Acute application of TNF stimulates apical 70-pS K channels in the thick ascend - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:43 |只看该作者 |倒序浏览 |打印

作者：Yuan Wei, Elisa Babilonia, Paulina L. Pedraza, Nicholas R. Ferreri,  Wen-Hui Wang作者单位：Department of Pharmacology, New York Medical College, Valhalla, New York10595
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      【摘要】
   TNF has been shown to be synthesized by the medullary thick ascending limb(mTAL) ( 21 ). In the presentstudy, 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 andincreased NP o [a product of channel open probability( P o ) 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 theROMK-like small-conductance K channels in the TAL. Thedose-response curve of the TNF effect yielded a K m value of 1 nM, a concentration that increased channel activity to 50% maximalstimulatory effect of TNF. The concentrations required for reaching theplateau of the TNF effect were between 5 and 10 nM. The stimulatory effect ofTNF on the 70-pS K channel was observed in the presence of N -nitro- L -arginine methyl ester. Thisindicated that the effect of TNF was not mediated by a nitric oxide-dependentpathway. Also, inhibition of PKA did not affect the stimulatory effect of TNF.In contrast, inhibition of protein tyrosine kinase not only increased activityof 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 thatthe TNF effect results from stimulation of PTP activity is supported by PTPactivity assay in which treatment of mTAL cells with TNF significantlyincreased the activity of PTP. We conclude that TNF stimulates the 70-pSK channel via stimulation of PTP in the mTAL. 6 u& z/ }% Q1 }8 |& u
      【关键词】 ROMK channel protein tyrosine kinase protein tyrosine phosphatase N G 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 indicatethat the TAL produces TNF in response to bacterial LPS, ANG II, and elevatedextracellular calcium ( 15, 21, 28 ). Incubation of the TALwith TNF in vitro inhibited ouabain-sensitive Rb uptake( 10 ), suggesting that TNF mayinhibit transcellular sodium transport in the TAL. However, the sites at whichTNF 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:Ca 2 activated, large conductance, and 30 and 70 pS( 3, 13, 29 ). It is generally acceptedthat the 70- and 30-pS K channels are major K channelsresponsible for K recycling( 30 ). In addition, it ispossible that ROMK is an important component of both 70- and 30-pSK channels because neither K channel can be found inROMK knockout mice ( 18 ). Weshowed that apical K channels are regulated by a variety of signalingpathways, including nitric oxide (NO), protein kinase C, and protein tyrosinephospatase (PTP) ( 12, 19, 32 ). TNF has been shown toactivate these same pathways( 1, 5, 16, 20, 35 ). Thus the main goal of thepresent study is to investigate the acute effect of TNF on apicalK channels in the mTAL and delineate the mechanism by which TNFregulates 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. Animalswere kept on normal rat chow and had free access to water for 7 days beforeuse. The rats were killed by cervical dislocation, and the kidneys wererapidly removed. Several thin (0.5-1 mm) slices were cut from the kidney andplaced in ice-cold NaCl Ringer. The medullary TAL (mTAL) tubules were isolatedwith two watch-making forceps and placed on a 5 x 5-mm cover glasscoated with polylysine (Collaborative Research, Bedford, MA). The cover glasswas 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 MgCl 2, 1.8 CaCl 2, 5glucose, and 10 HEPES (pH = 7.4). We used a sharpened pipette to open the mTALto gain access to the apical membranes. The tubule was superfused with NaClRinger, and the temperature was maintained at 37°C.0 Y" ]# n7 `/ d

Patch-clamp technique. Electrodes were pulled with a Narishige model PP83 vertical pipette puller and had resistances of 4-6 M whenfilled with 140 mM NaCl. The channel current recorded by an Axon200Apatch-clamp amplifier was low-pass filtered at 1 kHz using an eight-poleBessel filter (902LPF, Frequency Devices, Haverhill, MA). The current wasdigitized by an Axon interface (Digitada1200), collected by an IBM-compatiblePentium computer (Gateway 2000) at a rate of 4 kHz, and analyzed using thepClamp software system 6.04 (Axon Instruments, Burlingame, CA). Channelactivity was defined as NP o, a product of channel openprobability ( P o ) and channel number ( N ). The NP o was calculated from data samples of 60-s duration inthe steady state as follows1 s7 ~0 i. j1 ]2 z' P$ v
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where t i is the fractional open time spent ateach of the observed current levels. Because three types of K channels have been identified in the mTAL( 3, 6, 13, 29 ), we measured the channelcurrent at three different membrane potentials in each patch to estimate theconductance of the K channel in the patch.% }0 B3 h  r+ D6 g7 E* w

