Functional characterization of three intercalated cell subtypes in the rabbit outer cortical collecting duct

Functional characterization of three intercalated

cell subtypes in the rabbit outer cortical

collecting duct.

C Emmons, I Kurtz

J Clin Invest.

1994;

93(1)

:417-423.

https://doi.org/10.1172/JCI116976

.

The distribution of Na(+)-independent Cl(-)-HCO3- exchange was studied in individual

intercalated cells from in vitro perfused rabbit outer CCDs using dual excitation laser

scanning confocal microscopy by measuring the pHi response to sequential removal of

Cl-from both sides of the tubule. Three patterns of intracellular pH (pHi) response were

observed. 39% of intercalated cells had only apical Cl(-)-HCO3- exchange (beta cell), 4%

had only basolateral Cl(-)-HCO3- exchange (alpha cell), and 57% had both apical and

basolateral Cl(-)-HCO3- exchange (gamma cell). Valinomycin-high K+ voltage clamping

had no effect on the pHi response of intercalated cells with bilateral Cl(-)-HCO3- exchange.

Although the mean rates of dpHi/dt following apical Cl- removal were similar in beta cells

compared to gamma cells, a wide range of apical rates was seen among individual beta

and gamma intercalated cells. Neither the apical nor the basolateral Cl(-)-HCO3- exchanger

in gamma cells was inhibited by 0.5 mM H2DIDS. Binding of apical peanut lectin was seen

both in beta cells and in gamma cells. In 41% of CCDs with four to seven intercalated cells

studied, all intercalated cells were of the same subtype. We conclude that the majority of

intercalated cells from the rabbit outer CCD have both apical and basolateral

Na(+)-independent Cl(-)-HCO3- exchangers (gamma cells), which are stilbene-insensitive.

Intercalated cells with only basolateral Cl(-)-HCO3- exchange are very uncommon […]

Research Article

Find the latest version:

Functional

Characterization

of Three Intercalated Cell

Subtypes

in

the

Rabbit Outer Cortical Collecting

Duct

CherylEmmons*" and Ira Kurtzt

NephrologyDivision, DepartmentofMedicine, *WadsworthVeteransAdministration Medical Center,LosAngeles, California 90024; and

PUniversityofCaliforniaatLosAngelesCenterforthe HealthSciences, LosAngeles, California90024

Abstract

Thedistributionof

Na'-independent

Cl --HCO3 exchange was studied inindividual intercalatedcells from in vitro perfused rabbit outer CCDs using dual excitation laser scanning confocal microscopy by measuring the pH1 response to sequential re-movalof Cl- from both sides of the tubule. Three patterns of intracellular pH(pH1)response were observed. 39% of interca-lated cells hadonlyapical Cl--HCO3 exchange

(,B

cell), 4% hadonly basolateral Cl--HCO- exchange (a cell), and 57% had bothapicaland basolateral Cl --HCO3 exchange(aycell). Valinomycin-high K+ voltage clamping had no effect on thepH1

response of intercalated cells with bilateral Cl --HCO3 ex-change. Althoughthemean ratesofdpH1/dtfollowing apical

Cl-removalweresimilar in

,8

cells compared toycells, awide rangeofapical rates was seen among individual

iB

andy interca-lated cells. Neither theapicalnorthe basolateralCI--HCO3

exchanger in ycellswasinhibitedby0.5mMH2DIDS.

Bind-ing of apicalpeanutlectinwas seenboth in

_ft

cells and inycells. In41% ofCCDs with four to seven intercalated cells studied, all

intercalated cells wereofthe samesubtype. We conclude that the majority of intercalated cells fromthe rabbitouterCCD

have bothapicaland basolateral

Na'-independent

Cl--HCO3

exchangers (y cells), which are stilbene-insensitive. Interca-lated cells with only basolateral

CI--HCO3

exchange are very

uncommonin therabbitouterCCD. There isatendencyforall

intercalatedcells in agiven rabbitouterCCDtobe of the same

subtype(eitherall 6 cells or all ycells), suggestingthe

pres-ence of CCD intertubule heterogeneity at the same cortical

level.This finding mayaccountfor intertubule differencesin

transepithelial H+-base transport. (J. Clin. Invest. 1994. 93:417423.) Keywords:acid-base*intracellular pH*

interca-lated cells *confocalmicroscopy*anionexchange

Introduction

Thecorticalcollecting duct

(CCD)'

is a heterogeneous epithelium

composed oftwomaincelltypes: principal cells and intercalated

Portions ofthis workwerepresentedatthe annualmeetingofthe

Amer-icanSocietyof Nephrology, 20-21November, 1991,Baltimore,MD, and were published in abstract form (1991. J. Am. Soc. Nephrol. 2:699).

