PTH stimulates a Cl-dependent andEIPA-sensitive current in chick proximal tubul - 《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 13:36 |只看该作者 |倒序浏览 |打印

作者：GaryLaverty, ColleenMcWilliams, AmandaSheldon,  Sighvatur S.Árnason作者单位：1 Department of Biological Sciences, University ofDelaware, Newark, Delaware 19716; and Department ofPhysiology, University of Iceland, IS-101 Reykjavík, Iceland 2 l8 u) g0 L, O$ ~

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      【摘要】: Z9 {; C/ w, o3 H  O+ ~, ?$ C1 `
   Theelectrophysiological effects of parathyroid hormone (PTH) were studiedin 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-circuitcurrent ( I sc )] response, with an average 2-minpeak response of 14.30 ± 1.58 µA/cm 2 over thebaseline I sc, followed by a slow decay. The PTHresponse was dose dependent, with a half-maximal response at 5 × 10 9 M and maximal response at 5 × 10 8 M. Forskolin and dibutyryl-cAMP also stimulated I sc, as did the phosphodiesterase inhibitorIBMX. In contrast, the phorbol ester PMA inhibited baseline I sc. The PTH response was nearly abolished byapical addition of 100 µM EIPA, an inhibitor ofNa /H exchangers, and partially blocked by theCl channel blockers5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 100 µM) andglibenclamide (300 µM). Higher doses of EIPA or NPPB alone (500 µM)were almost fully effective, with no or slight additional effects ofNPPB or EIPA, respectively. The anion exchange inhibitor DIDS (100 µM) and the Na channel blocker amiloride (10 µM) hadno effect. Bilateral reduction of Cl in the buffer, from137 to 2.6 mM, abolished the PTH response; increasing Cl concentration restored the I sc response, with ahalf-maximal effect at 50 mM. These data suggest that, in the chickproximal tubule, PTH activates both an Na /H exchanger and a Cl channel that may be functionally linked.
      【关键词】 avian kidney shortcircuit current chloride channels cysticfibrosis transmembrane regulator glibenclamide
   INTRODUCTION

PARATHYROID HORMONE (PTH) is known to affect a number of proximal tubule (PT) transportsystems. In rats, rabbits, and other mammalian species, PTH inhibitsNa  , fluid, and bicarbonate reabsorption in vivo and invitro (2, 4, 16, 29; for a review, see Ref. 15 ). A majorpart of this effect is linked to inhibition of an apicalNa /H exchanger (NHE) isoform, identified asNHE3 ( 10, 14, 16, 54 ). On the basis of recovery ofintracellular pH from an imposed ammonium chloride acid load, PTHtreatment inhibits an amiloride- and EIPA-sensitive exchanger in bothnative tissues and in the proximal-like opossum kidney (OK) cell line( 10, 17, 40 ). Furthermore, studies with brush-bordermembrane vesicles (BBMV) have shown that PTH treatment reduces theactivity of pH gradient-driven 22 Na uptake ( 14, 16, 20 ). PTH was also shown to decrease the V max for 22 Na uptake by OK cells( 30 ). These findings are suggestive of a PTH-induceddecrease in transporter number. In support of this, more recentimmunoblotting studies and subcellular fractionation experiments haveshown a PTH-induced internalization of NHE3 from the apical membrane toa subapical, intracellular compartment ( 10, 14, 16, 18, 54 ). PTH also reduces NHE3 activity via phosphorylation ofcytoplasmic domains of the transporter, thus indicating a dualmechanism of regulation ( 10, 14 ). These effects of PTHappear 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 (P i ) cotransport system, resulting in increased urinaryexcretion of P i ( 17, 54 ).7 R# z2 }2 q1 \9 P+ `