Cell cultures and measurement of protein tyrosine kinase and PTPactivity. Cultured mTAL cells were used to measure the activity ofprotein tyrosine kinase (PTK) and PTP before and after treatment with TNF. Themethod for isolation and primary culture of mTAL cells has been previouslydescribed ( 28 ). The TAL cellswere incubated in the presence or absence of TNF (10 nM) for 10 and 15 min. Wefollowed the method described previously to measure PTK( 31 ). Cells were lysed in 100µl 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, 5mM 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, 5µg/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 containing200 µM[ 32 P]ATP (1 cpm/fmol), 12 mM magnesium acetate, 2 mMMnCl 2, 0.3 mM dithiothreitol, 0.5 mM sodium orthovanadate, 0.5 mMammonium molyb-date, and 2 mM R-R-Src peptide(Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Gly). Reactions were quenched byadding 200 ml of 75 mM phosphoric acid, and 15 µl of cocktail were spottedon phosphocellulose paper. After several washes, the amount of 32 Pincorporated into R-R-Src peptide was assessed using a liquid scintillation counter.8 V) J2 d$ W2 C7 F

We used 32 P-labeled myelin basic protein described originally byTonks et al. ( 27 ) as asubstrate for the measurement of PTP activity. The release rate of 32 P from the basic protein is used as an index of PTP activity, andthe value obtained in the presence of vanadate is used as background. Theactivity of PTP was measured with a BioLabs PTP assay kit following the instruction provided by the vender (Bio-Rad, Hercules, CA). Briefly, culturedmTAL cells were incubated in the presence or absence of TNF (10 nM) for 10min. After treatment, cells were lysed in 100-µl buffer solution containing50 mM NaCl, 50 mM Tris·HCl (pH 7.4), 2.5 mM EDTA, 2.0 mMdithiothreitol, 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 1mM 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 32 P-labeled myelin basic protein were added to the sample followed by incubation for 10 min at 30°C. Reactions were terminated byadding 200 µl of ice-cold 20% trichloride acetate into the tube. The samplewas centrifuged for 5 min at 12,000 g, the resultant supernatant wascarefully taken, and the amount of 32 P released from the peptidewas measured using a liquid scintillation counter.& o: j* Y4 C0 l* W% Z

Solution and statistics. The pipette solution was composed of (inmM) 140 KCl, 1.8 Mg 2 Cl, and 5 HEPES (pH = 7.4).N G -nitro- L -arginine methyl ester ( L -NAME) andphenylarsine oxide were purchased from Sigma (St. Louis, MO), whereas H8 andherbimycin A were obtained from Biomol (Plymouth Meeting, PA). Herbimycin Awas dissolved in DMSO and the final concentration of DMSO was thathad 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 stocksolution. The data are means ± SE. We used paired and unpairedStudent's t -tests to determine the statistical significance. If the P value was

RESULTS

We first examined the effect of TNF on the 70-pS K channel activity in a cell-attached patch. Figure1 is a representative recording showing that addition of 10 nM TNFincreased NP o from 0.20 ± 0.02 to 0.97 ± 0.1( n = 7). The effect of TNF on channel activity is concentrationdependent: addition of 50 and 500 pM TNF increased NP o from 0.20 ± 0.02 to 0.33 ± 0.04 ( n = 6) and 0.45± 0.05 ( n = 10), respectively. The stimulatory effect of TNFwas maximal at concentrations between 5 and 10 nM( Fig. 2 ). Therefore, the calculated K m value of TNF, a concentration that increased channel activity to 50% the maximal value, was 1 nM( Fig. 2 ). In addition to the70-pS K channel, a ROMK-like 30-pS K channel was alsoexpressed in the mTAL ( 3, 18, 29 ). We examined the effect of10 nM TNF on the ROMK-like 30-pS K channel. To determine theeffect of TNF on the 30-pS K channel, patches containing the 30-pSK channels with a relatively low P o wereselected. Figure 3 is arecording made in a cell-attached patch demonstrating that TNF did not affectthe activity of the 30-pS K channel ( n = 3) because NP o = 0.51 ± 0.05 (TNF) was not different from 0.50± 0.05 (control).
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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 andthe pipette holding potential was 0 mV. Top : time course of theexperiment and the 2 parts of data indicated by numbers are extended to showthe fast time resolution. C-, channel closed.+ j5 @0 ~5 i# D: D; s% W2 {9 _
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Fig. 2. Dose-response curve of TNF effects. The experimental number is from 6 to 10for each dose.- s* I2 r) _& r& f1 [/ b
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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 tracesare 1-min channel activity recorded in the absence (control) or presence ofTNF. Two parts indicated by numbers are extended to show the fast timeresolution. The experiment was conducted in a cell-attached patch and theholding potential was 0 mV.) u- v" h+ p: u# c
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As the dose-response curve of TNF was established, 5 nM TNF was used inexperiments designed to study the mechanism by which TNF stimulates the 70-pSK channel. Several factors that have been shown to stimulate the70-pS K channel include protein kinase A (PKA), theNO-cGMP-dependent pathway, and PTP( 12, 19, 29 ). Therefore, we examinedthe effect of TNF on the 70-pS K channel in the presence of PKAinhibitors ( Fig. 4 ). Inhibitionof PKA did not significantly affect channel activity. Moreover, inhibition ofPKA did not alter the TNF effect because application of TNF further increasedchannel 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 doesnot result from stimulation of PKA.