AddresscorrespondencetoDr.Cheryl Emmons, Nephrology

Divi-sion,UCLACenter for the HealthSciences, 10833 Le Conte Boule-vard, Factor 7-453, Los Angeles,CA90024.

Receivedforpublication 7April1993and inrevisedform11August

1993.

1. Abbreviations used in thispaper:BCECF-AM, acetoxymethylester

of

2',7'-bis-(2-carboxyethyl)-5-(and-6

)carboxyfluorescein; CCD,

cor-TheJournal ofClinicalInvestigation,Inc. Volume93, January 1994, 417-423

cells( 1). Itis the lattergroupof cells that is believedto mediate transepithelial CCD acid-base tranwsport. Intercalated cells have

been

traditionally

divided intotwotypesbasedontheir

morphol-ogy,antibody binding

properties,

andacid-base

tranwsport

charac-teristics. Bicarbonate secretion is attributedto j3intercalated

cells,

which have been modeled with an apicalNa+-independent

Cl--HCO--exchanger,abasolateralH+-ATPase,and a basolateralCP channel. a intercalated cells arethoughttoeffect CCD HCO- ab-sorption and are modeled with an apical H+-ATPase,abasolateral

Na+-independent Cl--HCO- exchanger and a basolateral CP channel(2-8).Inaddition, apical lectin bindinghasbeen

consid-ered acharacteristic marker for

#

cells (3, 9, 13). Although

_f#

cells

donotbindantibodiestotheerythrocyte anion exchangerband3,

acells doexhibit basolateral staining (9, 15, 19).However, recent

morphologic and immunocytochemical studies have identified "hybrid" cells with features attributedtobothintercalated cell

sub-types(14-17,29). Thefunctionalsignificance ofthesefindingsis unknown.

Muchofthecurrentunderstanding ofCCD acid-base trans-portcharacteristics is derived from whole tubule studies. The

heterogeneous natureoftheCCDmakesit difficulttoascertain function on asingle-cell level. Optical approaches havebeen used tostudysingle-cellH +-basetransport;however, the cylin-dricalgeometryofthetubulehasmadeoptical studiesof indi-vidualcells troublesome due to theacquisition ofout-of-focus information. This difficultycan be overcome using confocal microscopy (10).The present study was undertaken to func-tionally characterize the distributionofNa+-independent

C1--HCO- exchangeinsingle intercalatedcellsintherabbitouter

CCDusing dual excitation laser scanning confocal microscopy.

The resultsindicatethat the majority of rabbitouterCCD in-tercalated cells donotfiteither of thetwoclassicintercalated cell models.

Methods

Tubuleperfusion.Male New Zealand Whiterabbits weighing4lbsand with free access torabbit chow (Prolab; Agway Country Foods, Inc., Syracuse, NY)werekilled by cervical dislocation. The leftkidneywas

removed,cutin coronalsections,andplaced in chilled solution 1 (Ta-bleI). Corticalcollectingductsweredissected from theouter mmof cortex, with themostsuperficialendstartingatthemostdistal

connect-ing tubule arcade into thecollectingduct (11).The tubulewas

trans-ferredto a 100-Mllaminar flow chamber and theperitubularbathing

solutionwasexchangedat a rateof 2ml/min.

Solutions. The solutions used in thestudyarelisted in TableI.All

wereNa'-free(tetramethylammonium replacement)toeliminate any

contribution ofNa'-dependenttransport processestoalterations in

pH,.

SolutionI wasusedfor the dissection solution. Solution 2wasthe initialperfusingandbathingsolution,exceptwherespecified.Solution 3wasfree of

CP-

(gluconatereplacement). Solutions4and5wereused tical collecting duct; 6-CF,

6-carboxylfluorescein; H2DIDS,

4,4'diisothiocyanatodihydrostilbene-2,2'-disulfonic

acid;

pHi,

Table I. ListofSolutions Usedinthe Study

Solutions 1 2 3 4 5 6

Tetramethylammoniumhydroxide 0 0 115 0 0 0

Tetramethylammonium chloride 140 115 0 20 0 0

Gluconic acid lactone 0 0 115 0 20 0

Tetramethylammonium bicarbonate 0 25 25 25 25 0

KCl 0 0 0 95 0 115

K2HPO4 2.5 2.5 2.5 2.5 2.5 2.5

K-gluconate 0 0 0 0 95 0

CaCl2 1 1 0 1 0 1

Ca-gluconate 0 0 3.5 0 3.5 0

MgCl2 1 1 0 1 0 1

Mg-gluconate 0 0 1 0 1 0

Hepesacid 5 0 0 0 0 5

L-alanine 5 5 5 5 5 5

Glucose 5 5 5 5 5 5

Allconcentrationsareinmmol/liter.

for thevalinomycinexperiments,andsolution 6wasusedfor calibra-tion of

_pHi

with 10

AM

nigericinadded. TheHCO--containing solu-tions were bubbled with 6.5%CO2and 93.5%02,while the