There is much less known about PTH function in the avian PT. There issome evidence for an apical NHE in chickens ( 35 ), and PTHin vivo leads to increased whole animal urinary flow rates, Na excretion, and urinary pH, suggesting PTH inhibition ofthis system ( 26 ). However, this has not been demonstrateddirectly. Moreover, there are a number of known differences in proximal transport characteristics between birds and mammals. For example, wepreviously demonstrated that superficial PTs of the European starlingdo not acidify the urine ( 24 ); i.e., there was nomeasurable pH gradient between the tubule lumen and peritubular bloodand therefore no preferential bicarbonate reabsorption, as seen in mammalian PTs ( 15 ). We and others were also unable todetect, by histochemical methods, PT carbonic anhydrase activity,whereas distal tubule and collecting duct segments were clearlypositive ( 25, 37 ). Martinez et al. ( 28 ) foundthat superficial, nonlooped nephrons of chickens seem to possess noneof the known mammalian basolateral acid-base transporters.. C1 b! }2 f3 j8 P
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The avian PT also possesses specific systems for secretory transport(basolateral to apical) of both urate and P i, although there is little known about these systems ( 3, 7, 13, 36, 52 ). Such secretory processes may have evolved in part tocompensate for the lower filtration rates found in nonmammaliannephrons ( 7, 36 ). Thus PTH in birds is thought to increaseP i excretion by both inhibition of a reabsorptive flux, asin mammals, and by stimulation of a secretory flux ( 13, 36, 52 ).
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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 permeablemembrane filters become highly polarized and exhibit transepithelialtransport, measurable by classic electrophysiological methods. Usingthis approach, we undertook these studies to investigate PTH effects onthe avian PT. The results demonstrate a novel effect of PTH on thissystem involving stimulation of a Cl -dependent andEIPA-sensitive short-circuit current ( I sc ).
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MATERIALS AND METHODS

Reagents and supplies. The growth media and supplements, collagen, Percoll, PTH [bovinePTH-(1-34) fragment], and all other agonists andantagonists 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 inDMSO or water.8 j1 {4 h# d" h) l* n; D
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Cell culture. Chick PT cultures were prepared as described previously( 45 ), using methods similar to those for mammalian primarycultures ( 12, 19, 48 ). Briefly, 4- to 7-day-old WhiteLeghorn chicks were killed by cervical dislocation (approved by theInternational Animal Care and Use Committee), and kidney tissue wasremoved asceptically. Pieces of tissue were pooled from five to seven chicks in ice-cold Hanks' balanced salt solution (HBSS) withpenicillin and streptomycin. The pooled tissue was then minced andenzymatically disaggregated in a solution containing 1 mg/mlcollagenase A and 0.6 U/ml Dispase for 30 min at 37°C. This digestedmaterial was then triturated with a 10-ml pipette and sieved through astainless steel screen (30 mesh, 0.52-mm openings). The filtrate, atthis point, consisted of short, intact tubule fragments of~100-200 µm in length. Following the techniques described forisolation of rat PTs ( 48 ), the tubule suspension waswashed multiple times in HBSS by low-speed centrifugation and thenplaced in a 1:1 mixture of Percoll and 2× Krebs-Henseleit buffercontaining (in mM) 240 NaCl, 8 KCl, 2 KH 2 PO 4,30 NaHCO 3, 2.4 CaCl 2 · 2H 2 O, 2.4 MgSO 4 · 7H 2 O, 10 glucose,and 20 HEPES.8 ?6 ^+ g( m% m! }3 G! y
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The suspension was centrifuged through the Percoll density gradient at15,000 g for 30 min (4°C). For chick kidney, this process resulted in two major tissue bands at low and high densities. Thehigh-density band, designated as the "PT band," consisted almostentirely of short PT fragments, as assessed by microscopic appearanceand marker enzyme enrichment ( 45 ). The PT band was removedfrom the Percoll and washed several times in HBSS and one time ingrowth media. These washing steps and all subsequent work was done inthe absence of antibiotics. A final suspension of tubules was preparedin 3-4 ml of growth media and used for seeding culture inserts.* m' ?; r; U6 _$ ]2 X