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 acell-attached patch and the holding potential was 0 mV. Top : channelactivity in the presence of H8 (control) and TNF H8 with slow timeresolution. Two parts of the data are demonstrated with extended timecourse.; {. [% [6 m  k. L8 r3 C, b

Stimulation of TNF receptors has been shown to increase NO production ( 5, 16 ). Because the NO-dependentcGMP pathway can stimulate activity of the 70-pS K channel, weinvestigated the role of NO in mediating the effect of TNF on the 70-pSK channel in the presence of L -NAME, an inhibitor of NOsynthase (NOS). Inhibition of NOS decreased the NP o of the70-pS K channel to 0.13 ± 0.02 ( n = 5). This isconsistent with the previous observation that L -NAME inhibitedactivity of the 70-pS K channel( 19 ). However, L -NAME did not block the effect of TNF because it increased NP o from 0.13 ± 0.02 to 0.9 ± 0.1( n = 5) in the presence of 0.5 mM L -NAME ( Fig. 5 ).

Fig. 5. Effect of 5 nM TNF on the 70-pS K channels in the presence of0.2 mM N G -nitro- L -arginine methyl ester( L -NAME). The experiment was carried out in a cell-attached patchand the holding potential was 0 mV. Top : channel activity in thepresence of L -NAME and TNF L -NAME with slow timeresolution. Two parts of the data are demonstrated with extended timecourse.
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Several studies demonstrated that stimulation of the TNF receptor increasesPTP ( 1, 26 ). We previously observedthat the 70-pS K channel is regulated by PTK and PTP( 12 ). If the effect of TNF wasthe result of stimulation of PTP, which leads to enhancement of tyrosinedephosphorylation, inhibition of PTK should mimic the effect of TNF such thatthe effect of TNF on the 70-pS K channel activity should be absentin 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 thetubule treated with herbimycin A. Figure6 is a recording showing the effect of TNF on channel activity inthe presence of herbimycin A. We confirmed the previous finding thatinhibition of PTK significantly stimulates the 70-pS K channel( 12 ) and increases NP o 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 becauseTNF did not significantly increase channel activity ( NP o =1.25 ± 0.1). This indicates that the effects of TNF and herbimycin Aare not additive.

Fig. 6. Effect of 5 nM TNF on the 70-pS K channels in the presence ofherbimycin A (Herb; 2 µM). The experiment was carried out in acell-attached patch and the holding potential was 0 mV. Top : channelactivity under control conditions, in the presence of herbimycin A, and in thepresence of TNF herbimycin A with slow time resolution. Three parts of thedata are demonstrated with extended time course.# j2 d# w2 G: z* ^