Hepes-con-tainingsolutionswerebubbled with 100%02.Tetramethylammonium

bicarbonatewasmadebybubbling tetramethylammonium hydroxide

with 100%CO2. Glass tubing, surrounded by heated water jackets, connectedthesolution reservoirs and theperfusionchambertoprevent anyCO2ortemperature loss. ThepH of all solutionswas7.4,and all

experimentswereperformedat370C.

pHi

measurements/confocal

imaging. TomeasurepHi, 5

AtM

ace-toxymethylesterof2',7'-bis(2-carboxyethyl)-5(and

-6)carboxyfluore-scein(BCECF-AM)wasaddedtotheappropriateperfusatesolution for 15min, resultinginselective dyeloadingofonlytheintercalated cells andconfirmed by "rough" surface appearance with transmission imaging (12). Theperfusatewasthen changedtotheappropriate

dye-free solution for 10 min before any datawasrecorded. Thefluorescent

measurements were accomplished with the use ofadual-excitation

laser-scanninginverted confocal fluorescentmicroscope,aspreviously

described(10).Thepreviousmethodologywasmodifiedasfollows.A

laser-scanning system (model MRC600; BioRadLaboratories, Rich-mond,CA)wascoupled to theside port ofaDiaphotinverted micro-scope(Nikon Inc., Melville, NY).Anargonlaser(model5424A; Ion LaserTechnology, Inc., Salt LakeCity, UT) was used for the 488-nm excitation and a helium-cadmium laser (model 4214NB; Liconix,

SantaClara,CA)providedthe 442-nmexcitation.A40xfluorite

ob-jective(N.A. 0.8; Nikon Inc.)wasusedin all experiments.

_pHi

was measuredfromanarea ofcytoplasm of - 3 to 5

Am

in diameter in up

to 7intercalated cells along a

250-Am

lengthof tubule (UMANS soft-ware;BioRad Laboratories). A zoom factor of 2.5 was used to obtain images of the tubule (final magnification of 800). A computer-driven electronic shutter (Vincent Associates, Rochester, NY) in front ofeach laserlimited the duration of laser exposure to 1 s per ratio. Excitation ratio measurements were taken every5s after asolution change and the rateof change of

_pHi

_(dpHi/dt)wasevaluated in the initial 20 s.

Materials. 4,4'diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS) and BCECF-AM were from Molecular Probes, Inc. (Eu-gene,OR). Tetramethylammonium hydroxide was from Fisher Scien-tific (Fair Lawn, NJ) and all other chemicals were from Sigma Chemi-calCo. (St. Louis, MO).

Statistics. All results are reported as mean±SEM. Student's

un-pairedt testwasused to compare results from two group means ob-tainedfrom separate tubules. Student's paired t test was used to com-pare results ofinhibitor studies in the same tubule. Statistical

signifi-cancewasaccepted at the P<0.05level.

Results

Patterns

of

Na+-independent Cl--HCO5 exchange in CCD in-tercalated cells. In the initial experiments, CF- was replaced

with gluconate (solution 3) sequentiallyonboth sides of the tubule. This protocol wasexamined in 309 intercalated cells

from 72 CCDs. Threepatternsof

pHi

response wereseen(Figs.

1-3). Onepatternis illustrated in Fig. 1. In this particular cell, the initial

pHi

was6.92. Removal of Cl- from the lumen

re-sulted inanincrease in

pHi

toasteady-state value of 7.05.

pHi

wasnotaltered further with removal of CF- from the bath. This

patternofCl--base exchangewasseenin 39% of the interca-latedcells studied with this protocol and is consistent with the existing model of the intercalated cell, an intercalated cell with only apical anion exchange. In these intercalated cells with only apical Cl--base exchange, mean baseline

pHi

was

6.94±0.06, mean dpH,/dt after apical Cl- removal was

1.16±0.17pH/min, andmean

ApHi

was0.33±0.04 pH units.

Asecondpatternof pH, responseisseenin Fig. 2. pH, was

7.1

7.0

pHj

6.9

-- L Cl

"Vf

- B Cl

M1f

4 6 Time (min)

8 10

Figure 1.

_pHi

response tosequential lumen and basolateral C1- re-moval (pattern 1). Rere-moval of lumenCl- results in intracellular

alkalinization, withnofuther change in

_pHi

from removal of bath C1-. Theseresults arecompatible with the classic , cell model.

7.4

-7.3

-

7.2-7.1

-7.0

-6.9

-- B Cl + B Cl

I

F

+ L Cl

41

7.2 pH.

7.1

-

7.0-15 1 7 19 21 23

Time (min)

Figure2.

pHi

responsetosequentiallumen and basolateral Cl-

re-moval(pattern 2).