Before each preparation (1 day), 12 Nunc tissue culture inserts werecollagen coated by soaking the filters in a 20:1 dilution of type Icalfskin collagen, removing excess solution and allowing the filters tocompletely air-dry. The inserts were prewetted with growth mediumseveral hours before seeding. The growth media used was serum-free andantibiotic-free DME/F-12 (1:1) supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenite, 5 × 10 8 Mhydrocortisone, and 20 µM ethanolamine.
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Six to seven drops of the final tubule suspension were seeded in eachprepared filter insert; they were placed in individual wells of a24-well culture dish with 0.5 ml growth media added to both the outerwells and inner cup. Cells were grown in a 37°C incubator with anatmosphere containing 5% CO 2 and were fed every secondday. Under these growth conditions, monolayers typically reachedconfluence 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 apicalmicrovilli and proximal-like electrophysiological characteristics,including glucose-stimulated I sc ( 45 ).& d+ G) ?3 h* k

Electrophysiology. Filter inserts with intact monolayers were mounted in modifiedUssing chambers with adapters fitted for the Nunc 10-mm cups (WarnerInstrument, Hamden, CT). An "O" ring sealed the outside of the cupwithin the adapter. Thus the epithelial monolayer formed an intactbarrier between circulating apical and basolateral Ringer solutions,with no edge damage. A transport buffer containing (in mM) 130 NaCl, 4 KCl, 1.3 CaCl 2, 1 MgSO 4, 5 HEPES, and 25 NaHCO 3 was circulated on both sides and gassed with 5%CO 2 -95% O 2 (pH 7.5). For Cl substitution experiments, NaCl and KCl were partially or completely replaced with gluconate salts on both sides. Heated reservoirs kept thebuffers (16 ml on each side) at 37°C. The monolayers wereshort-circuited with an automatic two-channel voltage clamp (DVC 1000;WPI) with correction for fluid resistance compensation. Ringer-agarbridges were used to electrically couple the apical and basolateralsolutions to a matched pair of calomel half-cells for measurement ofthe potential difference (PD). A second set of bridges was connected toa pair of Ag/AgCl wires for passing current. I sc was measured continuously and displayed on a strip-chart recorder, withintermittent measurement of the open-circuit PD. Transepithelialresistance (TER) was also monitored continuously by current deflectionsin response to 2-s changes in the clamping voltage (to 1 mV) every 5 min.& X0 Z6 r  i3 G. {6 x6 x/ E
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Experiments were always run in pairs on monolayers selected for similarresistances from the same culture. For each experimental group (e.g.,EIPA inhibition), data were collected from at least four differentcultures. We have observed that most of the variation in responsesoccurs between cultures, with a high degree of consistency betweenmonolayers from the same culture. Once a stable baseline I sc was obtained, glucose was added to bothapical and basolateral solutions to a final concentration of 5 mM. Theresulting increase in I sc was regarded as acheck on the proximal-like behavior of these cultures( 45 ). All other agents were added after the I sc had stabilized again, after glucose addition(postglucose baseline). PTH, agonists, and inhibitors were added fromconcentrated stocks, with a minimum of 15 min between additions (20 minafter PTH addition). Changes in the I sc from theprevious, extrapolated current values were calculated in units ofmicroamperes per square centimeter (0.5 cm 2 growth surfaceon the Nunc 10-mm inserts). For the antagonist studies, one monolayerof a pair was chosen at random to serve as a control. After postglucosestabilization, the antagonist was added to one monolayer and an equalvolume (16 µl) of appropriate vehicle to the other. This was followed15-20 min later by PTH addition (10 9 M) to thebasolateral side of both the control and antagonist-treated monolayers.
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The following agents were used at the indicated final concentrations,derived from various published studies (see DISCUSSION ): channel/transporterblockers: 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 someexperiments, the phosphodiesterase inhibitor IBMX was added to bothsides at 100 µM. The dose response to PTH was tested in the range of10 10 to 1.4 × 10 7 M, with a"standard" concentration of 1.0 × 10 9 M usedfor inhibitor and Cl substitution studies. According toour dose-response studies, this concentration gave ~40% of themaximal response.
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Immunoblotting. Standard Western blotting methods were used to investigate thepossible 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). Totalprotein (30 µg) was loaded on 8% SDS-polyacrylamide gels and probedwith 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). Neitherantibody 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 incurrent at 2, 10, and 20 min after addition. Effects of PTH in thepresence or absence of inhibitors were analyzed at 2 and 10 min afterhormone addition. Two-minute peak responses to PTH were used for thePTH dose-response and Cl substitution series. Significantdifferences between groups ( P paired and unpairedStudent's t -tests.
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RESULTS
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Baseline electrophysiological characteristics. Table 1 presents baselineelectrophysiological measurements and I sc responses to glucose addition for all groups used in this study.Baseline I sc was somewhat variable, ranging frommean values of 7 to 15 µA/cm 2, but with no statisticallysignificant differences among groups. All monolayers grown under theseconditions and tested with normal Cl transport bufferconsistently displayed low PDs and a modest TER, ranging from 0.6 to1.9 mV and 63 to 150 · cm 2,respectively. There were some significant differences in TER amonggroups in Table 1, mostly compared with the higher TER seen in themonolayers tested at the lowest Cl concentrations(; 2.6 mM). All monolayers also consistentlydisplayed a glucose-stimulated increment in I sc,attributable to a Na -glucose luminal cotransporter andcharacteristic of the vertebrate PT ( 13, 15, 45 ).$ h' s. N# {7 ^
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Table 1. Baseline electrophysiological characteristics

