channel
(17) and other ion
transporters in the mTAL (7),
an increase in PGE2 production following stimulation of the
Ca2+-sensing receptor should decrease Na+
transport in the mTAL. Therefore, COX-dependent metabolites of arachidonic
acid are likely involved in mediating effects of a sustained stimulation of
the Ca2+-sensing receptor. Although TNF stimulates the
apical 70-pS K+ channel, it is unlikely that the net effect of
prolonged stimulation of the Ca2+-sensing receptor would
increase channel activity. This is due to the fact that increases in TNF
production induced by raising extracellular Ca2+ may not
be high enough to significantly stimulate the 70-pS K+ channel. It
was calculated that the concentration of TNF in the mTAL incubated in a media
containing 2 mM Ca2+ was below 10 pM
(28). Because TNF at
concentrations lower than 10 pM does not affect channel activity
(Fig. 2), the net effect of
long-lasting stimulation of the Ca2+-sensing receptor
should be inhibiting the apical K+ channel.
The finding that TNF can be formed in the mTAL in which 20-25% of filtered
Na+ load is reabsorbed strongly suggests that TNF may have an
effect on membrane transport in the mTAL. This notion is supported by
observations that incubation of isolated mTAL in TNF containing media for 24 h
decreased ouabain-sensitive Rb+ uptake
(10). TNF-induced decreases in
ouabain-sensitive Rb+ uptake suggest that TNF inhibits the active
Na+ transport in the mTAL. In the present study, we observed that
TNF increases the 70-pS K+ channel activity. Because the 70-pS
K+ channel contributes significantly to the apical K+
conductance and K+ recycling
(30), it is expected that TNF
should increase Na+ influx across the apical membrane. This
apparent paradox may best be explained by considering that effects of TNF are
time dependent and biphasic: the acute effect of TNF is to stimulate, whereas
the chronic effect of TNF is to inhibit the active Na+ transport in
the mTAL. Also, the acute effect of TNF is mediated by stimulation of PTP.
This is a direct consequence of stimulation of TNF receptor-dependent
signaling without the involvement of gene transcription. In contrast, the
chronic effect of TNF on Rb+ uptake is the result of increasing
COX-2 expression and activity because blocking COX-2 attenuates the inhibitory
effect of TNF on Rb+ uptake
(10,
28). Therefore, the effect of
TNF on the transport in the mTAL depends not only on concentrations but also
on temporal factors. A similar phenomenon that the response of cellular
signaling to hormones depends on exposure time or pattern has been reported in
mediating the effect of growth factor
(2).
The role of TNF in the regulation of epithelial transport in the mTAL is
not clear. Because TNF production is stimulated by bacterial products and
cytokines, it is possible that TNF may be responsible for the abnormal renal
Na+ handling during pathological conditions. In this regard, it has
been reported that Na+ excretion is reduced, whereas TNF production
increased in diabetic rats (9).
Furthermore, TNF stimulated Na+ uptake in the distal tubule cells
isolated from diabetic rats
(9). Therefore, it is
conceivable that TNF may also increase Na+ transport under certain
circumstances in the mTAL by stimulation of K+ recycling. Moreover,
it has been reported that TNF-induced cell apoptosis is related to activation
of K+ channels, which leads to intracellular K+ loss
(23,
24). Indeed, inhibition of
K+ channels has been demonstrated to prevent cell death in the
proximal tubule induced by hypoxia
(25). However, further study
is required to examine whether activation of the apical K+ channels
is the early event of TNF-induced cell damage in the mTAL.
In conclusion, acute application of TNF stimulates the apical 70-pS
K+ channel and the effect of TNF is most likely mediated by
enhancing PTP activity in the mTAL.
 |
DISCLOSURES
|
---|
The work is supported by National Institutes of Health Grants DK-54983 (W.
H. Wang), HL-34300 (W. H. Wang), and HL-56432 (N. R. Ferreri).
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: W. H. Wang, Dept. of
Pharmacology, BSB Rm. 537, New York Medical College, Valhalla, NY 10595
(E-mail:
wenhui_wang{at}nymc.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.
 |
REFERENCES
|
---|
- Bassal S, Liu
YS, Thomas RJ, and Phillips WA. Phosphotyrosine phosphatase activity in
the macrophage is enhanced by lipopolysaccharide, tumor necrosis factor
, and granulocyte/macrophage-colony stimulating factor.