_pHi

wasunchanged afterremovaloflumen Cl

-andincreased inresponsetoremovalofbathCl-. Thisresultwas

rarelyseenandis compatible withtheclassicacell model.

- B Cl

'if

+ B Cli - L CI

20 22 24 26 28 30 32 34

Time (min)

Figure4.

_pHi

responsetosequential lumenandbasolateral C1- re-moval(pattern3). Removal of bath Cl- caused

_pHi

toincrease. Readdition of bath Cl- resulted inrecoveryofpHi.Removalof

lu-menCl- resultedinanincreaseinpHi.

notaltered withremovaloflumen Cl-.H

bath Cl-resulted inanincrease inpH, fror

pattern ofCl--base exchange is consister model of theaintercalated cell,onewith

ionexchange. ThispatternofCl--base ex4

only 4% of CCD intercalated cells, and

quently that this celltypecouldnotbestum

Athirdpatternof

pHi

responseisseenii

baseline

pHi

was7.02. Removal oflumen Cl

tionwith subsequent stabilization of

pHi

at

Fig. 1, removal of basolateral Cl- in this

alkalinizationto

pHi

7.30, withadecreaseir

readdedtothe bath. This tracing isnotco

thecurrentaor intercalated cell models.

(Fig. 4), bath Cl- was removed initially,

creasein

pHi

to7.20.Readdition of bath Cl

return toitsbaseline, 7.05. After removal(

- L Cl

pH.

B Cl

0 2 4 6 8

Time (min)

Figure 3.

pHi

responsetosequentiallumen and moval(pattern3).After removalof lumen Cl-,I lowedbyafurther cell alkalinization inresponse Cl-. These findings indicate thepresenceof bilat change.Thisnewintercalated cell subtype is

desig

lowever,removalof increasedto7.35.ThesequenceofC1-removal madeno differ-n6.92to7.20. This ence in the pHi response seen. 57% of the intercalated cells

it with the current studied demonstratedthispattern of an increase in pHi after only basolateralan- bothbath andlumen Cl-removal. These results arebest

ex-changewasseen in plained by an intercalated cell with bilateral

Cl--base

ex-occurred so infre- changers

(-y

cell).In theseintercalatedcells with bilateral

C1--died inanydetail. base exchangers, baseline pHi was 7.06±0.06. The dpHi/dt

nFig. 3. In thiscell, after apical Cl- removal was 1.25±0.18 with a ApHi of

-caused alkaliniza- 0.30±0.04. The dpHi/dt after basolateral

C1-

removal was 7.21. Incontrastto 0.86±0.15 with aApHi of0.29±0.05 (all NS vs. intercalated cell caused further cellswith onlyapical anionexchange).

pH1 when Cl-was Furtherexperiments were done in separate tubules to

elimi-nsistent with either nate thepossibilityof anyconductive pathwayas a cause for Inaseparatestudy theapical andbasolateral

Cl--induced

pHichanges.The proto-resulting in anin- col wasrepeatedinthepresence of 5 uM luminal and

basolat--causedthe

_pHi

to eral high K+-valinomycin solutions (solution 4, Table I) to of lumenC1-,

_pHi

clampthe apicalandbasolateralmembranepotential. The re-sults of such an experiment are seen in Fig. 5. In this cell,

removal of lumen Cl- (solution 5) resulted in intracellular alkalinization,asexpected inacell withapical anion exchange.

+ BCi Removal of bath Cl-resulted in further alkalinization, which

wasreversible with readdition of Cl-tothe bath. The

dpHi/dt

afterapical Cl- removal in thepresenceof high

K+-valinomy-cin solutions in 32 intercalated cells with bilateral Cl --base exchange from 12 tubules was 0.92±0.15 with a

ApHi

of

0.25±0.05. The

dpHi/dt

after basolateral Cl- removal was

1.13±0.16 witha

ApHi

of0.24±0.04.

Although themeanratesof

dpHi/dt

inresponsetoluminal Cl- removalaresimilarforboth and intercalated cell

sub-types, anarrayofrateswasseen, asdepicted in Fig. 6. These

datasuggest thatwithin a given intercalated cell subtype

(f3

cells withonlyapical Cl--base exchange and ycells with

bilat-eralCl --baseexchange),there isawiderangeofCl --base

trans-1'0 12 14 portrates.

Effect

of

H2DIDS

on Cl--base

exchange

in CCD

interca-basolateralCl- re- lated cells with bilateral

Cl--base

exchange.

To further

charac-pHb

increasedfol- terize the CCD

intercalated cells

with

bilateral

Cl--base

ex-toremoval of bath change, the effect of the stilbene inhibitorH2DIDSon these

eral Cl--baseex- transporterswasstudied inseparateexperiment.Inthis proto-Ynatedastheycell. col,agivenintercalated cellwasfirstsubtyped functionally by

- L Cl

7.2

-7.1

pHj

7.0

-6.9

- B Cl- + B Cl'

Bl

I

2 4 6 8 10

Time (min)

Figure 5.