PTH-induced current response. Figure 1 presents tracings from twoexamples of experiments on paired PT monolayers. As seen in Fig. 1 A, top trace, exposure of the chick monolayersto 1.0 × 10 9 M PTH resulted in a positive currentresponse that peaked at 2 min and slowly decayed thereafter. Data from43 PTH-treated monolayers (taken from the control monolayers from allantagonist groups combined) are summarized in Fig. 2. The peak response at 2 min averaged14.30 ± 1.58 µA/cm 2, falling thereafter to6.78 ± 0.63 and 3.38 ± 0.44 µA/cm 2 at 10 and20 min, respectively, still significantly different from the baselinecurrent ( P$ `8 e1 H- H' F! b4 p
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Fig. 1. Short-circuit current ( I sc )recordings from two sample experiments ( A and B ),each showing tracings ( top and bottom ) from amatched pair of proximal cell monolayers. All monolayers were initiallytested for glucose responsiveness ( a ). Spikes representcommand voltage steps for measurement of transepithelial resistance(TER). A : experiment showing positive I sc responses to 10 9 M parathyroidhormone (PTH; c ) and 10 µM forskolin ( d ) in acontrol ( top trace ) and EIPA-treated ( bottomtrace ) monolayer pair. EIPA (100 µM, apical) reduced thepostglucose baseline current ( b ) and nearly abolished bothPTH and forskolin-activated I sc responses. B : experiment showing opposite effects of 500 µM dibutyryl(DB)-cAMP ( top trace ) and 100 nM PMA ( bottomtrace ), activators of PKA and PKC signaling pathways,respectively. Whereas DBcAMP stimulated a slow increase in I sc, PMA reduced the baseline current. EIPA( d ) abolished both the cAMP-induced current and part of theresidual current in both monolayers.
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Fig. 2. Summary of agonist effects on I sc responses. I sc values (2, 10, and 20 min) areplotted for 10 9 M PTH ( n = 43), 10 µMforskolin ( n = 8), 500 µM DBcAMP ( n = 8), and 100 nM PMA ( n = 9). PMA and DBcAMP were addedto both apical and basolateral bathing solutions; PTH and forskolinwere added to the basolateral side only. Note marked overshoot (2-minresponse) to forskolin and decrease (negative change in I sc ) in response to PMA. Values are means ± SE. * Values significantly different from baseline, P t -test).
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Positive I sc responses were also produced by theadenylate cyclase activator forskolin, as demonstrated in Fig. 1 A, top trace, and by the membrane permeableDBcAMP (Fig. 1 B, top trace ). An early, rapid peakresponse to forskolin is clearly evident, followed by a sustained lateresponse, whereas the response to DBcAMP exhibits a slower,monotonic rise in I sc. In contrast, 100 nM ofthe phorbol ester PMA, an activator of protein kinase C (PKC), slowlyinhibits the current in these cells over the 20-min time course (Fig. 1 B, bottom trace ). The time courses for theseexperimental groups are summarized in Fig. 2. The marked 2-minovershoot after forskolin is clearly seen. The sustained responses at10 and 20 min were similar for forskolin and DBcAMP, averaging between9 and 12.5 µA/cm 2. In contrast, the slow decrease in I sc with PMA reached an average 20-min value of 7.28 ± 1.83 µA/cm 2.