Biochim Biophys Acta 1355:
343-352, 1997.[ISI][Medline]
- Bhalla US, Ram
PT, and Lyengar R. MAP kinase phospatase As a locus of flexibility in a
mitogen-activated protein kinase signaling network.
Science 297:
1018-1023, 2002.[Abstract/Free Full Text]
- Bleich M,
Schlatter E, and Greger R. The luminal K+ channel of the thick
ascending limb of Henle's loop. Pflügers Arch
415: 449-460,
1990.[ISI][Medline]
- Boim MA, Ho K,
Schuck ME, Bienkowski MJ, Block JH, Slightom JL, Yang Y, Brenner BM, and
Hebert SC. The ROMK inwardly rectifying ATP-sensitive K channel. II.
Cloning and intrarenal distribution of alternatively spliced forms.
Am J Physiol Renal Fluid Electrolyte Physiol
268: F1132-F1140,
1995.[Abstract/Free Full Text]
- Chong MMW,
Thomas HE, and Kay TWH. Suppressor of cytokine signaling-1 regulates the
sensitivity of pancreatic
cells to tumor necrosis facror. J
Biol Chem 277:
27945-27952, 2002.[Abstract/Free Full Text]
- Cornejo M,
Guggino SE, and Guggino WB. Modification of
Ca2+-activated K+ channels in cultured
medullary thick ascending limb cells by N-bromoacetamide.
J Membr Biol 99:
147-155, 1987.[ISI][Medline]
- Culpepper RM and Andreoli TE. Interactions among prostaglandin E2,
antidiuretic hormone, and cyclic adenosine monophosphate in modulating
Cl- absorption in single mouse medullary thick ascending limbs of
Henle. J Clin Invest 71:
1588-1601, 1983.[ISI][Medline]
- Diem R, Meyer
R, Weishaupt JH, and Bahr M. Reduction of potassium currents and
phosphatidylinositol 3 kinase-dependent AKT phosphorylation by tumor necrosis
factor-
rescue axotomized retinal ganglion cells from retrograde cell
death in vivo. J Neurosci 21:
2058-2066, 2001.[Abstract/Free Full Text]
- DiPetrillo K,
Coutermarsh B, and Gesek FA. Urinary tumor necrosis factor contributes to
sodium retention and renal hypertrophy during diabetes. Am J
Physiol Renal Physiol 284:
F113-F121, 2003.[Abstract/Free Full Text]
- Escalante BA,
Ferreri NR, Dunn CE, and McGiff JC. Cytokines affect ion transport in
primary cultured thick ascending limb of Henle's loop cells. Am J
Physiol Cell Physiol 266:
C1568-C1576, 1994.[Abstract/Free Full Text]
- Giebisch G.
Renal potassium transport: mechanisms and regulation. Am J Physiol
Renal Physiol 274:
F817-F833, 1998.[Abstract/Free Full Text]
- Gu RM, Wei Y,
Falck JR, Krishna UM, and Wang WH. Effects of protein tyrosine kinase and
protein tyrosine phosphatase on the apical K channels in the thick ascending
limb. Am J Physiol Cell Physiol
281: C1185-C1195,
2001.
- Guggino SE,
Guggino WB, Green N, and Sacktor B. Blocking agents of
Ca2+-activated K+ channels in cultured
medullary thick ascending limb cells. Am J Physiol Cell
Physiol 252:
C128-C137, 1987.[Abstract/Free Full Text]
- Hebert SC.
Extracellular calcium-sensing receptor: implications for calcium and magnesium
handling in the kidney. Kidney Int
50: 2129-2139,
1996.[ISI][Medline]
- Klahr S and
Morrissey J. Angiotensin II and gene expression in the kidney.
Am J Kidney Dis 31:
171-176, 1998.[ISI][Medline]
- Lamas S, Michel
T, Brenner BM, and Marsden PA. Nitric oxide synthesis in endothelial
cells: evidence for a pathway inducible by TNF-
. Am J
Physiol Cell Physiol 261:
C634-C641, 1991.[Abstract/Free Full Text]
- Liu HJ, Wei Y,
Ferreri N, Nasjletti A, and Wang WH. Vasopressin and PGE2
regulate the apical 70 pS K+ channel in the thick ascending limb of
rat kidney. Am J Physiol Cell Physiol
278: C905-C913,
2000.[Abstract/Free Full Text]
- Lu M, Wang T,
Yan Q, Yang X, Dong K, Knepper MA, Wang WH, Giebisch G, Shull GE, and Hebert
SC. Absence of small conductance K channel (SK) activity in apical
membrane of thick ascending limb and cortical collecting duct in ROMK
(Bartter's) knockout mice. J Biol Chem
277: 37881-37887,
2002.[Abstract/Free Full Text]
- Lu M, Wang XH,
and Wang WH. Nitric oxide increases the activity of the apical 70 pS
K+ channel in TAL of rat kidney. Am J Physiol Renal
Physiol 274:
F946-F950, 1998.[Abstract/Free Full Text]
- MacEwan DJ.