_pHi

response tosequential lumen and basolateralCl- re-moval inthe presenceof5,Mvalinomycin(lumen andbath).The

pHi

response toluminal and basolateral C1- removal did not differ from control cells,suggestingthatapical and basolateral Cl --base ex-changers, rather thanparallelCl-andH+ -base conductivepathways,

accountfor the results.

Cl- removal on both sides of the tubule. Then, 0.5 mM H2DIDS wasaddedtoeitherthe lumen orbathfor 20minand

the Cl- removal studieswererepeated inthe presence ofthe

stilbeneinhibitor.Fig.7shows the lack of effect of luminal 0.5 mMH2DIDSontheapical anion exchangerin arepresentative intercalated cellwith bilateral Cl--baseexchange.Baseline

_pHi

was6.79.Removalof lumen Cl- caused alkalinizationto

pHi

6.95. Cl- was returned to the lumen and 0.5 mMH2DIDSwas added for 20min.Then,luminalCl- wasagain removed, with asimilardegreeofalkalinizationresulting.The additional

alka-linization inresponse toremovalofbath Cl-indicates that this intercalated cellwas onewith bilateralCl--base exchange.

Simi-4.

.

a

7,, - L Cl + L0.5 mM H2 DIDS Cl

7.1

-I

- L Cl - B Cl

l'L-pHi

P~6.8X

6.5- , . . .

0 6 12 18 2 4 30 36 4 2

Time (min)

Figure 7.

_pHi

response of a -y intercalated cell in responsetoluminal Cl- removal before and after the addition of 0.5 mMluminal

H2DIDS. There was noeffectonthe intracellular alkalinization

re-sultingfrom removal of lumen Cl-after20-minexposureto0.5 mM luminal H2DIDS.

larstudies in40 intercalated cells(11 tubules)with bilateral Cl --baseexchangerevealed no difference in either theratesof

alkalinization in responsetoremoval of lumen Cl- after

stil-bene exposure or the resultant

ApHi

values

_(dpHi/dt

1.11±0.08 vs. 0.96±0.10,

ApHi

0.35±0.02 vs. 0.31±0.04,

paired, NS). These results demonstrate that the apicalanion

exchanger in CCD intercalated cells with bilateral anion

ex-changeis notH2DIDS-sensitive.

Fig. 8 shows the lack of effect of basolateral H2DIDSon the basolateral Cl --baseexchangerinanintercalated cell with

bilat-eral Cl --baseexchange.Removal of lumen Cl-causedpHito

increase,followedbyafurther cell alkalinization after removal of basolateral Cl-. After 20 min ofH2DIDSexposureon the basolateralside, sequentialCl- removalwasrepeated,withno

effect on theresulting degreeof alkalinization. Similar studies innineintercalated cells with bilateral Cl--baseexchange from

seventubules revealednodifference ineitherthe rates of

alka-linization in response to removal of basolateral Cl- after

stil-c;

0

E

ca

0

oC

ca

cm

a)

3- .

:

U

2

-

1-.

I

U

* * U

pHj

*

U.0

.

0 1 2 3 4 5

Apical Cl removal

Figure 6.dpHi/dtin response to luminal Cl-_{removal (x axis) vs. that}

observed with subsequent basolateral C1- removal (y axis) in indi-vidual intercalated cells. Rather than a clustering of values into two clearly separated groups, a wide range of results is evident. Note the

paucityofacells with onlybasolateralCl--base exchange.

0 4 8 12 1 6 20 24 28

Time (min)

bene exposure or the resultant

ApHi

values

(dpHi/dt

0.75±0.19 vs. 0.95±0.30,

ApHi

0. 12±0.02 vs. 0. 15±0.03, paired, NS). These results demonstrate that the basolateral

an-ion exchanger in CCDintercalated cells with bilateral anion

exchangeisnotH2DIDS-sensitive.

Lectinbinding in CCD intercalated cells. Binding ofpeanut

lectinto theapicalmembrane has beenused as a marker for the

classic(3intercalated cellmodel(3,9, 13). In eleven separate tubules,experimentswere done todeterminewhether interca-lated cells withbilateralCl--base exchange bind peanut lectin. The tubules were initially perfused with 50 ug/ml FITC-peanutlectin andcellsexhibitingapical peanut lectin binding were identified as seen in Fig. 9. Then, the perfusate was changed to onecontainingBCECF-AM. After dye loading, Cl-was removedsequentially from the apical and basolateral sides

ofthetubule, asdescribed previously.Of 44 CCD intercalated cells that boundapicalpeanutlectin, 48% had only apical an-ion exchange and 52% had both apical and basolateral anan-ion

exchange. Thus, CCD intercalatedcellswith bilateralCl--base

exchange do bind lectin. Therefore, apicalpeanutlectin

bind-ing is not aspecific marker for intercalated cells with

exclu-sively apicalCl--base exchange((3cells).