The forskolin overshoot was most likely because of a high rate of cAMPproduction, followed by secondary regulation by phosphodiesterase andpossibly other regulatory controls. This interpretation was supportedby 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 I sc by 7.53 ± 1.09 µA/cm 2 ( n = 15). Subsequent addition of0.2 µM forskolin further raised I sc by9.10 ± 1.62 µA/cm 2 without an overshoot. Raisingthe forskolin concentration to the standard level of 10 µM had nofurther significant effect on I sc (0.20 ± 0.11 µA/cm 2 ), indicating that IBMX and low-dose forskolinmaximally stimulated this transport system. These data, combined withthe observation that DBcAMP also stimulates I sc in a monotonic fashion, suggest that cAMP activates a single, coupledtransport process in these cells., A% _  E: Y) b; C
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The PTH stimulation of I sc was tested over arange of cumulative concentrations of the hormone, from10 10 to 1.37 × 10 7 M. Figure 3 shows a clear dose-dependent responseover a 100-fold range, with a threshold at 5 × 10 10 M, an apparent half-maximal activation of 14 µA/cm 2 at5 × 10 9 M, and a maximal response of 28 µA/cm 2 at 5 × 10 8 M PTH. The avianantidiuretic hormone arginine vasotocin had no effect on I sc in these cells, even at 10 6 M(data not shown).