TNF receptor subtype signaling: differences and cellular consequences.
Cell Signal 14:
477-492, 2002.[ISI][Medline]
- Macica CM,
Escalante BA, Conners MS, and Ferreri NR. TNF production by the medullary
thick ascending limb of Henle's loop. Kidney Int
46: 113-121,
1994.[ISI][Medline]
- McLarnon JG,
Franciosi S, Wang X, Bae JH, Choi HB, and Kim SU. Acute actions of tumor
necrosis factor-
on intracellular Ca2+ and
K+ current in human microglia. Neuroscience
104: 1175-1184,
2001.[ISI][Medline]
- Nietsch HH, Roe
MW, Fiekers JF, Moore AL, and Lidofsky SD. Activation of potassium and
chloride channels by tumor necrosis factor
. Role in liver cell death.
J Biol Chem 275:
20556-20561, 2000.[Abstract/Free Full Text]
- Penning LC,
Denecker G, Vercammen D, Declercq W, Schipper RG, and Vandenabeele P. A
role for potassium in TNF-induced apoptosis and gene-induction in human and
rodent tumour cell lines. Cytokine
12: 747-750,
2000.[ISI][Medline]
- Reeves WB and
Shah SV. Activation of potassium channels contributes to hypoxic injury in
proximal tubules. J Clin Invest
94: 2289-2294,
1994.[ISI][Medline]
- Sasaki CY and
Patek PQ. Transformation is associated with an increase in sensitivity to
TNF-mediated lysis as a result of an increase in TNF-induced protein tyrosine
phosphatase activity. Int J Cancer
81: 141-147,
1999.[ISI][Medline]
- Tonks NK, Diitz
CD, and Fischer EH. Purification of the major
protein-tyrosine-phosphatases of human placenta. J Biol
Chem 263:
6722-6730, 1988.[Abstract/Free Full Text]
- Wang D, Pedraza
PL, Abdullah HI, McGiff JC, and Ferreri NR. Calcium-sensing
receptor-mediated TNF production in medullary thick ascending limb cells.
Am J Physiol Renal Physiol 283:
F963-F970, 2002.[Abstract/Free Full Text]
- Wang WH.
Two types of K+ channel in thick ascending limb of rat kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
267: F599-F605,
1994.[Abstract/Free Full Text]
- Wang WH, Hebert
SC, and Giebisch G. Renal K channels: structure and function.
Annu Rev Physiol 59:
413-436, 1997.[ISI][Medline]
- Wang WH, Lerea
KM, Chan M, and Giebisch G. Protein tyrosine kinase regulates the number
of renal secretory K channel. Am J Physiol Renal
Physiol 278:
F165-F171, 2000.[Abstract/Free Full Text]
- Wang WH, Lu M,
Balazy M, and Hebert SC. Phospholipase A2 is involved in
mediating the effect of extracellular Ca2+ on apical
K+ channels in rat TAL. Am J Physiol Renal
Physiol 273:
F421-F429, 1997.[Abstract/Free Full Text]
- Wang WH, Lu M,
and Hebert SC. Cytochrome P-450 metabolites mediate extracellular
Ca2+-induced inhibition of apical K+ channels
in the TAL. Am J Physiol Cell Physiol
270: C103-C111,
1996.
- Wei Y, Bloom P,
Gu RM, and Wang WH. Protein-tyrosine phosphatase reduces the number of
apical small conductance K channels in the rat cortical collecting duct.
J Biol Chem 275:
20502-20507, 2000.[Abstract/Free Full Text]
- Wu CC, Hong HJ,
Chou TC, Ding YA, and Yen MH. Evidence for inducible nitric oxide synthase
in spontaneously hypertensive rats. Biochem Biophys Res
Commun 228:
459-466, 1996.[ISI][Medline]