Pattern ofCl--base exchange among CCDs. The ability to studyseveralintercalatedcellssimultaneouslyin the same tu-bule permitted comparison of intercalated cell Cl--base

ex-changepatternswithinagiventubulealong a250-Mmlength.

Of 49tubulesinwhich4 to 7 intercalatedcells were studied, 41%ofthesetubules hadintercalated cellswith only one pat-ternof anion exchange. Fig. 10 illustrates the distribution of

anionexchange in intercalated cells of individual tubules with

eitherfour, five, six,or sevenintercalatedcellsstudied in each

tubule. Of those tubules withonly one pattern of intercalated cellanionexchange,35%ofthe tubules had exclusively interca-lated cellswithapical anionexchange

(13

cells) and 65% of the tubules hadexclusively intercalatedcells with both apical and

Figure9.Fluorescent image ofacorticalcollectingductafterluminal perfusionwith50Ag/mlFITC-peanutlectin. Afteridentification of

intercalatedcells thatbound lectintotheirapicalmembrane, the

tu-bulewasluminallyloaded withBCECF-AM, and Cl--base exchange wasassessedonboth sides ofthetubule.Inthistubule, cells between theopen arrows werestudied. Cellsdesignated byadarkarrow

ex-hibitedbothapical and basolateral Cl--HCO- exchange(eycells), while the remainingcells had only apical Cl--HCO- exchange((3

cells).

0 co

= M 0.

0_

con

7ra tm

' S.

ZD e _Q

00 c_ c ='

o

3t a

on E

I Io

=

on

_

Cm

., cO 3

m .

60-50 40

30

-20 10

# of intercalated cells

n=16 n=12

studied per tubule

Figure10.Percentageof tubules with fourtosevenintercalated cells

demonstratingthesameanionexchangedistribution. Thexaxis in-dicates the numberofcells studied ina250-MMlengthofan individ-ual tubule. Thenumberwithin the bars indicates the number of tu-bules studied. Thesamedistribution of intercalated cellanion

ex-changewas seenin38% of the 16 CCDs with 4 intercalated cells studiedpertubule, 33% of the 12CCDs with 5 intercalatedcells studiedpertubule, 50% of the 12 CCDs with 6 intercalated cells studiedpertubule,and 44% of the 9 CCDs with7 intercalated cells studiedpertubule.Takentogether,anaverageof 41% of all these

CCDs(49 tubules)had thesamedistribution of intercalated cell an-ionexchange.

basolateral anionexchange(ycells). The findingthat thereare

CCDs withoneintercalated celltypemayaccountfor transepi-thelial Hf -baseand Cl-transportdifferences thatexistamong

CCDs dissected from thesamelevelintheoutercortex.

Discussion

Intercalated cells in the CCD are presently divided intotwo

subtypes:aand cells.Thepurposeofthe presentstudywasto

functionally characterize the distribution of CL--base exchange activity in single rabbit intercalated cells in the outerCCD.

Cl--base exchange activity was documented by the

pHi

re-sponsestosequential removal of apical and basolateralCl- in

thesamecell. Theapproach allowedafunctional

determina-tion of whetherthetraditionalsubdivisionof intercalated cell

subtypesinto twocategories hasbeen overly simplistic. The

results demonstratethat themajority of intercalatedcells from

the rabbitouterCCD have bothapical andbasolateral

Na'-in-dependent Cl--base exchangersthatarestilbene-insensitive. In

keeping with the nomenclature given initiallytothe carbonic

anhydrase-rich cells of the turtle bladder,and then extendedto

CCDintercalatedcellsonthe basis ofmorphologic similarities, (3, 24, 27) weproposethat thenewlyidentified CCD

interca-lated cells with bilateral

Na'-independent

Cl--HCO-

ex-changersbe called ycells. Whether these cells also existinthe

turtlebladder is unknown.Inaddition, intercalatedcells with

only basolateral Cl--base exchange (a cells)arevery uncom-mon in the rabbitouter CCD. Intercalated cells with exclu-sively apical Cl--base exchange

((l

cells) and cellswith bilateral

Cl--base exchange (ey cells)wereboth foundtobind peanut lectinontheapical membrane. Surprisingly,alarge percentage

of CCDs hadintercalatedcells ofonlyonesubtype.

n=12 n=9

Onescientificconcernmustbe addressedregarding theuse

of

NaI-free

solutions inthis study.Itis

possible

that the

magni-tudeofanion exchange could be decreased duetothe lower

pHi

inthepresenceofzeroNa+ solutions.ANa+ -freeprotocol was

chosen because the purpose ofthis study was to examine

Na'-independent

Cl--base exchange. Inthepresenceof

Na',

other

Na'-dependent

transport processescouldminimize

pHi

changes.Itisunlikely that either

apical

orbasolateral Cl--base exchangewas

significantly

underestimated in this study forthe

following reasons. 96% ofthe 309 intercalated cells studied demonstrated

apical

anion exchange.