Fig. 3. PTH dose-response curve for hormone-induced I sc. Changes in I sc areplotted as a function of sequentially increasing doses of PTH for 14 proximal monolayers (means ± SE). Half-maximal stimulation wasseen at 5 × 10 9 M PTH.
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Effects of transporter and channel blockers on the PTH-stimulatedI sc. A number of transport inhibitors were found to partially block thepostglucose I sc, as exemplified by EIPA, aninhibitor of NHEs (Fig. 1 A, bottom trace ). Table 2 summarizes the initial effects of theseinhibitors on post-glucose I sc and TER values. The NHE inhibitor EIPA, the Cl channel blockersglibenclamide and NPPB, and the Na channel blockeramiloride all significantly reduced I sc, with EIPA causing the greatest effect. DIDS, a blocker ofCl /base exchange, had no overall effect on I sc, although some of the individual monolayersshowed 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.+ A; d9 L2 \9 b& w
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Table 2. Effects of inhibitors on postglucose I sc and TER
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The effects of these inhibitors on monolayer responses to 1 × 10 9 M PTH are shown in Fig. 4. To preserve information about the PTHtime course, both 2- and 10-min responses are plotted. The open barsshow significant I sc responses to PTH in thecontrol monolayers of these five groups, with means ranging from 13.2 to 21.7 µA/cm 2 at the 2-min time points. The filled barsshow significant inhibition of this response in paired monolayers withapical addition of EIPA, glibenclamide, and NPPB (added 15-20 minbefore PTH addition). EIPA at 100 µM significantly reduced the 2-minPTH response from 14.81 ± 3.41 to 2.63 ± 0.92 µA/cm 2 and the 10-min response from 6.56 ± 1.24 to1.94 ± 0.64 µA/cm 2 ( P channel blockers glibenclamide and NPPB alsosignificantly attenuated the PTH response, although not as fully asEIPA. Average inhibition ranged from 45 to 65% at the 2- and 10-mintime points. Ten micromolar amiloride, a dose normally used toeffectively block electrogenic epithelial Na channels, wascompletely ineffective against the PTH response as was also the casewith 100 µM DIDS.
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Fig. 4. Summary of inhibitor effects on PTH-induced I sc responses. I sc responses (2 and 10 min) are shown for paired monolayers in the absence(open bars) or presence (filled bars) of various antagonists. Allinhibitors were added to the apical bathing solution at the followingconcentrations (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 valuessignificantly elevated over baseline I sc ( P t -test). * Significantdecreases in I sc responses with inhibitorcompared with matched control monolayers ( P t -test).$ D: t* w! E9 g) F( n
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To evaluate the inhibitor efficacy and additivity of theCl dependence and EIPA sensitivity of this system, aseparate series of experiments was performed with the NHE inhibitorEIPA and the Cl channel blocker NPPB. I sc was first maximally stimulated with IBMX and0.2 µM forskolin as described above. Addition of 100 µM EIPAreduced the I sc by 19.80 ± 3.68 µA/cm 2 from a stimulated baseline of 35.20 ± 4.44 µA/cm 2. Increasing the EIPA concentration in this series( n = 5) to 500 µM reduced I sc by an additional 11.70 ± 1.29 µA/cm 2. The combinedinhibition was 90% of the total stimulated current. When thesemonolayers were further treated with 500 µM NPPB, the I sc declined nonsignificantly by 0.20 ± 0.20 µA/cm 2. In a complementary set of experiments,monolayers were sequentially exposed to 100 µM NPPB, 500 µM NPPB,and 500 µM EIPA. From a stimulated I sc of34.10 ± 4.22 µA/cm 2, these treatments decreased I sc by 9.70 ± 1.55, 15.00 ± 1.35, and 3.80 ± 1.35 µA/cm 2, respectively( n = 5). The combined inhibition was 84% of the totalstimulated current. Thus these data demonstrate that higher doses ofeach inhibitor alone were almost fully effective on the transportcurrents in these monolayers and that the Cl -dependentand EIPA-sensitive components do not appear to be additive.5 }( C# c  c$ m% ], ]
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Dependence of the PTH response on. The PTH-induced I sc response was found to becompletely dependent on the presence of Cl (Fig. 5 ). Symmetrical reduction of in the bathing solutions to 2.6 mM essentiallyabolished the PTH response. Increments in  between25 and 137 mM restored the PTH response in a dose-dependent manner.Half-maximal stimulation was obtained at [Cl 65 mM.) d# Y: @# R' C" ], _/ V
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Fig. 5. Cl dependence of the PTH-induced I sc response. Peak responses (at 2 min) to PTH(10 9 M) are shown from separate groups of monolayersbathed 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) mMCl. Cl substitution was made with sodiumand potassium gluconate salts. Half-maximal stimulation occurred at 50 mM Cl concentration. Data are means ± SE.
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DISCUSSION5 E0 `! F9 U% R; l! G
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This study describes a novel effect of PTH on a chick PT culturesystem. This primary culture model has previously been shown to exhibitproperties characteristic of vertebrate PTs, including apicalmicrovilli and vectorial transport, exemplified by glucose-stimulated I sc ( 45 ). In the present study, PTHat 10 9 M consistently induced a positive increase in I sc that was EIPA sensitive. The I sc response peaked at 2 min and then decayedover a 10- to 20-min time course, possibly reflecting receptordesensitization (Figs. 1 A and 2 ). The I sc response was dose dependent over a 100-fold range, with a half-maximal effect at 5 × 10 9 M PTH.This dose-response relationship is similar to that seen for PTHinhibition of fluid reabsorption in vivo ( 4 ) and for stimulation of cAMP production ( 9 ) and inhibition of NHEactivity ( 17 ) in the OK PT cell line. The lack of responseto even high doses of the avian antidiuretic hormone arginine vasotocinindicates specificity of this PTH response in these chick PT cells.* G% H) L8 W$ x7 i