Although the observation that the effects of TNF and herbimycin A are notadditive 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 TNFcannot further increase channel activity because it is already at peakfollowing inhibition of PTK. Therefore, we examined whether inhibition of PTPcan reverse the stimulatory effect of TNF. Because addition of phenylarsine oxide (PAO), an inhibitor of PTP, decreased channel activity, we selected thepatches with a high NP o to study the effect of TNF in thepresence of PAO. Inhibition of PTP not only decreased NP o from 0.44 ± 0.04 to 0.21 ± 0.01 ( n = 5) but alsoabolished the effect of TNF on channel activity because NP o did not significantly alter and was 0.18 ± 0.01 ( Fig. 7 ). Moreover, we examinedthe effect of PAO on channel activity in the mTAL, which was challenged withTNF. 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 from0.92 ± 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.
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Fig. 7. Effect of 1 µM phenylarsine oxide (PAO) and 5 nM TNF PAO on theactivity of the 70-pS K channels. Data are summarized from 5cell-attached patches. * Data are significantly different from thecontrol value ( P
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Fig. 8. Recording illustrating the effect of PAO in the presence of TNF. Theexperiment was carried out in a cell-attached patch and the holding potentialwas 0 mV. Top : channel activity in the presence of TNF (5 nM), PAO (1µM) TNF, and wash-out with slow time resolution. Three representativeparts indicated by numbers are extended at fast time scale.
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To examine whether TNF treatment can stimulate the activity of PTP, we usedcultured mTAL cells ( 28 ) tomeasure the activity of PTP in the presence or absence of 10 nM TNF for 10min. Results summarized in Fig.9 show that treatment of mTAL cells with 10 nM TNF increased therelease of 32 P from the substrate from 2,050 ± 100 pmol/mgprotein (control) to 4,200 ± 300 pmol/mg protein ( n = 4). Theeffect 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 ± 2,100pmol/mg protein, n = 5) in mTAL cells.) ?) T& T8 j- l4 Q

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) orTNF for 10 min and the release rate of 32 P from the substrate, asan index of activity of PTP, was measured with the synthesized peptide. * Data are significantly different from the control value (withoutTNF), P/ U4 f0 y1 ~) g- H7 i- N
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DISCUSSION% F( H1 e/ u4 C" B- K+ h

The main findings of the present study are that acute application of TNFstimulates the apical 70-pS K channel in the mTAL and that theeffect of TNF on channel activity is blocked by inhibition of PTP and PTK.Similar observations that TNF acutely regulates K channel activityhave been reported in retinal ganglion cells( 8 ) and microglia cells( 22 ). In the present study, wesuggest that the effect of acute application of TNF on the 70-pS K channel is possibly mediated by stimulation of PTP activity. This conclusionis supported by four lines of evidence: 1 ) inhibition of PTKabolished the effect of TNF on channel activity; 2 ) the effect of TNFwas absent in the presence of PTP inhibitor; 3 ) application of PAOreversed the stimulatory effect of TNF; and 4 ) treatment of mTALcells with TNF increased PTP activity. We hypothesize that acute applicationof TNF stimulates PTP activity and leads to enhancement of tyrosine dephosphorylation, which increases the activity of the apical 70-pSK channel in the mTAL. However, when PTK has been inhibited byherbimycin A, the effect of TNF is absent because inhibition of PTK also leadsto increased tyrosine dephosphorylation.( a! T5 r1 d# v# }9 j5 a
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PTK and PTP have been shown to play important roles in the regulation ofthe 70-pS K channel: stimulation of PTK decreases, whereas anincrease in PTP activity augments activity of the 70-pS K channel( 12 ). The effect of PTK on the70-pS K channel is possibly the result of the stimulation oftyrosine phosphorylation of the 70-pS K channel or its closelyassociated proteins because addition of exogenous c-Src PTK could inhibit the70-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. Thissuggests that the 30-pS K channel is not directly regulated byPTP. The finding is consistent with previous findings that stimulation of PTKactivity inhibits only the 70-pS K channel but not the 30-pSK channel ( 12 ).Therefore, the mechanism by which PTP or PTK regulates the apical 30-pSK channel is different from that of the 70-pS K channel. In addition, the observation that inhibition of PTP decreased theROMK-like 35-pS K channel in the cortical collecting duct (CCD)( 34 ) indicates further thatROMK-like small-conductance K channels in the mTAL and CCD arealso differentially modulated by PTK and PTP. Because ROMK1 is in the CCDwhereas ROMK2/3 is in the TAL( 4 ), this strongly suggeststhat the mechanism by which PTK regulates ROMK1 or ROMK2/3 is different. Thephysiological 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 basallevel of apical K conductance, whereas the 70-pS K channel activity is subjected to modulation by a variety of factors such asTNF.
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TNF is a cytokine produced by a variety of cells including the epithelialcells in the mTAL ( 21 ). TNFproduction increases in response to inflammation, injury, and infection. Ithas been reported that IL-1 and -2 increase TNF production( 20 ). TNF plays an importantrole in inflammation, cell differentiation, and cell death( 20 ). The role of TNF in renalpathophysiological 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 hasbeen shown that stimulation of the Ca 2 -sensing receptor increases TNF generation ( 15, 28 ).