Therefore,

in

only

4%of the 309 intercalated cells would

apical

anion

exchange

have been missed. If basolateral anion exchange was

missed,

the

percentageofycells would beevengreaterthan57%.However, it is unlikely that basolateral anion exchange was underesti-mated dueto alow

pHi

effect

since,

after luminal C1- removal, the

starting

_pHi

increasedto - 7.20before basolateral Cl-

re-moval. Takentogether, it is unlikely that in

Na'-containing

solutions,

the percentages of intercalated cell

subtypes

would

havevaried

significantly

from the results reported in this

study.

Although, intercalated cellshavebeen

traditionally

divided intotwodistinct subgroups, severalrecentstudieshave identi-fied intercalated cells that donotfit into the conventional

cate-gories ofaand

_fl.

Basedoncellular

morphology

from transmis-sion electron

microscopy,

Ridderstraleand

colleagues

were

un-abletodivide rabbit CCDintercalated cells intotwo distinct

groups, but noted several ultrastructural manifestations that had

"4subtle

andgradual

differences,

withmanyintermediate variations"(14).Using antibodies

against

band 3 and

H+ATP-ase, Alperet al. found that 1% ofrat intercalated cells had

apical H'ATPase staining

with no band 3

labeling (15).

Schwartzetal.characterizedintercalated cells in

perfused

rab-bit CCDs with

6-carboxyfluorescein (6-CF) uptake,

peanut lec-tin

binding,

andluminal

endocytosis,

andfound5

categories

of cells: 6-CF

positive

only, 6-CF and lectin

positive

(consistent

with the 13 cell model), 6-CF and

endocytosis positive

(consis-tentwiththeacellmodel), lectinpositive

only,

and endocyto-sis positive only (29). A similar study by Schuster and co-workers combined lectin binding and antibodies against

H+ATPase

andband 3to

identify

subtypes of rabbit interca-lated cellsandfound that75%of intercalated cells had diffuse H+ATPase staining with eitherweak or bright apical lectin

caps,consistent withtheclassical,Bcellmodel.However, 13%

hadapical H+ATPase with bright apical lectin caps, 5% had

diffuse H+ATPase withnolectin

binding,

and4%had bright lectinstaining withno H+ATPase. Only 2%ofcells had

stain-ing characteristics compatible with the current a cell model,

apicalH+ATPasestaining with nolectinbinding(16). More

recently, Bastani et al. identified six patterns ofH+ATPase

immunocytochemistry inrat CCD intercalated cells:

well-po-larized apical staining,poorly polarized apical staining, diffuse,

apical and basolateral, poorly polarized basolateral staining, and

wvell

polarized basolateral staining ( 17). The present

re-suli-

provide the first functional evidence for an intercalated cell

tIiat

doesnot fit the classic a orf3 cell models. That the numberofCCD intercalatedcells with bilateral Cl--base

ex-change

('y

cells)identified by

pHi

measurements is greater than

the numberof "hybrid"cellsfound in the

immunocytochemi-calstudiesmay resultfromthelackofasuitable

immunocyto-chemicalmarkerfortheapical Cl --base exchanger.

Thedirection inwhich HCO- is transported by the CCD is

knowntobeafunction ofthe invivo acid-basestatusof the animal

(

18).

Basedonthe traditional intercalated cell

models,

theCl --base

exchanger

and

H+ATPase

inaand,Bintercalated cells arepresent on

opposite

cell membranes. Some authors

have

proposed

thata and A cellscan reversetheir

polarity

and

interconvert,

causing

a

change

inthedirection ofHCO-

trans-port

(3).

Remodeling

of the

apical

membrane with

endocyto-tic removal of the

apical

Cl--HCO-

exchanger

of 13 cells has

beendemonstrated inresponseto anin vitro acid

incubation,

although

development

ofnew basolateral transport systems

was not

investigated

inthis

study

(

19).

Thus,

reversalof func-tional

polarity

of intercalated cellswasnotdemonstrated.

There is evidence that suggests thata _{and a}intercalated cellsaredistinct celltypeswhich donotinterconvert. The

api-cal membraneof either rabbitor rat intercalated cells isnot

labeled with band 3

antibodies,

while the basolateral

mem-brane ofsomeintercalated cells does label with antibodiesto

band 3

(9).

HCO-

reabsorption

inrabbit CCDs

(presumably

anacell

function)

is blocked

by

basolateral stilbene

inhibitors,

but

apical

stilbenes donotinhibit HCO- secretion

(attributed

to13

cells)

as

reported

by

Schuster

(20).