Forskolin resulted in a similar I sc response toPTH. The large 2-min overshoot appeared to be because of secondaryphosphodiesterase activation, since separate experiments with IBMX and10-fold lower forskolin maximally stimulated I sc without an overshoot (see RESULTS ). The sustained current after forskolin showed little decay with time.The membrane-permeant cAMP analog DBcAMP also increased I sc, but with a slower onset and lack ofovershoot (Figs. 1 B and 2 ). These observations suggest thatPTH is acting on this transport system via the adenlylate cyclase/PKAsignaling pathway. Interestingly, the phorbol ester PMA, an activatorof PKC, caused a decrease in baseline I sc. Thusthese two signaling pathways, both of which are activated by PTH inproximal cells ( 1, 9 ), appear to have opposite effects onthis I sc response. In the OK cell model, it hasgenerally been observed that activation of either the PKA or PKCsignaling pathways inhibits NHE activity ( 1, 17 ). On theother hand, PKC stimulation resulted in increasedNa /H activity in both native tissues (rabbitBBMV) and primary cultures of rabbit PTs ( 19, 51 ). Thusthe potential role of PKC regulation of apical NHE activity isuncertain but may be related to different PKC isoforms present invarious cell types or under different assay conditions( 15 ).$ a; J" V7 [' K& ]

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 NHEactivity that is either linked to or dependent on a Cl transport process. Several observations support this conclusion. First,the I sc response to PTH was nearly abolished byapical addition of 100 µM EIPA (Figs. 1 A and 4 ). Thisamiloride analog is generally considered to be a selective inhibitor ofNHE transporters, although various isoforms may have differentinhibitory constants ( 18, 34 ). In contrast, amilorideitself, at a dose that inhibits electrogenic epithelial Na channel activity ( 32, 53 ), had no effect on thePTH-induced current response, although it did slightly decrease thebaseline I sc (Table 2 and Fig. 4 ). Thus it seemsunlikely 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 blockerssignificantly inhibited the PTH-induced response. Glibenclamide, asulfonylurea receptor inhibitor used to stimulate -cell insulinrelease, has also been widely used as a blocker of CFTRCl channels ( 41, 43 ). Similarly, thearylaminobenzoate NPPB is known as a potent inhibitor of a variety ofCl channels ( 33, 38, 41 ). Both of theseblockers, when added to the apical side, significantly reduced thePTH-induced I sc response (Fig. 4 ). In contrast,apical addition of 100 µM DIDS has no significant effect on this PTHresponse. DIDS is primarily used at these concentrations as a blockerof Cl /base or Cl /formate/oxalate exchangersof the PT ( 49 ). This compound also blocks some types ofCl channels, although typically at higher concentrations( 32-34, 38, 41 ). However, it is ineffective againstCFTR channels from the extracellular side ( 41 ). Thus thisresult also rules out such anion exchangers as a possible mechanismbehind the PTH-induced current response. It should be noted that, whenused at higher concentrations, both NPPB and EIPA eliminated~80-90% of the total stimulated current, and there was almostno additivity of these two blockers (see RESULTS ).

Bilateral reduction of  in the bathing solutions,from 137 to 2.6 mM, essentially abolished the I sc response (Fig. 5 ). Increasing over the range of 25-137 mM restored the fullhormone response, with a half-maximal response at 50 mM. These datasuggest that a low-affinity Cl transport process issomehow linked to the PTH response. Because, in the present study, wedid not measure isotope fluxes, it is not possible to identify theion(s) responsible for the I sc response. Giventhe 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 thatintracellular Cl levels in PT cells lie aboveelectrochemical equilibrium; i.e., increased apical Cl conductance would result in Cl exit from the cell, ratherthan entry ( 44 ). It should also be noted that, under theseshort-circuit conditions, passive (i.e., electrically driven) couplingof Cl fluxes is eliminated. Thus, taken together, thesedata suggest that PTH activates both NHE activity and aCl channel, one that is possibly CFTR related. Theseactivities appear to be functionally linked, either at the transportlevel or via a common regulatory pathway.
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Previous clearance studies in chickens have shown that, as in mammals,PTH administration results in whole animal diuresis, natriuresis, andurinary alkalinization ( 26 ), suggesting a conventional inhibitory action of PTH on NHE3 activity of the PT, in addition to thestimulated system seen in this study. This leads to the question ofwhether a PTH-induced I sc response may also bepresent in mammalian PTs, masked by the much larger inhibitory effect on Na reabsorption. To our knowledge, there are no otherreports of PTH-stimulated current in PT cells. However, severalobservations suggest that a similar system may be present in mammals.First, in primary cultures of human PT cells, forskolin caused asimilar positive I sc response to that seen inthe chick cultures ( 47 ). PTH was not tested in these humancells, however, and possible implications of the forskolin responsewere not addressed.