Stimulation of the Ca 2 -sensing receptor is expectedto decrease Na reabsorption in the mTAL( 14 ). One mechanism by which stimulation of the Ca 2 -sensing receptor inhibitsNa transport is to block K recycling across the apicalmembrane in the mTAL. We previously demonstrated that raising extracellular Ca 2 inhibits the apical 70-pS K channel byincreasing 20-HETE generation( 33 ). Because K recycling is important, inhibition of K channels should lead todecreased Na absorption in the mTAL( 11 ). However, this20-HETE-dependent mechanism may be responsible for acute stimulation of theCa 2 -sensing receptor, whereas the TNF-cyclooxygenase(COX)-dependent pathway is involved in mediating the effect of sustainedstimulation of the Ca 2 -sensing receptor in the mTAL. Ithas been shown that a prolonged stimulation of theCa 2 -sensing receptor by incubation of mTAL cells in amedia containing high Ca 2 for more than 3 h increasesCOX-2 expression and PGE 2 generation. TNF has been shown to play a key role in inducing COX-2 following stimulation of theCa 2 -sensing receptor( 28 ). Because PGE 2 has been shown to inhibit the apical K channel( 17 ) and other iontransporters in the mTAL ( 7 ),an increase in PGE 2 production following stimulation of theCa 2 -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 ofthe Ca 2 -sensing receptor. Although TNF stimulates theapical 70-pS K channel, it is unlikely that the net effect ofprolonged stimulation of the Ca 2 -sensing receptor would increase channel activity. This is due to the fact that increases in TNFproduction induced by raising extracellular Ca 2 may notbe high enough to significantly stimulate the 70-pS K channel. Itwas calculated that the concentration of TNF in the mTAL incubated in a mediacontaining 2 mM Ca 2 was below 10 pM( 28 ). Because TNF atconcentrations lower than 10 pM does not affect channel activity( Fig. 2 ), the net effect of long-lasting stimulation of the Ca 2 -sensing receptorshould be inhibiting the apical K channel.$ [6 E4 [& t" ^  h, ?; u

The finding that TNF can be formed in the mTAL in which 20-25% of filteredNa load is reabsorbed strongly suggests that TNF may have aneffect on membrane transport in the mTAL. This notion is supported byobservations that incubation of isolated mTAL in TNF containing media for 24 hdecreased ouabain-sensitive Rb uptake( 10 ). TNF-induced decreases inouabain-sensitive Rb uptake suggest that TNF inhibits the activeNa transport in the mTAL. In the present study, we observed thatTNF increases the 70-pS K channel activity. Because the 70-pSK channel contributes significantly to the apical K conductance and K recycling( 30 ), it is expected that TNFshould increase Na influx across the apical membrane. Thisapparent paradox may best be explained by considering that effects of TNF are time dependent and biphasic: the acute effect of TNF is to stimulate, whereasthe chronic effect of TNF is to inhibit the active Na transport inthe mTAL. Also, the acute effect of TNF is mediated by stimulation of PTP.This is a direct consequence of stimulation of TNF receptor-dependentsignaling without the involvement of gene transcription. In contrast, thechronic effect of TNF on Rb uptake is the result of increasingCOX-2 expression and activity because blocking COX-2 attenuates the inhibitoryeffect of TNF on Rb uptake( 10, 28 ). Therefore, the effect ofTNF on the transport in the mTAL depends not only on concentrations but alsoon temporal factors. A similar phenomenon that the response of cellularsignaling to hormones depends on exposure time or pattern has been reported inmediating the effect of growth factor( 2 ).

The role of TNF in the regulation of epithelial transport in the mTAL isnot clear. Because TNF production is stimulated by bacterial products andcytokines, it is possible that TNF may be responsible for the abnormal renalNa handling during pathological conditions. In this regard, it hasbeen reported that Na excretion is reduced, whereas TNF productionincreased in diabetic rats ( 9 ).Furthermore, TNF stimulated Na uptake in the distal tubule cellsisolated from diabetic rats( 9 ). Therefore, it isconceivable that TNF may also increase Na transport under certaincircumstances in the mTAL by stimulation of K recycling. Moreover,it has been reported that TNF-induced cell apoptosis is related to activationof K channels, which leads to intracellular K loss( 23, 24 ). Indeed, inhibition ofK channels has been demonstrated to prevent cell death in theproximal tubule induced by hypoxia( 25 ). However, further studyis required to examine whether activation of the apical K channelsis the early event of TNF-induced cell damage in the mTAL.+ y- A( V2 N: @1 [5 j( F3 y

In conclusion, acute application of TNF stimulates the apical 70-pSK channel and the effect of TNF is most likely mediated byenhancing 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).
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