Differences have also been noted in carbonic

anhydrase

II

staining

between

type

A

and type Brat CCD intercalated cells

_(22).

_Recently,

Satlin

andSchwartz foundadecrease innetCCD HCO- secretion in

response to an in vitro acid

incubation,

but no

change

in

HCO-

absorption,

suggesting

that reversal of functional

polar-ity

didnot occurbetweenaand 13 cells

_(23).

Theidentification

of

-yintercalated cells in the present

study

makes the issue of reversal of

polarity and/or

remodeling

ofaand 13 intercalated

cellsevenmore

complex.

That therate of

apical

vs. basolateral

Cl--base

exchange

varies

widely

amongycells under control

conditions

(see

Fig.

6)

suggeststhe

_possibility

that these

_cells,

as a

population,

may

not have a uniform contribution to

transepithelial

He-base

transport. The cell

currently

feltto be

responsible

for CCD HCO

secretion,

the :

cell,

sharesseveral characteristics with theycell. CCD HCO- secretion is unaffected

_by

luminal

stil-benes

(20).

The

apical

CL--HCO-

exchanger

of 13 andy inter-calated cells isnotinhibited

by

stilbenes.In

addition,

the

apical

membrane ofboth celltypesbinds peanut

lectin.

These similar-ities suggestthe

possibility

that both cells maycontributeto

CCD HCO- secretion.A ycellcouldmediate HCO- secretion ifits

apical Cl--HCO-

exchanger

functionedat agreaterrate

thanits basolateral

CL--HCO-

exchanger

and ifother transport

processes

(i.e.,

H+ATPase)

mediated

efflux

of

acid-equivalents

acrossthebasolateral membrane.

Immunocytochemical

studies suggest that 30% of rabbit CCD intercalated cellsareof thea

subtype (9). Using

luminal

endocytosis

as anacell

probe,

Schwartzetal.

found

that 21% ofintercalated cells in therabbitmid CCDwereacells

(20).

However,

the presentstudiessuggest thatcells with

only

baso-lateralCl--base

exchange,

consistent withthetraditionalacell model,aremuch lesscommonin therabbitouterCCD.This is

in

keeping

with the

findings

of Weinerand Hamm,

although

theanatomical location ofthe CCDsexaminedwasnot

speci-fied

(21

).

As

such,

thecell

responsible

for HCO-

absorption

in therabbitCCDisnotknownatpresent.A ycellcouldmediate

HCO-

absorption

if the basolateralCl--baserateexceeded the

apical

rate, and ifan

apical

transport

pathway

for efflux of

Cl--base exchangers. Net HCO- absorption is mediated by other apical H + efflux and basolateral base efflux pathways (28). Whether similar transport processes are present on they

cell is yet to be determined. Against the notion that y cells mediate HCO- absorption isthe findingthat the basolateral exchanger of these cells is unaffectedbystilbenes. CCD

HCO3

absorption, however, is stilbene sensitive. Further studies are needed todistinguishthe

pHi

regulatoryrole, the volume regu-latory role, and thetransepithelial He-base transportfunction oftheapicalandbasolateralanion exchangersin ycells.

Previousreportsof variability in CCD Cl-tracerfluxhave beenattributedtoaxialheterogeneity of intercalatedcell sub-typesalongtherabbit CCD. fi intercalated cells arethoughtto

providethemajorpathwayforCCDtransepithelial Cl- trans-port and are more common in the outer cortex, the region

where Cl- tracer flux isgreater (11, 24). However, another

explanation is provided by the findingin the present study that,

inmanyCCDs, theintercalatedcells are allofthe same sub-type. It is feasible that similar

H'-base

transport properties

amongintercalated cellsinthesametubuleaccountfor whole

tubule H+-base transport differences. The mechanism(s) of intertubuleheterogeneity isnotclear.DifferencesinCO2

ten-sionare not alikely explanation, given the known axial

gra-dient of CO2 inthe cortex(25, 26).Inaddition, it is unlikely

thatlocal hormonaldifferences could exist between individual

tubuleswithin thecortex.Furtherstudiesarerequired to better understandthis phenomenon of interCCD heterogeneity.

In summary,themajority of rabbitouterCCDintercalated cells

(y

cells) have both

apical

and basolateralCl

--HCO3

ex-changers which are stilbene insensitive. Apical peanutlectin bindstoboth f3 cells and y cells. Inaddition, inmanyrabbit

outerCCDs, intercalatedcellsinagiventubule areofthe same

subtype,

suggesting

thepresenceofintertubule heterogeneity.

Acknowledgments

This workwassupported byaVeteransAffairsCareer Development

Research Associate Award(toC.Emmons) and by a National Insti-tutesof Health grant(85 1-IG-4toI. Kurtz). Dr. Kurtzisan

Estab-lished Investigator of the AmericanHeartAssociation.

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