Second, patch-clamp studies in primary cultures of rabbit PTs revealeda PTH-activated Cl channel ( 46 ). ThisCl conductance could also be activated by forskolin, by acatalytic subunit of PKA, and, interestingly, also by PKC exposure.cAMP- and PKA-activated Cl channels were also observed inprimary cultures of rat PTs ( 12 ). Furthermore, PTH andcAMP have been shown to increase Cl membrane permeabilityin rat kidney BBMV ( 27 )./ W$ X) ^$ j, ~( G
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Regarding the possibility of CFTR localization to the PT, severalstudies have demonstrated CFTR mRNA in PT segments or in cultured cells( 21, 31, 38 ), whereas expression at the protein level hasbeen observed by some ( 6, 11 ), but not other,investigators ( 38 ). In the present study, attempts weremade to detect CFTR protein in monolayer extracts using two differentanti-human CFTR antibodies. Although these attempts were unsuccessful,this could indicate that the antibodies lacked sensitivity or that thetransporter activity is species specific or distinct from the humanCFTR protein, despite pharmacological similarities. Nevertheless, therecent realization that CFTR controls a wide variety of membranechannels and transporters ( 22, 42 ) and that a family ofintracellular signal complex proteins, known asNa -H exchanger regulatory factors (NHERFs),bind both NHE and CFTR via PDZ domains ( 50 ) provides apossible model for linkage of NHE and CFTR function.
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Although the transport mechanism behind the PTH-induced I sc response is unknown, an intriguingpossibility is suggested by recent studies of a novelCl -dependent NHE found in rat distal colon crypt cells( 33, 34, 39 ). As determined by pH gradient-stimulated 22 Na 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, butlower concentrations, used to inhibit anion exchangers, had no effect( 33, 34 ). Moreover, an antibody to CFTR also partiallyblocked Cl-NHE activity ( 33 ). Recently, this transporterwas cloned from rat distal colon crypt cells and shown to have homologywith NHE1, but with a markedly shortened and novel COOH-terminal domain( 39 ). Of particular interest, cDNA probes for thistransporter revealed widespread distribution of specific mRNA inseveral rat tissues, including kidney. This again raises thepossibility of a system similar to that described in the present studyin mammalian PTs. In this regard, Choi et al. ( 8 ) haveshown that 50% of EIPA-sensitive proximal NHE activity remains intactin NHE2 plus NHE3 double-knockout mice, suggesting the presence of anas yet undefined NHE activity.
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It is unclear what physiological function this system might have in theavian and/or mammalian PT or how its activation by PTH fits in with theother known effects of this hormone. Because these studies wereperformed in a tissue culture environment, in vivo implications need tobe considered with caution. Among other factors, hormone releasepatterns (pulsatile vs. continuous), stability, and concentration inthe whole animal will be different. Furthermore, it is possible thatthe observed current response represents only one manifestation of amultistep process in vivo. Nevertheless, it is interesting to considerseveral avian PT transport systems that are affected by PTH. In birds,PTH is known to stimulate a secretory component of P i transport ( 52 ) in addition to its inhibition ofreabsorptive transport ( 13, 36 ). PTH was also shown toincrease urate clearance in birds, although the mechanism of thisresponse was not determined ( 23 ). It is also of interest that Cl secretion has been correlated with net fluidsecretion in vertebrate PTs, clearly demonstrated in teleosts( 5 ) and hypothesized to exist in other vertebrates( 44 ). Potential interactions among these transport systemsdeserve further study.4 x/ Z- Q) \5 Q% l0 D7 I

ACKNOWLEDGEMENTS
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This study was funded by National Science Foundation GrantIBN-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).0 g: m9 |! }  I6 u( X, b
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